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3
T cells, or T lymphocytes, develop in the thymus, and
are a subfamily of circulating leucocytes that play an
important role in the adaptive immune response and
furthermore serve as crucial effector cells through antigen-specific cytotoxic activity and the production of
soluble mediators (cytokines/chemokines). The characteristic feature of all T cells are clonal antigen-specific heterodimeric receptor molecules on the surface
(T cell receptor; TCR). The accessory molecules CD4
and CD8 define the effector function and MHC restriction of T cells. Some T cells migrate to various locations throughout the body and interact with antibody
production of B cells. Subsets of T cells (CD4+) have
been classified as type 1 or type 2 T helper cells,
depending on the cytokines they produce. Another
subset is formed by the (CD8+) cytotoxic T cells.
Cancer and the Immune System
Streptococcus Infection and Immunity
Mast Cells
case letters (a, b). In the WHO nomenclature, human
locus and gene are given a combination of capital
letters and numbers (e.g. (TCR)BV8S1). Roman fonts
indicate gene products and italics the genes. The antigen-binding sites of the chains are formed by protein
loops called complementarity determining regions
(CDR), which are connected by conserved framework
regions. CDR1 and CDR2 and a fourth hypervariable
loop, which is involved in superantigen binding, are V
gene encoded. The CDR3 are VDJ or VJ encoded. The
high variability of the CDR3 results from the combination of the V and J , or the V, D, and J genes, as well
as additional events such as introduction of so-called
N- or P-nucleotides, and imprecision of recombination. The number of different TCR which can be created by these mechanisms exceeds that of lymphocytes
in the body.
Superantigens
Chronic Beryllium Disease
Mucosa-Associated Lymphoid Tissue
Cell-Mediated Lysis
Cytotoxic T Lymphocytes
Helper T Lymphocytes
Lymphocyte Proliferation
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T Cell
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3
3
3
3
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T Cell Antigen Receptor (TCR)
T Cell Antigen-Specific Receptor
The TCR is a molecule on surface of T cells composed
of two polypeptides (α and β chains) of nearly equal
molecular weights. Similar to antibody molecules the
TCR has N-terminal variable amino acid sequences
which combine to provide the individualized specificity (idiotype) shared by all TCR of a single cell of a
single clone; the C-terminal portion is common to all
α and β chains of the TCR. The antigen-specific receptor recognizes and binds peptides of thymus-dependent antigens (proteins) when presented by
class I and II molecules which are encoded in the
major histocompatibility complex (MHC). The peptides are generated by antigen-processing and antigen-presenting cells like macrophages, dendritic cells
and B cells.
Metals and Autoimmune Disease
Idiotype Network
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The clonally distributed antigen receptor of T cells is
composed of a heterodimer consisting of α and β or γ
and δ polypeptide chains, each containing one N-terminal variable (V) domain and one plasma membrane
inserted constant (C) domain. Both heterodimers are
expressed in association with the signal transducing
CD3 chains. Most T cells are CD4+ or CD8+, and
express the αβ TCR. They recognize foreign peptides
presented by self - MHC molecules. A small number
of T cells express the γδ TCR, which recognizes different types of mostly unknown ligands. Antigen recognition by γδ bearing T cells is not MHC restricted.
The antigen-binding domain of α or γ chains is encoded by the V (variable) and J (joining) genes, that of
β or δ chain by V, D (diversity) and J genes, which are
recombined during T cell development. The gene
names are usually numbered, with a Greek letter as a
suffix for the chain (e.g. Vβ 8.1 for the variable gene
8.1 of the β chain). Alleles are designated by lower-
T Cell-Dependent Antibody Response
Assays for Antibody Production
3
T Cell-Dependent Antigen
An antigen that requires the presence of T cell help to
stimulate the B cell to secrete antibody. Such antigens
do not elicit a productive antibody response by B cells
unless the B cell receives help from a CD4 T cell. Help
is generally supplied in the form of both a contactdependent signal via CD40 plus specific T cell-secreted cytokines. Generally, protein antigens are T-dependent antigens.
Plaque-Forming Cell Assays
Memory, Immunological
Immunoassays
The other six chains form the CD3 complex and consist of CD3γ which forms a heterodimer with CD3ε,
CD3δ which also forms a heterodimer with CD3ε and
a homodimer of CD3ζ. The CD3 chains are the targets
for kinase phosphorylation. It is the CD3 complex that
propagates the signal from the TCR complex to downstream signaling cascades.
Signal Transduction During Lymphocyte Activation
T Cell Selection
3
T Cell-Dependent Antibody Response
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Thymus: A Mediator of T Cell Development and
Potential Target of Toxicological Agents
3
3
An antigen that can stimulate B cells to secrete antibody in the absence of T cell help.
Plaque-Forming Cell Assays
3
T Cell Oligoclonality
This describes the restriction of an antigen-specific
T cell response to one or several T cell receptor configurations. Following HLA–antigen–TCR interaction,
this limited number of responder T cells undergo clonal expansion and increase the size of their TCR specific subpopulations.
Chronic Beryllium Disease
T cell receptors are composed of two different polypeptide chains, α and β chains (αβT cells) or, in a
minor population of T cells, of γ and δ chaisn (γδT
cells). The maturation of T cells occurs either in the
thymus or, under special circumstances, (γδT cells)
extrathymically. The first critical stage in T cell maturation is the successful rearrangement of the TCR β
chain (pre-TCR complex). A functional TCR β chain
initiates the arrangement of the TCR α locus and expression of both the CD4 and CD8 molecules
(CD4+CD8+ double positive thymocytes). In contrast
to these subsets of αβ T cells only little is known of
the physiological effector function or antigen specificity of γδT cells. The receptor of these T cells is much
more homogenous compared to αβ T cells. They are
found in epithelia from where they do not recirculate.
One hypothesis is that they participate in the pre-adaptive immune response.
Cancer and the Immune System
3
3
T Cell-Independent Antigen
γδT Cells
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T Cell Receptor (TCR)
Antigen Presentation via MHC Class II Molecules
T-Dependent Antibody-Forming Cell
Response
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T Cell Receptor (TCR) Complex
T Helper 1 Cells
3
The T cell receptor (TCR) complex consists of eight
transmembrane chains that are expressed on the surface of T cells. The TCRα and TCRβ chains bind
antigenic peptide when presented to them in the contact of MHC (major histocompatability) molecules.
Plaque Versus ELISA Assays. Evaluation of Humoral Immune Responses to T-Dependent Antigens
Helper T lymphocytes
T Lymphocyte
CD4 T cells that produce cytokines such as interleukins IL-4 and IL-5 but not IL-2 and interferon IFNγ.
By this they direct humoral immune responses.
Helper T lymphocytes
Flow Cytometry
3
Cytokines such as IFN-α and IL-2, are autocrine and
paracrine signaling molecules produced by CD4+
T cells in response to MHC class II antigen stimulation, and stimulate growth and activation of immunocytes and other inflammatory cells.
Chronic Beryllium Disease
T Helper 2 (Th2) Cells
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T Helper 1 Cytokines (Th1)
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T Helper 2 (Th2) Response
CD4 T cells that produce cytokines such as interferon
(IFN)γ, and interleukin IL-2 but not IL-4 and IL-5. By
this they direct cellular immune responses.
Helper T lymphocytes
Flow Cytometry
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T Helper 1 (Th1) Response
A reaction mediated by CD4-positive T cells that
serves to activate macrophages and promote digestion
of intracellular bacteria. Th1 cells secrete cytokines
such as interferon-γ (which activates macrophages)
and lymphotoxin-α (which activates macrophages, inhibits B lymphocytes, and is directly cytotoxic to some
cells).
Lymphocytes
Chronic Beryllium Disease
A reaction mediated by CD4-positive T cells that kill
infected cells and direct the destruction of extracellular
pathogens by activating B cells. Th2 cells secrete cytokines such as the interleukins IL-4 and IL-5 (which
activate B lymphocytes) and IL-10 (which inhibits
macrophage activation).
Lymphocytes
3
T Helper 1 (Th1) Cells
T Helper Cell
CD4+ helper cell subgroups that are defined by a different pattern of cytokine release. The Th1 subgroup
produces a cytokine profile to induce inflammation
and cell-mediated immunity. The Th2 subgroup produces a cytokine profile to induce antibody synthesis.
Both subgroups act antagonistically to each other to
secure an enhanced, but balanced immune response.
Cytokines
Maturation of the Immune Response
Leukocyte Culture: Considerations for In Vitro Culture of T cells in Immunotoxicological Studies
Food Allergy
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3
3
3
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T Helper 1–T Helper 2 Balance
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Helper T lymphocytes
Maturation of the Immune Response
T Helper Lymphocyte
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T Helper 2 Cells
T Helper Cell Polarization
3
Balance in immune response after contact with antigen, especially important for response to allergens
where T helper activity drives immune response either
to cellular (delayed-type hypersensitivity, Th1) or antibody (IgE, Th2) mediated allergic reaction. Rats
prone to a T helper 1 reaction (Lewis rats) showed
more resistence to Salmonella infection compared to
rats prone to a T helper 2 reaction (Brown Norway
rats).
Salmonella, Assessment of Infection Risk
Trace Metals and the Immune System
T Lymphocyte
White blood cell with characteristic appearance, cellsurface markers, and function. They undergo differen-
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3T3 Neutral Red Uptake (NRU) Test
tiation in the thymus. T lymphocytes control most
aspects of the immune response, and are involved directly in attack on virus-infected cells and aberrant
cells, such as malignant cells and cells originating
from a different individual (as in a transplanted organ).
CD Markers
Canine Immune System
Delayed Type Hypersensitivity
Tachypnea
Increased number of breaths per minute.
Septic Shock
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3
3
TAPA-1 (Target of an Antiproliferative
Antibody-1)
Trace Metals and the Immune System
3
Tachycardia
Heart rate above 100 beats per minute.
Septic Shock
The cytotoxic activity of immune cells is targeted towards specific cell types, which vary depending on the
cytotoxic cell type involved.
Limiting Dilution Analysis
Target Cell Killing
Cell-Mediated Lysis
Targeted Mutant Mouse
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T Suppressor Lymphocyte
Target Cell
3
T regulatory or suppressor T cells play important roles
in the regulation of immune responses and mediate a
dominant immunologic tolerance. The mechanisms by
which naturally occurring Tregs are able to suppress
CD4+ and CD8+ T cell proliferation are not yet
known. The CD4+CD25+ Tregs represent a subset of
suppressor T cells and have been shown to play a
critical role in the prevention of organ-specific autoimmunity and allograft rejection.
Transforming Growth Factor β1; Control of T cell
Responses to Antigens
A DNA probe (labeled with a fluorescent reporter dye
and a fluorescent quencher) used to detect specific
sequences in PCR products. When amplification occurs the Taqman probe is degraded by the 5' exonuclease activity of Taq DNA polymerase, thus separating
the quencher from the reporter. The increase of reporter dye fluorescence is used to determine the presence
of specific gene sequences.
Polymerase Chain Reaction (PCR)
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T Regulatory Cells (Tregs)
Taqman
3
The in vitro 3T3 neutral red uptake (NRU) phototoxicity test was developed and validated in a joint EU/
COLIPA project (1992–97). The aim was to establish
a valid in vitro alternative to the available in vivo tests.
The parameter for the detection of cell viability and for
measuring the total activity of a cell population is
based on the uptake of the vital dye neutral red into
cellular lysosomes of living murine BALB/c 3T3 fibroblasts.
Three-Dimensional Human Skin/Epidermal Models
and Organotypic Human and Murine Skin Explant
Systems
TAPA-1 (CD81) is a 26 kDa surface protein expressed
on the surface of B cells as well as T cells. It binds
several different integrins and is believed to be involved in activation, cell adhesion and migrations.
Signal Transduction During Lymphocyte Activation
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3T3 Neutral Red Uptake (NRU) Test
Knockout, Genetic
3
Tests for Autoimmunity
TbAT1 Genes
Trypanosomes are unable to synthesize purines de
novo and rely on nucleoside transporters. The Trypanosoma brucei adenosine transporter 1 (TbAT1),
also described as the trypanosomal P2-transporter, enables adenosine uptake. In addition, it confers susceptibility to antitrypanosomal drugs such as arsenicals.
Various point mutations have been identified in the
TbAT1 gene of resistant trypanosomes.
Trypanosomes, Infection and Immunity
629
tures. 2,3,7,8-TCDD has been assigned a toxic equivalency factor (TEF) of 1.0. TEF values for individual
congeners of dioxins, furans, and biphenyls in combination with their concentration can be used to calculate
the total TCDD toxic equivalents concentration
(TEQs) contriubted by all dioxin-like congeners in
the mixture using appropriate equations. Compounds
are included in the scheme and assigned a TEF if they
show structural relationships to PCDD or PCDF, bind
to the aryl hydrocarbon receptor, elicit aryl hydrocarbon receptor mediated biochemical and toxic responses, and persist and accumulate in the food chain.
Dioxins and the Immune System
3
3
TCDD
Dioxins and the Immune System
Teratogen
3
Any substance or exposure that causes birth defects.
Birth Defects, Immune Protection Against
3
Telomeres
3
TEQ/TEF
The complex nature of polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls, which are usually
generated together in occupational or environmental
exposure complicates the risk evaluation for humans.
This is a concept introduced to facilitate risk assessment and regulatroy control of exposure to these mix-
Testosterone
3
Telomeres are the physical ends of chromosomes.
They are specialized nucleoprotein complexes that
have important functions, primarily in the protection,
replication, and stabilization of the chromosome ends.
In most organisms telomeres contain repeated simple
DNA sequences composed of a G-rich strand and a Crich strand (called terminal repeats). These terminal
repeats are highly conserved—in fact all vertebrates
appear to have the same simple sequence repeat (up
to 2000 times) in telomeres (TTAGGG)n. After each
cell division, telomere shortening takes place. Telomere length is therefore indicative for the numbers
of divisions a cell has been through. Critically short
telomeres trigger replicative senescence and cell cycle
arrest. The innate immune system provides the first
line of defence against many microorganisms and is
essential for the control of common bacterial infections. It comprises macrophages, neutrophils, and natural killer cells. These cells of the innate immune response play also a pivotal role in the initiation of a
subsequent adaptive immune response.
Aging and the Immune System
Steroid Hormones and their Effect on the Immune
System
Tests for Autoimmunity
Raymond Pieters
Head Immunotoxicology
Institute for Risk Assessment Sciences (IRAS)
Yalelaan 2
P.O. Box 80.176
3508 TD Utrecht
The Netherlands
Short Description
A considerable number of chemicals, including many
drugs, are capable of inducing autoimmune-like diseases in man (1–3).
Autoimmunogenic chemicals rarely induce similar
clinical adverse effects in test animals and are hardly
ever identified in general toxicity testing. Hence, autoimmune-like symptoms often become apparent only
after introduction to the market. In combination with
the fact that these symptoms can induce very serious
or life-threatening conditions, the autoimmunogenicity
of chemicals, and drugs in particular, poses a huge
problem to certain sectors of society—patients, clinicians, pharmaceutical companies, and governmental
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Tests for Autoimmunity
agencies. Conceivably, there is an urgent need for
screening tests to identify such chemicals.
The main reason for the inability to assess a chemical's
potential to cause autoimmune-like diseases is that the
underlying mechanism is very complex, and can involve the interplay of many predisposing factors. An
important factor is the genetic make-up, the major
histocompatability complex (MHC) haplotype, nonMHC regulatory genes, metabolic polymorphisms,
and gender; but many environmental factors (such as
ongoing infections and food ingredients) are also
known to co-influence autoimmune phenomena (Figure 1).
The complexity of the etiology may be the reason that
only a few drug-using patients develop autoimmunelike derangements, but also explain why symptoms
suddenly appear after a long period of symptom-free
drug usage.
A set of rational criteria to establish autoimmune etiology of diseases in man was postulated in 1962 and is
reviewed in (4). One of the criteria requires the presence of circulating antibodies or cell-mediated autoimmunity. Others require that the corresponding autoantigen should be identified, and—more importantly
—that the disease can be reproduced by passive transfer of that antibody or the self-reacting cells or by
immunization with the self antigen.
Today, it is realized that autoreactivity (both autoreactive B as well as T cells) is a normal and necessary
property of a healthy immune system and that only
few self-reactive autoantibodies or autoreactive lymphocytes may be considered pathogenic (i.e. directed
against a pathologically relevant autoantigen and capable of causing tissue damage and reproducing disease
in experimental animals). The frequency of these
pathogenic autoreactive antibodies or lymphocytes
may be significantly higher in diseased compared to
control population (2,3).
Tests for Autoimmunity. Figure 1 Representation of
risk factors that are possibly involved in development of
autoimmune derangements. Adapted from (7).
Although autoreactivity is a healthy phenomenon and
changes in autoimmune-linked parameters do not necessarily result in an autoimmune disease, it is important to note that changes in such parameters may be
used to flag a chemical as possibly autoimmunogenic.
Characteristics
At present no clearly defined screening tests for autoimmunity in animals exist. The popliteal lymph node
assay (PLNA) is a simple straightforward local lymph
node assay that may be useful to screen for initial
immunostimulating capacity of chemicals. But this
assay can only be regarded as a first screening test
for immunosensitizing potential and to indicate that a
chemical might induce autoimmune-linked symptoms.
It is preferable for screening tests for autoimmunity to
use relevant exposure routes and demonstrate systemic
changes in parameters indicative of autoimmunelinked responses. Diagnosis of autoimmune-linked
diseases in test animals, like rats or mice, may be
based on a combination of general well-being, routine
clinical tests and (immuno)histology. Clinical investigations should include general hematology (e.g. to
check for anemias) as well as tests for complement
activity, or acute-phase proteins, and for erythrocyte
sedimentation. Liver and renal impairment should be
monitored biochemically (3). Morphologically, a wide
range of organs should be checked for indications of
inflammation, overt apoptosis (in the thymus in particular). Peripheral immunologic organs should be
checked for indications of activation (e.g. hyperplasia
or formation of germinal centers) (5).
Morphological indications of tissue inflammation, activated immune organs or immunomodulation (e.g.
thymus atrophy) should be followed up by more thorough investigations into alterations of autoimmune
parameters (5). Because development of actual autoimmune disease depends on a complex interplay of
(non)inherent factors (see Figure 1), relevant changes
in any of the animals should be considered as an alert
to pursue further investigations. This is certainly the
case in outbred animals which are used for evaluation
of toxicity, but also in inbred animals which are also
not always 100% responsive.
The initial focus in follow-up studies should be on
detection of autoantibodies, which can be directed
against a wide spectrum of autoantigens (2). In case
the target autoantigen is not yet known, and particularly for screening purposes, the indirect immunofluorescence (IF) technique may be useful. The immunofluorescence technique, which is also used in the clinic, has been used successfully in animal studies.
Briefly, cryosections or isolated cells grown on microscopic slides (for instance HepG2 tumor cells for antinuclear antibodies (ANA) or freshly isolated granulocytes for antineutrophil cytoplasmic antibodies
Tests for Autoimmunity
(ANCA)) are an incubated with serum suspected to
contain autoantibodies followed by a incubation with
fluorochrome-labeled second-step antibody. Interestingly, the immunofluorescence technique can be applied to cryosections of a range of relevant organs
(such as kidney, thyroid, liver, skin, adrenals, and
sex organs), although false-positive staining (perhaps
as a result of antibody binding to Fc receptors) particularly in inflamed tissue has to be taken into account.
When the specificity is known, autoantibodies can be
detected by various other techniques (most notably
enzyme-linked immunoassay, ELISA). In many
cases, it may suffice to perform an ELISA for ANA.
Compound-specific lymphocyte transformation tests
(LTT) can be used in cases of drug allergy or chemical
exposure (see for instance Schnyder et al 2000) but
detection of autoreactive T cells in case of chemicals is
much more difficult. This is mainly due to the fact that
the relevant autoantigen (chemically altered or previously cryptic epitopes) is hardly ever known, and
also because specific autoreactive T cells are relatively
scarce even in clinical situations. A solution would be
to immortalize selected self-reactive T cell clones, but
this is not easy to incorporate in a general testing
model for autoimmunity.
Pros and Cons
All of these methods may at best provide circumstantial evidence for autoimmune effects and/or etiology in
animals. The advantage of these methods is that they
can all be used in animal toxicity studies without interference with the study per se, and only ask for more
extensive analyses of samples (blood, serum and organs) that are already to be isolated at dissection. But,
importantly, as the immunological effects depend
greatly on genetic make-up, autoimmune effects may
be easily missed when small groups of outbred test
animals are used. So animal tests for autoimmunity
(including the parameters discussed here) should be
done with larger test groups and should be performed
over relatively long periods of exposure (> 90 days).
Importantly, adverse effects which are indicative of
autoimmunity—even if they occur in only one animal—should already be taken as an alert to execute
follow-up studies with inbred animal strains, such as
the frequently used high-IgE-responding Brown Norway (BN) rat. To date, only a limited number of compounds (e.g. HgCl2, gold salts, d-penicillamine, nevirapine, hexachlorobenzene) have been shown to induce
autoimmune-like phenomena in this rat strain.
Relevance to Humans
Chemical-induced autoimmune effects detected in animals can be predictive for the human situation. However, as in humans the prevalence of autoimmune effects will be low in (outbred) animals as well. Studies
631
with particularly sensitive rat strains, for instance, such
as the BN rat, may identify much better the hazard of
autoimmunogenic potential of a chemical. Notably,
such a sensitive rat has to be regarded as a representative of very susceptible humans.
Regulatory Environment
At present guidelines for detection of autoimmunogenic capacity do not exist. It should be realized that
none of the present animal models—including the BN
rat model—is capable of detecting autoimmunogenic
potential of a wide range of different chemicals. The
popliteal lymph node assay (PLNA) is an animal
model that may indicate whether a chemical is immunostimulatory. Immunostimulation may result in sensitization of the immune system and is considered one
of the prerequisites for inducing autoimmunity.
A number of the parameters proposed here, however,
could be easily or are already incorporated in existing
guidelines. For instance, the OECD guideline 407 includes the hematology, clinical biochemistry and pathology of a series of organs. But without further analyses of (auto)antibody levels, larger test groups of
inbred animals and long exposure periods
(> 90 days) a chemical's potential to induce autoimmunity will hardly ever be detected in these toxicity
studies.
So, future research to design predictive protocols and
screening models is greatly needed. This could be initiated by thorough research into the relevance of the
above-mentioned parameters in repeated-dose studies
over a relatively long period with inbred strains of rats
(e.g. BN and Lewis strains) as well as mice (e.g. SJL
and C3H/He strains), but also in outbred animals that
are normally used in toxicity studies. Such studies
should first be performed in a limited number of
well-equipped laboratories, and should be followed
by more extensive ring studies.
References
1. D'Cruz D (2000) Autoimmune diseases associated with
drugs, chemicals and environmental factors. Toxicol
Letters 112–113:421–432
2. Verdier F, Patriarca C, Descotes J (1997) Autoantibodies
in conventional toxicity testing. Toxicology 119:51–58
3. D'Cruz D (2002) Testing for autoimmunity in humans.
Toxicol Letters, 127:93–100
4. Shoenfeld Y, Isenberg D (eds) (1990) The Mosaic of
Autoimmunity, Factors Associated with Autoimmune
Disease. Introduction. Research Monographs in Immunology, Volume 12. Elsevier, Amsterdam
5. Frieke Kuper C, Schuurman H-J, Bos-Kuijpers M,
Bloksma N (2000), Predictive testing for pathogenic
autoimmunity: the morphological approach. Toxicol
Letters 112–113:433–442
6. Schnyder B, Burkhart C, Schnyder-Frutig K et al. (2000)
Recognition of sulphamethoxazole and its reactive
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2,3,7,8-tetrachlorodibenzo-p-dioxin
metabolites by drug-specific CD4+ T cells from allergic
individuals. J Immunol 164:6647–6654
7. Kammüller ME, Bloksma, N, Seinen W (1989) Immune
disregulation induced by drugs and chemicals. In:
Kammüller ME, Bloksma N, Seinen W (eds) Autoimmunity and Toxicology. Elsevier, Amsterdam, pp 3–25
Three-Dimensional Human Skin/
Epidermal Models and Organotypic
Human and Murine Skin Explant
Systems
Hans-Werner Vohr . Eckhart Heisler
2,3,7,8-tetrachlorodibenzo-p-dioxin
Dioxins and the Immune System
PH-PD, Toxikology
Bayer HealthCare AG
Aprather Weg 18
D-42096 Wuppertal
Germany
3
Synonyms
Tetravalent Vanadium
Tetravalent vanadium is the ionic form of vanadium
when four outer shell electrons (that is, two from 4s
and two from 3d orbitals) have been shed, thereby
giving the atom an overall charge of +4.
Vanadium and the Immune System
3
TGF-β1
Transforming Growth Factor β1; Control of T cell
Responses to Antigens
human skin recombinants, reconstructed human skin/
epidermis, 3-D human skin/epidermal equivalents, in
vitro engineered skin/epidermal substitutes, artificial
skin/epidermis, organotypic murine or human skin explant system, MSE, HSE, hOSEC
Definition
Human full-thickness skin models and reconstituted
epidermal equivalents are in vitro-engineered tissue
cultures that provide a three-dimensional architecture
which is biochemically, morphologically and functionally comparable to human epidermal tissue/skin in
vivo. Organotypic skin explant systems are based on
ex vivo skin removed from humans or mice and subsequently cultured in toto. All the models were shown
to be useful in screening for topically applied irritating, corrosive or photocytotoxic compounds. Results from experiments with systemically applied compounds have already been published with such models, too. Furthermore, in recent studies it was demonstrated that 3-D skin models also provide the capacity
to further characterize and screen for substances with a
sensitizing potential.
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3
Characteristics
Reconstructed Human Epidermal Models
Reconstructed human epidermal models are built up
from proliferating, differentiating and cornifying keratinocytes which are airlift-cultured on a porous polymeric membrane. The design of the cell culture conditions (air-liquid interphase and medium/ingredients)
drives the cells to differentiate and form a three-dimensional (3-D) epidermal multilayer with a functional and stratified surface. Most of the key structural
elements of native epidermis like keratins, transglutaminase and lipid composition that characterize
the status of keratinocyte differentiation are present
in 3-D human epidermal equivalents.
3
An important mechanism in the immune regulation
involves homeostasis between the T helper 1 (Th1)
and T helper 2 (Th2) activity of CD4+ T helper cells
expressing different cytokine patterns. T helper cells
showing Th1 activity are more prone to induce a cellmediated immunity whilst T helper cells obtaining
Th2 activity are more prone to induce a humoraltype immune response. T helper cells showing either
Th1-type or Th2-type reactivity are exclusively characterized by differences in cytokine expression.
Briefly, Th1 reactivity is predominantly connected to
interferon (IFN)-γ, IL-2, and IL-12 secretion. In contrast Th2 cells express mainly IL-4, but also IL-5, IL6, IL-10 and IL-13. The Th1/Th2 balance is integrated
in the immune regulation in a dynamic and reversible
manner, depending also on kinetics and dose–response
of the immune response.
Cancer and the Immune System
3
Th1/Th2 Balance
3
Three-Dimensional Human Skin/Epidermal Models and Organotypic Human and Murine S
633
Three-Dimensional Human Skin/Epidermal Models and Organotypic Human and Murine Skin Explant
Systems. Figure 1 Two different reconstructed tissues: H&E stained sections of untreated reconstructed human
full thickness skin model (Advanced Cell Systems, AST-2000) and epidermal model (Skinethic RHE ). Both models
are comercially available. (Picture of AST-2000 by kind permission of Advanced Cell Systems, St. Katharinen,
Germany).
Organotypic Skin Explant Systems
Organotypic skin explant systems from human, rats or
—to a lesser extent—mice have also been established
for evaluating percutaneous absorption and penetration. However, in comparison to reconstructed skin
models the explant cultures naturally provide a physiological cell composition and micro-architecture including immunocompetent cells (e.g. Langerhans
cells).
For toxicological and immunotoxicologic research,
both topical treatment (application of compounds to
the dry stratum corneum) as well as systemic-like
treatment (application of substances directly into the
cell culture medium) are possible using 3-D skin mod-
els. Hazard identification is based on the measurement
of decreased cell viability and changes in tissue morphology after treatment (histological examination; see
below). In recent studies it was also shown that topical
and systemic-like treatment of 3-D skin models with
hazardous compounds often results in induced expression and/or release of immunomodulating proteins
(cytokines, chemokines, matrix metalloproteinases,
growth factors, and other parameters which are involved in a variety of biochemical pathways; see
below). The determination of these parameters gives
a detailed overview of the cell status which can additionally confirm the results from viability testing and
histological examinations (multiple endpoint analysis;
MEA).
Screening for Irreversible Cutaneous Toxicity
(Corrosion)
Screening for Acute Irritation
Both artificial skin models and organotypic skin explant systems are suitable for screening for dermal irritation induced by topically applied irritating or
photo-irritating compounds and formulations. Most
likely in this situation is that in vivo substances with
a strong irritant potential provoke severe destruction
of the reconstructed or explanted tissues and affect the
integrity of residential cells. The use of these systems
to test chemicals, compounds, or formulations according to their irritant properties depends on the measurement of cell viability after topical treatment with compounds and additional time-related incubation. Cytotoxic and photocytotoxic effects cause a significant
3
Full-Thickness Human Skin Substitutes
Full-thickness human skin substitutes additionally provide a dermal layer that usually consists of a collagen
matrix which is populated by living fibroblasts. In an
early state of research the use of de-epidermized
human dermis as the backbone of full-thickness skin
equivalents has been discussed as well. In comparison
to single-cell culture systems, the most predominant
feature of these artificial skin models is the existence
of a physiological and functional barrier (the stratum
corneum) that regulates percutaneous absorption/penetration of compounds as well as transepidermal water
loss. Although the barrier functions of artificial skin
models are different from the situation in vivo, the
results from studies evaluating the penetration properties of various reference test compounds have shown a
good correlation to in vivo data.
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Three-Dimensional Human Skin/Epidermal Models and Organotypic Human and Murine S
decrease in cell viability. For this reason, determination of cell viability is essential for the assessment of
compound biocompatibility using artificial skin models or organotypic skin explant systems. However, in
most of the published test protocols MTT conversion
is used as a single endpoint parameter for the determination of cell viability and consequently the degree of
cytotoxicity caused by irritation and photoirritation.
Recently the identification of more specific parameters
allows a multiple endpoint analysis (cell viability, histological examination, release of IL-1α;).
Expression of Immunomodulating Proteins and
Screening for Dermal Sensitization
Both irritation and sensitization of the skin are related
to the expression and release of immunomodulating
proteins such as cytokines, chemokines and cell surface proteins, especially within the epidermis. The
local immune system of the skin in vivo is based on
the interactions between epidermal keratinocytes, epidermal Langerhans cells, and dermal fibroblasts. Once
activated by antigen uptake and processing, Langerhans cells undergo morphological changes and start to
migrate to the local draining lymph nodes. There
T cells become activated upon successful antigen presentation. In cases of cutaneous irritation causing epidermal cell damage, keratinocytes release a cocktail of
proinflammatory proteins from their intracellular reservoirs. This finally results in a non-specific activation of the skin’s immune system (see also contact
hypersensitivity section).
Considerable efforts have been made to integrate Langerhans cells into reconstructed human skin models.
However, there is still no complex in vitro system
available that provides functional antigen-presenting
cells in the epidermis or dermis. Nevertheless, keratinocytes are also thought to be involved in the initial
steps of irritation and sensitization. Topical treatment
of artificial skin models with irritating compounds
leads especially to the release of interleukins IL-1α
and IL-8 by keratinocytes. Furthermore, the subsequent analysis of cell culture supernatants by different
ELISA techniques (enzyme-linked immunoassay) additionally show an induced release of different chemokines and cytokines as shown in Table 1.
The profile of released proteins depends on the kind of
model used for the experiments. In comparison to reconstructed epidermal models, full-thickness skin
models provide a set of parameters that are related to
the interaction between epidermal keratinocytes and
dermal fibroblasts. In recent studies carried out with
sensitizing substances the ratio between IL-1α and IL8 release after topical treatment with the compounds
revealed promising results that suggest that reconstructed human skin models are capable of discriminating sensitizers from compounds with an exclu-
sively irritant potential. Other studies identified promising parameters (increased release of the chemokines monocyte chemoattractant protein 1 (MCP-1)
and interferon-inducible protein (IP-10) from a
human full-thickness skin model AST-2000 after treatment with the standard sensitizer (oxazolone) that certainly can contribute to a successful discrimination
between sensitizers and irritants in vitro.
From an immunological point of view, however, it is
of prime importance for sensitization testing to analyze
parameters ( MIG, Langerin, TARC, etc.) that are
characteristic for the cross-talk between keratinocytes,
fibroblasts and antigen-presenting cells in their natural
setting. For this reason, research on skin sensitization
(screening, mechanistic) is particularly focussed on the
use of organotypic skin explant systems as well as the
development of skin recombinants that incorporate
functional antigen-presenting cells.
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634
Pros and Cons
Experimental Strategies
Methods used in in vitro dermal toxicology are often
based on single-cell culture systems, which in turn are
built up from either freshly isolated primary cells derived from cosmetic surgery, foreskins or well-established cell lines. Methods for cytotoxicity and photocytotoxicity testing, like the 3T3 neutral red uptake
(NRU) test, have been successfully validated. However, test principles based on single-cell cultures are subject to some limitations due to their lack of a physiological barrier. For this reason they are usually restricted to soluble substances and therefore fail when
it comes to testing hydrophobic compounds or formulations. Furthermore, the concentrations of compounds
inducing irritation in single-cell cultures are significantly lower than those determined in in vivo experiments. Due to the absence of a stratified surface, false
positive results may also occur, because substances
may be classified as (photo)cytotoxic by 3T3 NRU
although they are physicochemically unable to pass
through the physiological barrier (the stratum corneum).
By using 3-D skin models it is possible to overcome
these problems, and they offer a promising test system
for topical and systemic-like compound administration. Furthermore, artificial skin models and organotypic skin explant systems may be suitable for screening for sensitizing properties of compounds in vitro.
With respect to this last point research is still in progress, but a convincing system may be available in the
near future.
Test Principles
As already mentioned, MTT testing is often used as a
single-endpoint parameter for predicting the irritant
potentials of substances, although cytotoxicity is not
3
3
Three-Dimensional Human Skin/Epidermal Models and Organotypic Human and Murine S
635
Three-Dimensional Human Skin/Epidermal Models and Organotypic Human and Murine Skin Explant
Systems. Table 1 Expression/release of immunomodulating proteins from 3-D skin models
Parameter
Expression/Release
Release Inducible
Interleukin-1α
++ (a,b,c)
Yes
Interleukin -1β
+ (b,c)
Slightly
Interleukin -6
+++ (+a,b,c)
Yes
Interleukin -8
+++ (+a,b,c)
Yes
Tumor necrosis factor-α
+ ((a),b,c)
Slightly
Monocyte chemoattractant protein MCP-1
+++ (+b,c)
Yes
MIG
+ (c)
Slightly
Interferon-inducible protein IP-10
+ (b,c)
Slightly
Macrophage inflammatory factor MIP-3α
+ (c)
Slightly
Matrix metalloproteinase MMP-3
++ (+b,c)
Slightly
Matrix metalloproteinase MMP-9
++ (b,c)
Yes
a, epidermal model; b, full thickness skin model; c, organotypic skin explant system; +, low level; ++, medium level; +++, high
level; (+), high background.
a sufficient stand-alone parameter for predicting cutaneous irritation. In vitro testing associated with MTT
conversion is always subject to some limitations, because the test principle is based on a chemical redox
reaction which may also run without any participation
of living cells. This may lead to false positive results.
Another problem with MTT, especially concerning 3D skin models, was observed when test results were
compared to histological examinations of reconstructed skin models after compound treatment. Due
to cellular activity, formazan crystals were found to be
formed especially in the cells from the basal layer. For
Three-Dimensional Human Skin/Epidermal Models
and Organotypic Human and Murine Skin Explant
Systems. Figure 2 H&E stained section of Skinethic
RHE after treatment with 0,4% SDS and 24 hours of
incubation (5% CO2, 37°C, max hum.) The area
marked with the red arrow shows massive destruction
of cells in the upper epidermal layers. However, the
basal layer (blue arrow) is not affected. Here, MTT test
gave false negative results. Although cell viability was
correctly determined, the integrety of the cells in the
upper epidermal layer was hardly affected. This effect
however, was undetectable by MTT alone (By kind
permisson of SkinEthic Laboratories, Nice, France).
this reason, it is not possible to detect undesired compound-related effects on cells from the stratum spinosum or stratum granulosum by MTT (see Figure 2).
Other test principles for the determination of cell viability are based on the quantitative analysis of enzymes from the cytosol of cells. When cells lose
their integrity through damage to the plasma membranes, the leakage of these proteins can be recorded
and quantified by bioluminometric or other optical enzymatic test systems.
In this context the measurement of lactate dehydrogenase (LDH) and/or adenylate kinase leakage is often
discussed as a defined parameter for the analysis of
substance-related cytotoxic effects on in vitro cell systems.
Finally, the induced release of proinflammatory mediators like IL-1α additionally serves as a good parameter for the characterization of skin irritation, because
IL-1α was found to be released from cells which are
influenced by irritating chemicals. Although MTT is a
reliable and valid parameter for the analysis of cell
viability, the results should be supplemented and verified additionally by multiple endpoints, such as expression and release of proinflammatory mediators,
decrease of the barrier function determined by transepidermal water loss (TEWEL) and/or evaluation of
morphological changes (histologic examination).
Comparison to In Vivo Test Principles
The replacement of in vivo methods for corrosivity
and irritancy according to Draize by in vitro reconstructed skin models is often discussed, especially
from an ethical point of view. In addition, the use of
3-D skin models is less time-consuming than in vivo
T
636
Three-Dimensional Human Skin/Epidermal Models and Organotypic Human and Murine S
testing, and if the costs for animal health and care are
taken together, reconstructed skin models are less costeffective than animal testing, too. However, a ranking
between strong irritation and weak or mild irritation
based on experimental results from testing with reconstructed skin/epidermis still seems to be questionable.
The establishment of in vitro test methods for sensitization is not that easy, although protein fingerprinting
of cells from organotypic skin explant systems and
reconstructed epidermal/skin models revealed promising results that contribute to the in vivo situation. In
recent studies it was shown that the expression and
release of immunomodulating proteins (Table 1)
serve as good parameters for the characterization of
compounds with sensitizing properties. However, the
use of these parameters as criteria for predicting sensitization has not been validated so far. For this reason,
guinea pig assays like those described by Buehler or
Magnusson and Kligman are still the most reliable
methods for sensitization testing, even though they
are based on visible subjective parameters like the formation of erythema. In this context, another valid
method is LLNA/IMDS (local lymph node assay/integrated model for the differentiation of (chemical)-induced skin reactions) which characterizes sensitizing
compounds with the help of cellular parameters, but is
still based on animal treatment.
Predictivity
Irritation of the skin caused by exposure of individuals
to different kinds of hazardous compounds or formulations is the most common non-specific immune reaction observed in human skin. In vivo (animal) test
principles according to the methods of Draize are frequently used for the identification of substances with
irritant potential. For several reasons, however, these
test methods are questionable. The analysis of substances according to Draize testing is mainly based
on the evaluation and scoring of macroscopic parameters such as overcasting of the rabbit eye cornea
or redness of the skin after treatment with the compounds being tested. As far as this point is concerned,
it has recently been shown that the choice of endpoints
for the assessment of acute skin irritation according to
international standards (methods according to Draize)
may lead to misclassification of substances. Furthermore, the transfer of established data from animal testing to the human situation in vivo is still controversially discussed. For this reason the human patch test
was established. This ideally meets the requirements,
but patch testing in human is restricted to weak or
moderate irritating compounds. These pragmatic disadvantages of in vivo animal and human testing for
skin corrosion or acute skin irritation are furthermore
accompanied by the discussion of the ethical justification of animal testing in toxicological research. With
the use of reconstructed tissue models it is possible to
overcome most of the problems described above.
From multiple endpoint analysis (see Characteristics)
reliable parameters are available that are simple to
determine, while the output is more stringent than visual evaluation of results.
Furthermore, artificial skin models were proven to be
reproducible in intra- and interlaboratory multicenter
studies. As mentioned above, the predictivity of reconstructed tissue models is limited. In comparison to
human in vivo skin the different physiological barrier
function of the reconstructed stratified surface may
cause problems because the risk of false positive results cannot be totally excluded. In addition, distinguishing between weak and moderate irritating compounds is sometimes not easy. However, research is
focussing on new parameters that could help to solve
these problems. Despite this early state of affairs, it is
possible to state that human reconstructed tissue models exhibit acceptable predictivity in screening for corrosive compounds (sensitivity and specificity > 80%).
Although the validation and catch-up validation studies for acute irritancy of topical applied formulation
and/or raw materials are still in progress, a high correlation of sensitivity has already been estimated by
the relevant ECVAM Task Force.
Another main topic of interest concerns alternative in
vitro models for skin sensitization. At present, no reconstructed tissue model is available that meets the
guideline criteria for adequate screening. However,
considerable efforts have been made to search for
parameters (cytokines, chemokines) which specifically
characterize the complexity of the processes leading to
skin sensitization (skin penetration, formation of protein-hapten complexes, antigen uptake and processing,
migration of LC to the local draining lymph nodes,
presentation of antigen to T cell populations in the
draining lymph nodes). In the light of this complexity,
the use of organotypic skin explant systems seems to
be very promising, because they provide the same
micro-architecture and the same cell composition as
in vivo skin and are therefore potent tools for mechanistic studies.
Relevance to Humans
The test results from animal testing for irritancy and
corrosion according to Draize are controversially discussed among toxicologists. In cases of acute irritation
these test methods have never been validated and they
principally depend on a collection of cross-connected
empirical clinical and preclinical data. For this reason,
the use of reconstructed human tissues is of particularly great value, because the cells used for these skin
constructs are of human origin. Although some differences in the characteristic barrier function have been
Three Rs
described, the experimental design closely matches the
human situation in vivo.
Unfortunately, screening for sensitization in vitro is
even more complex because artificial tissue structures
are necessary which must in addition provide immunorelevant cross-talk activities. For hazard identification, on the other hand, fingerprinting of proteins released from 3-D in vitro skin models has already been
evaluated and some of these parameters were shown to
hold key positions in immunological pathways (IL-8,
MCP-1, IL-1α, IL-6, etc.). These may therefore help
to screen for compounds with a sensitizing potential in
vitro. As mentioned earlier, human skin explant systems in particular are believed to be very suitable
models for further characterization of immunorelevant
parameters. In the heat of discussion about testing for
sensitization, one should keep in mind that in vivo
animal testing (guinea pig assays or the (modified)
local lymph node assay) or human patch testing, as
well as all possible in vitro models which are going
to be established and validated in the future, are not
capable of taking all parameters influencing the induction of skin sensitization into account (individual parameters such as genotype, age, sex, side of contact/penetration and of course the overall condition of the
skin).
Regulatory Environment
Skin Irritation/Corrosion
The international standards for skin irritation and corrosion are still based on in vivo test principles according to the methods of Draize et al. (1944). However,
the considerable efforts of organizations like ECVAM,
ICCVAM, COLIPA, the Steering Committee on Alternatives to Animal Testing (SCAAT) have had a favorable and lasting influence on the establishment of in
vitro test methods of international guidelines. The use
of several in vitro human skin models for skin corrosion was validated by ECVAM in 2000. For acute skin
irritation, however, a first prevalidation study failed
but the process of improving the use of reconstructed
tissue models especially in this field of toxicologic
research is strictly ongoing.
Guidelines for Determination of Substance-Induced
Skin Corrosion
* OECD Guideline 402: Acute Dermal Tox.
* OECD Guideline 404/405: Acute Dermal/Occular
Tox Irritation and Corrosion
* OECD Guideline 410: Repeated Dose Dermal Tox.
* OECD Guideline 430: In Vitro Skin Corrosion—
Rat TER (Trans Epidermal Resistance) Test
* OECD Guideline 431: In Vitro Skin Corrosion—
Human Skin Models
* Annex V of Directive 67/548/EEC (1997)
* US Code of Federal Regulations (1991)
637
Sensitization
Up to now no in vitro screening model has been available to correctly predict exclusively sensitizing properties of compounds. From an immunological point of
view this is not surprising because of the lack of antigen-presenting cells in most of the reconstructed
human tissues. However, the induced release of immunomodulating proteins indicates promising parameters for successful discrimination between irritating
and sensitizing substances. As long as none of the
reconstructed or organotypic models match the criteria
for a successful prevalidation study, immunotoxicologic research must rely on guinea pig test principles
according to Bühler, Magnusson and Kligmann, or
on a refined test assay like the LLNA or the integrated
model for the differentiation of (chemical)-induced
skin reactions (IMDS).
Guidelines for Determination of Substance-Induced
Sensitization
* OECD Guideline 406: Skin Sensitization (1992)
* OECD Guideline 429: LLNA (2002)
* U.S. EPA-OPPTS Harmonized Test Guideline
870.2600 on Skin Sensitization (1998)
* FDA (CDER) (Draft) Immunotoxicology Evaluation of Investigational New Drugs (2001)
* CPMP/SWP/398/01 (Draft) Note for Guidance on
Photosafety Testing (2001) (as modified LLNA)
References
1. Botham PA, Earl LK, Fentem JH, Roguet R, van de Sandt
JJM (1998) Alternative methods for skin irritation testing:
the current status. ECVAM Skin Irritation Task Force
Report 1. ATLA 26:195–211
2. Zuang V et al. (2002) Follow-up to the ECVAM
prevalidation study on in vitro tests for acute skin
irritation. ECVAM Skin Irritation Task Force Report 2.
ATLA 30:109–129
3. Spielmann H et al. (2003) Report of the Second
SkinEthic Workshop: In Vitro Reconstructed Human
Tissue Models in Applied Pharmacology and Toxicology
Testing, Nice, France
4. Coquette A, Berna N, Vandenbosch A, Rosdy M, De
Wever B, Poumay Y (2003) Analysis of interleukin-1
alpha (IL-1 alpha) and interleukin-8 (IL-8) expression
and release in in vitro reconstructed human epidermis for
the prediction of in vivo skin irritation and/or sensitization. Toxicol In Vitro 17:311–321
5. Heisler E, Ahr HJ, Vohr HW (2001) Local immune
reactions in vitro: Skin models for the discrimination
between irritation and sensitization. Exp Clin Immunobiol 204:1–2
Three Rs
Reduction (fewer animals), refinement (less severe
T
Thrombin
Thrombin is a multifunctional serine protease that has
procoagulant activities when diffusable in the blood
stream. But it loses this ability and initiates a potent
anticoagulant pathway when bound to its endothelial
cell receptor thrombomodulin, thereby mediating generation of the anticoagulant enzyme-activated
protein C. The cellular activities of thrombin on platelets, endothelial or smooth muscle cells are mediated
through G protein-coupled protease-activated receptors (PAR) that are initially cleaved by thrombin before a newly generated peptide motif of the receptor
can serve as an internal tethered ligand for initiation of
cell signaling.
Blood Coagulation
Thymus: A Mediator of T Cell Development and
Potential Target of Toxicological Agents
Thymocyte Education
Thymus: A Mediator of T Cell Development and
Potential Target of Toxicological Agents
Thymocyte Selection
3
3
Thrombin
Thymocyte Development
3
procedures), and replacement (in-vitro alternatives) of
animal experiments, first proposed by Russel and
Burch in 1959.
Canine Immune System
3
638
Thymus: A Mediator of T Cell Development and
Potential Target of Toxicological Agents
Thymus
3
C Frieke Kuper
Thrombocytopenia
Thrombocytopenia is a condition in which the normal
concentration of platelets (thrombocytes) in the blood
is decreased. A significant shortage of platelets can
result in bruising and easy bleeding.
Leukemia
Antiglobulin (Coombs) Test
Toxicology and Applied Pharmacology
TNO Food and Nutrition Research
Zeist
The Netherlands
Synonyms
Thymus, thymus gland, sweetbread (when used as
food)
3
3
Definition
Thrombocytopenic Purpura
A rare autoimmune disorder characterized by a shortage of platelets, leading to bruising and spontaneous
bleeding. Approximately half of the cases are idiopathic (unknown cause). Other cases are caused by drugs,
infections or autoimmune disorders such as lupus erythematosus.
Interferon-γ
3
Thymic Hypoplasia
The thymus is a primary lymphoid organ in vertebrates; in mammals it is located in the cranioventral
mediastinum and lower part of the neck. The prime
functions of the thymus in mammals are the development of immunocompetent T lymphocytes from bonemarrow-derived stem cells, the proliferation of mature
naive T cells to supply the circulating lymphocyte pool
and peripheral tissues and the development of immunological self-tolerance. The thymus elaborates a number of soluble factors (thymic hormones) which regulate several immune processes, including intrathymic
and post-thymic T-cell maturation, and neuroendocrine processes such as the synthesis of neuroendocrine hormones by the central nervous system.
Characteristics
An immunodeficiency that selectively affects the
T lymphocyte limb of the immune response. There is
lymphopenia with diminished T cell numbers.
Trace Metals and the Immune System
Anatomy and Histology
The thymus is located in the cranioventral mediastinum and lower part of the neck, whereas small islands
of thymic tissue may be present near the thyroid and
3
Thymus
parathyroid glands. In young animals it is roughly
pyramid-shaped with its base located ventrally. The
gland consists of two lobes, fused in the midline by
connective tissue. The two thymic lobes are enclosed
by a fibrous capsule from which septa traverse into the
organ, dividing it into lobules. The lobules have basically the same architecture, with a subcapsular area, a
cortex, corticomedullary junction and a medulla.
The cortex is easily recognizable in hematoxylin and
eosin(H&E)-stained sections by its high density of
thymocytes (immature lymphocytes) and therefore
darker appearance when compared with the less densely populated medulla. The framework of the thymus
is formed of epithelial reticular cells in which the
bone-marrow-derived lymphoid (thymocytes/lymphocytes) and non-lymphoid cells (macrophages, dendritic
cells) are packed. The vast majority of lymphocytes
are T cells, but accumulations of B cells do occur.
Epithelial aggregates with centrally located cell debris,
the so-called Hassall’s bodies, are a characteristic
feature in the medulla.
The different thymic compartments are associated with
different T cell maturation processes, namely early
(cortical) maturation and late (medullary) maturation,
which in turn are associated with differences in the
marker expression and cytology of epithelial cells,
lymphocytes, macrophages and interdigitating cells
(Figure 1).
Moreover, the capacity of epithelial cells to synthesize
thymic hormones differs, the major site of hormone
synthesis being the medullary epithelium (1). A characteristic and unexplained microenvironment is
formed by the cortical and medullary areas which
are devoid of epithelial cells but full of thymocytes,
the so-called epithelial-free areas or EFAs (2). The
function of these EFAs is unknown, although medullary EFAs may be associated with autoimmune diabetes. Foci of myelopoiesis are found in the connective
tissue septa, within the lymphoid tissue at the outer
rim of the lobules, and at the corticomedullary zone.
Hemoglobin-containing cells can be found among the
myelocytic series in the interlobular septa, at the outer
rim of the lobules. In the medulla no erythroid precursors have been observed. Blood vessels enter the
lobules via the interlobular trabeculae/septa and
branch at the corticomedullary area to supply the cortex and medulla. Postcapillary venules in the corticomedullary region have a specialized cuboidal epithelium similar to that of the high-endothelial venules of
the lymph node, which allows passage of lymphocytes
into and out of the thymus. Sheaths of connective tissue and an epithelial cell layer with its basement membrane are found around the blood vessels. The space
between the epithelial basement membrane and the
vessel lining is often quite broad around the corticomedullary vessels and is called the perivascular space.
639
This space may contain all kind of blood cells and
most often contain fine lymphatics. Nerves course
along the blood vasculature.
During ontogeny, hematopoietic progenitor cells migrate into the thymic epithelial primordium between
days 11–13 of fetal life in mice. Small lymphocytes
can be found in the thymic primordium at about
day 14 (mouse) or day 15 (rat) of fetal life. The thymus is fully developed, meaning a cortex and medulla
can be distinguished, at day 17 of fetal life in the
mouse and by days 19–21 in rats, and the organ
grows considerably immediately after birth. This
growth is caused by the immense postnatal antigen
stimulation; at that time large numbers of mature
T cells are demanded. The thymus starts to involute
after adulthood is reached. With age, the two thymic
lobes diverge caudally and in old animals are almost
completely separated; the thymus is then restricted to
the area cranially to the aortic arch. The number of
lymphocytes decrease, especially in the outer cortex.
Although areas with different lymphocyte density,
suggesting the presence of cortex and medulla, are
often present in advanced age, the general arrangement
of the cortex enclosing the medulla is not strictly
maintained. This gives the thymus an irregular appearance. The expanding perivascular connective tissue
meshwork and increasing perivascular lymphocyte accumulations may further disturb the normal pattern.
The septa and capsule harbor increasing numbers of
adipose cells, which eventually invade the thymic parenchyma.
In addition to the expansion of the connective tissue
component, epithelial cords and tubules are large and
numerous in the old thymus and the epithelial Hassall’s bodies become relatively more prominent though
in absolute numbers they decrease. Adrenergic innervation of the gland is maintained in old animals. Thymic involution may be related to changes in the hormonal status of the individual; circulating thymic hormone is reduced to very low levels in adults. The
consequences of age-related involution are obvious:
the emigration of lymphocytes from the thymus
shows a dramatic decrease. Apparently, the persistent
generation of new antigen-recognition repertoire in the
T cell population of adults is not needed. Instead, the
body can defend itself using the established repertoire
and extra-thymic self renewal of the T cells. Pregnancy
in rodents results in radical, but reversible changes.
After an initial rise in thymic weight in early pregnancy, involution starts with lymphocyte cell death in the
cortex. In wild populations, cyclical enlargement and
regression is documented. For instance, most birds
showed an involuted thymus at the time of mating
and laying, whereas on subsequent egg incubation
the thymus size is increased.
T
3
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640
Thymus
Thymus. Figure 1 Schematic presentation of a thymus lobule with cortex, corticomedullary region, medulla and
an epithelial-free area (EFA). In the lobule, a simple overview of thymocyte maturation is presented: round cells
representing T lymphocytes (T) with their membrane markers CD4 and/or CD8
T-Cell Maturation
T cells reside in the thymus during their maturation
from progenitor cells to immunocompetent T cells.
The process of T-cell maturation includes a number
of steps which are associated with location in different
microenvironments (3). (See Figure 1).
The immature cells, which enter the lobules by the
blood vasculature at the corticomedullary junction,
first move to the outer subcapsular cortex, where
they appear as large lymphoblasts. They then pass
through the cortex where the cells become small lymphocytes with scanty cytoplasm. Finally, the cells
move to the medulla, where they appear as mediumsized lymphocytes. These translocational stages in development can be monitored by the immunologic phenotype: cells change from CD4−CD8− (double negative) at a very immature stage into a CD4+CD8+ (double positive) phenotype, which is characteristic for almost all lymphocytes in the cortex. In the medulla,
T cells have the phenotype of relatively mature cells,
with distinct CD4+CD8− (about 70%) and CD4+CD8+
(about 30%) populations. This phenotypic change is
accompanied by a crucial aspect of intrathymic T-cell
maturation: the genesis of the T cell receptor (TCR)
consisting of the alpha-beta heterodimer (4). The DNA
genomic organization encoding these chains is in
germ-line configuration, with a variety of gene segments encoding the variable part of the receptor molecule. Before transcription and translation into TCR
becomes possible, combinations have to be made of
gene segments encoding the variable and constant
parts of the TCR.
This process of gene rearrangement requires the thymus microenvironment. The cell can synthesize the
receptor after completion of this gene rearrangement.
The receptor is then expressed on the cell membrane
with the CD3 molecule, which acts as the transmembrane signal-transducing molecule after TCR stimulation. Even when the TCR has not yet been synthesized, this CD3 molecule is already present in the cytoplasm of the cell. T cells at this stage of maturation
Thymus
can be recognized by cytoplasmic staining with CD3
reagents.
TCR gene rearrangement is similar to the rearrangement of genes encoding immunoglobulin heavy and
light chains that takes place in the bone marrow microenvironment. However, after surface expression of
the TCR, the cell undergoes a process unique to
T cells, namely, specific selection on the basis of recognition specificity. First, the cell is examined for its
affinity for its own major histocompatibility complex
(MHC; self restriction). T cells with an intermediate
affinity for self MHC peptides are allowed to expand
(positive selection). Secondly, T cells with a high affinity for self MHC are deleted (negative selection). In
this way, the random pool of antigen-recognition specificities of T cells is adapted to the host's situation.
The T cell repertoire in germ-line configuration cannot
be fully expressed but is influenced by the individual's
own MHC haplotype.
It is generally accepted that the epithelial microenvironment of the thymic cortex plays a major role in
positive selection. This microenvironment expresses
MHC class I and class II products and morphologically (at electron microscopic level) shows close interactions with lymphocytes. This close interaction is reflected by the complete inclusion of lymphocytes inside the epithelial cytoplasm (thymic nurse cell).
Negative selection has been ascribed to either the epithelial compartment or the medullary dendritic cells.
The cortex can be considered a primary or central
lymphoid organ because of its antigen-free microenvironment. In contrast, antigens can move relatively
freely into the medulla and encounter antigen-presenting dendritic cells as well as antigen-reactive T cells.
Thus the medulla has properties of a secondary lymphoid organ.
3
Preclinical Relevance
The dynamics of the thymus with ongoing reactions of
cell proliferation and differentiation, and gene amplification, transcription and translation makes it highly
susceptible to toxic insults. Compounds that interfere
with these processes are often immunotoxic. Therefore, a decrease of thymus weight in preclinical studies
is often a first indicator of toxic action of a xenobiotic
agent on the immune system, although some compounds, like cyclosporine, profoundly alter thymic histophysiology, without apparent effect on thymus
weight. The dynamic nature of the immune system
provides it with great regenerative capacity: the original architecture of the thymus is restored rapidly following involution induced, for example, by irradiation, or treatment with glucocorticosteroids or organotin compounds.
Thymus in aged or immunocompromised animals may
hardly be visible. For histology adipose and connec-
641
tive tissues from the cranioventral mediastinum, which
contains thymic tissue, should then be collected. The
thymus is also very susceptible to acute (glucocorticoid-related) stress (5). It is conceivable that with age
the thymus becomes less sensitive to toxic insults and
that toxic effects on the thymus with age have less
functional importance, because of age-related thymic
involution. However, the components that constitute
the various thymic compartments are still present in
healthy old animals, as was shown by reconstitution
studies. Therefore, a decreased sensitivity to toxic
compounds may not be a general property of the involuted thymus in aged animals.
Relevance to Humans
The use of data obtained in laboratory animal species
for man presents difficulties when species differ in
organ anatomy and histophysiology and sensitivity.
The thymus is present in all vertebrates, possibly
with few exceptions, and there are only a few structural differences between the species (6). Anatomical
differences relate to thymus location and number of
thymic lobes, the prominence of epithelial aggregates
with centrally located cell debris, the so-called Hassall’s bodies, and the presence of B cell follicles. During the third month of gestation the thymic primordium becomes colonized by marrow-derived stem cells.
When these stem cells are indeed thymocyte precursor
cells, their migration into the thymic primordium at
that time is considerably earlier—relative to gestation
time—in humans than in mice or rats. Differences in
immunotoxicity between laboratory animals and man
appear to depend predominantly on differences in toxicokinetics and metabolism of substances. Moreover,
the interindividual differences and the age-related intraindividual variations are probably more marked
than interspecies differences. It should be emphasized
that the “normal” architecture of the thymus, as known
from textbooks, can be expected only between the late
gestational period and young adulthood, and before
pregnancy.
The universality of the immune system observed in
mammals and the data obtained so far indicate that
data from laboratory animals can be extrapolated
quite well to humans.
Regulatory Environment
Regulatory toxicity testing, which uses immune parameters, is still under development. This applies to
pharmaceuticals and industrial substances as well.
Nevertheless, most guidelines recognize the importance of the thymus. For instance, the European
Union guidelines on repeated-dose toxicity testing
with pharmaceuticals require the macroscopic and microscopic examination of the spleen, thymus, and
some lymph nodes with respect to the immune system.
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Thymus: A Mediator of T Cell Development and Potential Target of Toxicological A
Moreover, a multilaboratory, 28-day oral toxicity
study (OECD guideline 407) with the model immunotoxicants azathioprine and cyclosporine demonstrated
that the most consistent effects were observed in the
thymus (7).
References
1. Dabrowski MP, Dabrowski-Bernstein BK (1990) Immunoregulatory Role of the Thymus. CRC Press, Boca
Raton
2. Bruijntjes JP, Kuper CF, Robinson J, Schuurman H-J
(1993) Epithelium-free area in the thymic cortex of rats.
Dev Immunol 3:113–122
3. Van Ewijk W (1991) T-cell differentiation is influenced
by thymic microenvironments. Ann Rev Immunol 9:591–
615
4. Werlen G, Hausmann B, Naeher D, Palmer E (2003)
Signaling life and death in the thymus: Timing is
everything. Science 299:1859–1863
5. Godfrey DI, Purton JF, Boyd RL, Cole TJ (2000) Stressfree T-cell development: glucocorticoids are not obligatory. Immunol Today 21:606–611
6. Zapata AG, Cooper EL (1990) The immune system:
comparative histophysiology. In: The Thymus. John
Wiley, Chichester, pp 104–150
7. International Collaborative Immunotoxicity Study (ICICIS) Group Investigators (1998) Report of validation
study of assessment of direct immunotoxicity in the rat.
Toxicology 125:183–210
Thymus: A Mediator of T Cell
Development and Potential Target of
Toxicological Agents
Michael Laiosa
NIAID/NIH
Bethesda, MO 20897
USA
Allen Silverstone
Upstate Medical University
166 Irving Ave.
Syracuse, NY 13210
USA
Synonyms
T-cell development, thymocyte development, T-cell
selection, thymocyte selection, thymocyte education,
positive selection, negative selection, thymus, thymus
atrophy, thymus involution
Definition
T-cell development is the process by which hematopoietic progenitor cells from the bone marrow home to
the thymus and undergo a complex process of differentiation, proliferation and selection to become mature
T-cells that will emigrate from the thymus to peripheral lymphoid organs such as the spleen and lymph
nodes. Additional maturation and differentiation into
T-helper (Th) type 1 and Th type 2 subsets occur in the
periphery and are discussed elsewhere.
Characteristics
The thymus is the central organ for T-cell development in the body, and the principle function of the
thymus is to regulate T-cell recognition of self antigens
presented by the body to insure that useless or selfreactive T-cells do not mature. T-cell development is
characterized by progenitor cells that originate in the
fetal liver or bone marrow and enter the thymus
through the blood stream (1). The thymocytes then
undergo a highly regulated process of differentiation,
proliferation, selection, and maturation to become Tcells. The stages of murine thymocyte differentiation
can be distinguished by differentially expressed surface molecules stained with fluorochrome-labeled
antibodies and detected using flow cytometry. The
thymocyte subpopulation that appears earliest is identified by expression of the lymphoid homing receptor
CD44 and cKit, the receptor for the stem cell factor,
(CD44+CD25−, DN1) (1). Subsequently, the high affinity interleukin receptor IL-2α (CD25) and the heat
stable antigen (HSA, CD24) are upregulated and the
proliferation rate of this population also increases
(DN2) (1). Following expression of CD25, CD44 is
down modulated leading to the next stage of differentiation, CD44−CD25hi (DN3) (1). In the DN3 population, the αβ and γδ T-cell antigen receptor (TCR)
lineages begin to diverge as recombination activating
gene products 1 and 2 (RAG1, RAG2) begin somatic
gene rearrangement of the TCR β locus (1). Successful rearrangement and surface expression of a functional TCR β chain in a complex with the pre-Tα
protein results in a burst of proliferation and the gradual reduction of CD25 expression on the cell surface
(DN4) (1). Subsequent to successful expression of
TCR β, rearrangement of TCR α begins and the
CD8 and CD4 molecules are expressed on the cell
surface(1). It has been calculated that it takes 3–
4 days for a DN3 cell to differentiate into the DP
stage of T-cell development (1).
Once TCRα rearrangement is complete, the
CD4+CD8+ double positive (DP) thymocytes begin a
rigorous selection process by engaging their αβ TCR
with complexes of self peptides bound to major histocompatibility complex (MHC) class I and II proteins
(1), expressed by epithelial, myeloid, and dendritic
antigen presenting cells (APCs) in the cortex of the
thymus (2). The TCR-MHC interaction leads to one of
three possible outcomes depending on the nature of
3
642
Thymus: A Mediator of T Cell Development and Potential Target of Toxicological A
which can interrupt or inhibit various stages of T-cell
development, which ultimately leads to atrophy of the
thymus. Agents that have been shown to cause thymic
atrophy in vivo include corticosteroids, estrogens and
estrogen-like compounds, polychlorinated biphenyls
(PCBs), and polychlorinated dibenzodioxins and dibenzofurans (PCDD and PCDF). Representative
agents that are known to induce thymic atrophy and
possible mechanisms by which they can induce atrophy are listed in Table 1 (4,5).
Evidence of thymic atrophy after toxicant exposure
has a relatively strong correlation to predicting if an
agent will be immunotoxic as defined by classic immunotoxicity assays such as delayed-type hypersensitivity (DTH), and the sheep red blood cell (SRBC)
challenge assay (6). However, linking immunotoxicant-induced defects in thymic development to deficiencies in a functional response has been a major
obstacle in the field of immunotoxicology. Relating
thymic atrophy to alterations of functional responses
have suffered from a lack of data and agreement on the
type of assays, kinetics, and dosing protocols to be
used.
Relevance to Humans
The thymus has been shown to be essential for development of T-dependent immune responses. Indeed,
DiGeorge syndrome who
patients with the rare
lack a thymus present with a severe immunodeficiency
associated with a complete lack of T-cells. The DiGeorge T-deficiency can be completely restored by
the transplantation of an allogeneic thymus graft (7).
The essential role for the thymus in T-cell development has been further appreciated in recent clinical
studies. These studies show that despite the longstanding observation of thymus atrophy with increasing
age, the adult thymus is fully capable of producing and
selecting new T-cells following periods of systemic Tcell depletion. Following chemotherapy, production of
new thymic-derived naive T-cells has been observed
(7). Additionally, infection with HIV has been shown
to cause a dramatic thymic pathology characterized by
thymic atrophy and a block in T-cell development at
the CD3−CD4−CD8− stage of development. However,
thymopoieis can be restored in some HIV patients
undergoing highly active antiretroviral therapy
( HAART) (7). Finally, evidence of TCR gene rearrangement in recent thymus emigrants has been observed in normal adults of at least 60 years of age (7).
These data strongly support an active and dynamic
role for the thymus organ in mediating new T-cell development throughout an individual’s life.
The effect of immunotoxicants as mediators of thymic atrophy in humans has been controversial and
difficult to assess for some time. The lack of consensus
on whether a particular toxicant can cause thymic at3
3
3
3
the interaction. TCRs with no or weak affinity for
MHC will die by neglect. In comparison, potentially
self-reactive TCRs with too high or strong affinity for
the peptide MHC complex undergo negative selection.
Only TCRs with the appropriate affinity for peptide
MHC complexes will undergo maturation, CD4
(class II MHC) or CD8 (class I MHC) lineage commitment and positive selection (1).
The signal transduction that results in positive selection begins with phosphorylation of the intracellular
portion of the TCRζ chain by the src kinase Lck.
Phosphorylation of TCRζ results in the subsequent
recruitment of Zap70, which becomes activated and
phosphorylates the linker of activated T-cells (LAT).
The phosphorylated LAT acts as a docking complex,
which recruits and activates a number of molecules
involved in TCR signal transduction and calcium ion
(Ca2+) flux (3). The generation of a Ca2+ flux has been
shown to depend on phospholipase C γ (PLCγ),
which generates inositol-3-phosphate (IP3) and diacylglycerol (DAG) (1). IP3 is responsible for the increase
in intracellular Ca2+ and leads to the activation of the
calcineurin pathway and the NFAT family of transcription factors (1). In contrast DAG is involved in activating protein kinase C (PKC) family members and
can be a mediator in activation of the Ras pathway. In
DP thymocytes it is thought that DAG activates the
guanine nucleotide exchange factor RasGRP1 leading
to activation of the extracellular signal-related kinase
(ERK) (1). ERK activation in thymocytes undergoing
positive selection is thought to be involved in activating the early growth response-1 (EgR-1) nuclear transcription factor (1). The positively selected DP thymocytes then upregulate Bcl-2 and mature to become
either class II restricted (CD4+; T helper) or class I
restricted (CD8+; T cytotoxic) single positive thymocytes. Additional selection occurs in the medulla of the
thymus before final maturation and emigration of the
SP T-cells into the periphery (1).
Although negative selection results in a profoundly
different outcome (cell death rather than maturation)
many of the signaling pathways utilized are the same
or similar. Most current data on thymocyte selection
favor a model where the affinity between a TCR and
self peptide-MHC complexes determines whether a
thymocyte will be positively selected or deleted.
High affinity interactions with TCR and self peptideMHC may activate additional signaling pathways such
as the Jnk pathway, which ultimately lead to apoptosis
(1). In comparison, TCRs with weak or no affinity for
self peptide-MHC complexes will die by neglect in the
thymus within 1–3 days (1). Only thymocytes possessing the appropriate affinity and duration of binding
between a TCR and self peptide-MHC complexes can
be positively selected (1).
A number of toxicological agents have been identified
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T
3
3
644
Thymus: A Mediator of T Cell Development and Potential Target of Toxicological A
Thymus: A Mediator of T Cell Development and Potential Target of Toxicological Agents. Table 1 Agents
known to cause thymic atrophy and mechanism of atrophy induction1
Agent
Mechanism
Androgens
Loss of DP thymocytes; mediated by androgen receptor
Cisplatin
Apoptosis in proliferating thymocytes
Cyclosporin A
Prevents Ca++ mobilization; inhibits positive selection; delayed
negative selection
Dexamethasone (and other corticosteroids) Apoptosis in DP thymocytes
Dibutyl and tributyltin
Possible apoptosis; inhibition of proliferation of DN thymocytes
Diethylstilbestrol (DES), estradiol, estrogens and estrogen-like chemicals
No evidence of apoptosis, possible effects on progenitors and cell
cycle; estrogen receptor-mediated
Ethylene glycol monomethyl ether
Reduction in DP thymocytes, but no evidence of apoptosis
Reduction of lymphocyte progenitor capacity
Ethanol
Apoptosis; increase in CD4+ mature cells, loss of CD25+ DN cells;
evidence of Ca++ increase and protein kinase C activation
Malnutrition, vitamin deficiency
Increase of glucocorticoid levels; apoptosis of DP thymocytes
2,3,7,8, tetrachlorodibenzo-p-dioxin
No evidence of apoptosis in vivo; inhibition of bone marrow
progenitors; inhibition of cell proliferation in thymic DN cells; all
effects mediated by the aryl hydrocarbon receptor
T-2 toxin and other mycotoxins
Elimination of putative lymphocyte progenitor cells in fetal liver; no
evidence of apoptosis induction.
1
Adapted from Luster et al. (4) and Silverstone (5)
rophy is due in part to the obvious ethical considerations with human studies. Moreover, the vast majority
of immunotoxicity assays that have been developed
are in rodent models that possess inherent flaws
when attempting to determine dose, pharmacokinetic,
and risk assessment comparison models to humans.
The challenges of relating risk assessment models to
humans should be overcome in the future as immunotoxicologists begin to develop nonhuman primate
models, novel in vitro models and comparative toxicogenomic studies to fill in the gaps in knowledge
about particular toxicants as related to T-cell development and immunotoxicity (6,8,9).
3
Regulatory Environment
Regulatory agencies in the USA have recently started
to stress the importance of understanding how immunotoxicants affect the developing immune system in
children. The need to understand the effects of immunotoxicants in children is particularly important because of the possibility that during the period when
the immune system is most actively developing, it may
be especially sensitive to the effects of an immunotoxicant. Moreover, immunotoxicant exposure in children
may lead to more severe effects and/or a higher risk
for long-term deleterious outcomes when compared to
doses determined for adults (9). Although there are
currently limited data comparing adult and child re-
sponses to immunotoxicants on the developing immune system, several possibilities for differences
exist. An immunotoxicant may affect the developing
immune system of a child but not an adult. Furthermore, an immunotoxicant may affect the developing
immune system of a child at a lower dose than in an
adult (9).
In an attempt to get the full picture about childhood
exposure to immunotoxicants and the effect of exposure on the developing immune system of children,
several EPA sponsored workshops have listed the
need for expanding exposure studies in very young
animals as a high priority. These workshops include
the EPA sponsored workshop on endocrine disruptors
held in 1995, and the EPA sponsored workshop by the
Risk Science Institute of the International Life
Sciences Institute held in 1996. More recently, the
EPA added a recommendation to the two-generation
reproductive study (OPPTS 870-3800), stating: for F1
and F2 weanlings that are examined macroscopically,
the following organs should be weighed for one randomly selected pup per sex per litter: brain, spleen and
thymus (9). The recommendation to use thymus and
spleen weights was made because numerous studies
have concluded that thymic and splenic weight may
be immunotoxicant predictors (6).
In 2001 the EPA created a developmental immunotox-
Tight Junctions
Thymus-dependent antigens (TD) are protein antigens
which only can induced an antibody response with the
help of thymus-derived T helper cells. This T cell help
is also essential for the class switch observed during
TD immune responses.
Idiotype Network
Thymus Gland
The thymus is a primary lymphoid organ, the site of Tcell development. It is situated in the anterior superior
mediastenum, behind the breastbone. The organ, in
particular its epithelial cells and connective tissue provide the microenvironment wherein thymocytes proliferate, rearrange their T-cell receptor genes, and undergo positive and negative selection. The thymus
slowly atrophies after puberty, but can become fully
functional again in clinical situations like radiation
therapy and stem cell transplantation.
Thymus
Dioxins and the Immune System
Thymus: A Mediator of T Cell Development and
Potential Target of Toxicological Agents
Systemic Autoimmunity
3
3
3
Thymus Involution
T
3
1. Starr TK, Jameson SC, Hogquist KA (2003) Positive and
negative selection of T cells. Ann Rev Immunol 21:139–
176
2. Anderson G, Jenkinson EJ (2001) Lymphostromal
interactions in thymic development and function. Nat
Rev Immunol 1:31–40
3. Germain RN, Stefanova I (1999) The dynamics of T cell
receptor signaling: complex orchestration and the key
roles of tempo and cooperation. Ann Rev Immunol
17:467–522
4. Luster MI, Dean JH, Germolec DR (2003) Consensus
workshop on methods to evaluate developmental immunotoxicity. Environ Health Persp 111:579–583
5. Silverstone AE (1997) T cell development. In: Sipes G,
McQueen CA, Gandolfi AJ (eds) Comprehensive Toxicology, 1st edn. Elsevier Science, New York, pp 39 ff
6. Holladay SD, Blaylock BL (2002) The mouse as a model
for developmental immunotoxicology. Hum Exp Toxicol
21:525–531
7. Spits H (2002) Development of alpha-beta T cells in the
human thymus. Nat Rev Immunol 2:760–772
8. Buse E, Habermann G, Osterburg I, Korte R, Weinbauer
GF (2003) Reproductive/developmental toxicity and
immunotoxicity assessment in the nonhuman primate
model. Toxicology 185:221–227
9. Holsapple MP (2003) Developmental immunotoxicity
testing: a review. Toxicology 185:193–203
Thymus-Dependent Antigen
3
References
Loss of thymocyte weight and cellularity after exposure to an immunotoxicant.
Thymus: A Mediator of T-Cell Development and
Potential Target of Toxicological Agents
3
Lastly, in 2003, the National Institute of Environmental Health Sciences (NIEHS) and National Institute for
Occupational Safety and Health (NIOSH) cosponsored
a consensus workshop on methods to evaluate developmental immunotoxicity. This workshop made several recommendations for immunotoxicant screening
assays as well as assays that needed further validation
and assays for research development (4). The recommended screening assays for developmental immunotoxicants were the primary antibody response (T-dependent), delayed-type hypersensitivity response,
complete blood count (CBC), and weights of thymus,
spleen and lymph nodes. Assays that require additional validation include phenotypic analyses, macrophage function and natural killer cell activity. Finally,
stem cell functional assays were listed as assays that
require additional research and development (4).
Thymus Atrophy
3
icology working group. The mission of the this group
is to determine:
* the state of science to support the creation of a
guideline for developmental immunotoxicology
* what should be included in such a guideline
* how this guideline would be validated
* when a developmental immunotoxicology guideline would be used (9).
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Thymus: A Mediator of T-Cell Development and
Potential Target of Toxicological Agents
Tight Junctions
An intercellular junctional structure, typically found in
epithelia and endothelia. In the tight junction the two
membranes of neighboring cells are brought into close
proximity through binding of specific transmembrane
proteins. This results in a selectivity barrier that seals
the apical lumen from the basolateral intercellular
646
Time-Resolve Fluorometry
space and also establishes cellular polarity by preventing membrane-linked molecules from freely diffusing
between the apical and the basolateral cell surface.
Cell Adhesion Molecules
3
Time-Resolve Fluorometry
An instrumental design to collect emission at a certain
time interval after the pulsed excitation and to improve
the detection sensitivity by means of a temporal rejection of background.
Cytotoxicity Assays
3
Tissue Factor
This cellular receptor for factor VII/VIIa is constitutively expressed on cells of the media and adventitia of
the vessel wall. When it is exposed to plasma clotting
factors at sites of vascular injury it serves as a potent
(extrinsic) cofactor for the activation of factor X. Tissue factor is also associated with platelets and microparticles and is responsible for intravascular activation
of blood clotting in the absence of tissue damage.
Blood Coagulation
3
Tm Mouse
Knockout, Genetic
3
TNF-α
Tumor Necrosis Factor-α
3
Tolerance
Anke Kretz-Rommel
Principal Scientist
Alexion Antibody Technologies
Suite A, 3958 Sorronto Valley Rd
San Diego, CA 92121
USA
Synonyms
Immunological unresponsiveness
Definition
The primary function of the immune system is to protect the host from foreign materials while at the same
time ensuring that no attack against self proteins occurs. Immunological tolerance is the absence of immunological responsiveness to specific antigens, encompassing unresponsiveness to self antigens, but also tolerance to therapeutics such as antibodies, recombinant
proteins and conventional drugs. Breakdown of immune tolerance is defined by the appearance of Tcells or antibodies to self antigen or the therapeutic
entity. The result may be autoimmune disease or allergic or anaphylactic reactions. Furthermore, an immune
response to a drug may reduce its efficacy.
Immune tolerance is an active process at both the
B cell and T-cell level, involving processes taking
place in central lymphoid organs (thymus and bone
marrow) and peripheral lymphoid organs (blood,
spleen, lymph node, mucosal immune system). The
underlying mechanisms are subject to a continuous
debate involving clonal deletion, anergy, regulatory
T cells and regulatory dendritic cells. In this chapter
these concepts will be outlined with reference to drugs
affecting various tolerance mechanisms, and the interested reader is referred to more in depths reviews.
Characteristics of T Cell Tolerance
Central Mechanisms
T cells develop in the thymus. Recombination of gene
segments creates the two chains that make up the
T cell receptor (TCR) resulting in a large repertoire
of receptor specificities. To ensure the export to the
periphery of T cells that recognize peptides in the
context of self major histocompatiblity complex
(MHC), but do not strongly react to self antigens,
the cells have to undergo positive and negative selection processes as outlined in Figure 1.
Selection is a rigorous process that results in the death
of approximately 95% of T cells. T cells first have to
undergo positive selection on self peptide presented in
the context of self MHC. Successful signaling through
the TCR has been suggested to raise the threshold of
activation of these T cells possibly through the production of negative regulators (1). If the T cells still can be
activated in a subsequent encounter of self peptide
presented by MHC the T cell will undergo clonal deletion by apoptosis, a process termed negative selection. This leaves only T cells to be exported to the
periphery with a threshold of activation that can not
normally achieved by self peptides. Interference with
negative selection in the thymus has been proposed as
a mechanism for the induction of autoimmunity.
TCDD and cyclosporine have been evoked to affect
both positive and negative selection processes. The
reactive metabolite of the antiarrhythmic procainamide
hydroxylamine (PAHA) has been shown to interfere
Tolerance
647
Tolerance. Figure 1 Central tolerance mechanisms. After migration from the bone marrow to the thymus, T cells
first undergo selection on self peptides presented by thymic epithelial cells. Cells productively interacting with the
presented peptide proceed to negative selection resulting in deletion of cells with high affinity for self peptide.
TEC=thymic epithelial cell; APC=antigen presenting cell.
with positive selection in the thymus, resulting in the
export to the periphery of autoreactive T cells and autoantibody production similar to that observed in patients with drug-induced lupus.
Peripheral Mechanisms
T cells leaving the thymus still might respond to self
antigens if the antigens are present in such high concentration that they can bind to “weak” receptors or if
they did not encounter the self peptide in the thymus
which might be the case for certain tissue-specific
antigens. A number of peripheral mechanisms can
control these potentially self-reactive cells (2) as summarized in Figure 2.
Lack of Costimulation
Activation of T cells not only requires interaction of
the TCR with peptide presented by MHC on antigenpresenting cells (APC), but also a second signal (costimulation). Among the most important of these costi-
mulatory molecules are members of the B7 family,
interacting with CD28 on the T cell. Ligation of
CD28 by either B7-1 or B7-2 lowers the threshold
of TCR signaling needed to induce T-cell activation
and increases the effect of that signal by promoting
T cell expansion and proliferation. Recently, additional
members of the B7-CD28 family involved in the development or maintenance of immune tolerance have
been identified such as ICOS which is expressed by
activated T cells. Ligation of ICOSL by ICOS prolongs T cell activation. If a T cell receives a signal
through the TCR in the absence of costimulation,
cells are unresponsive to subsequent stimulation by
the peptide in context of MHC in the presence of
costimulation—a process termed anergy. While this
phenomenon has only been demonstrated in vitro, it
recently has been recognized that naive T cells (T cells
that have not been stimulated before) in the periphery
require frequent interaction with peptide presented by
MHC in order to survive. This has been suggested to
T
648
Tolerance
Tolerance. Figure 2 Peripheral tolerance mechanisms. A: Only T cells with high affinity for the antigen presented
by antigen presenting cells (APC) will proliferate. Since thymic emigrants have been tuned to have a threshold of
activation generally above that achieved by most self peptides, T cell interaction with self peptide presented by
major histocompatability complex (MHC) does not result in proliferation. B: If a T cell sees antigen in the context of
MHC in the absence of costimulatory signals, anergy can be induced. The T cell is subsequently unresponsive to
challenge with the cognate antigen by APCs, even in the presence of costimulatory molecules. C: Death molecules
such as fatty acid synthetase (FAS) and tumor necrosis factor (TNF) get upregulated in the course of a T cell
response to limit proliferation and cytokine production. T cells involved in the response to antigen will undergo
activation-induced cell death (AICD) by apoptosis. D: A number of immunoreceptors downregulate the T cell
response. Some of them are upregulated during the T cell response to limit it, and some of them are constitutively
expressed on tissues to prevent damage by T cells. E: Tolerogenic dendritic cells can induce Treg which control the
response of other T cells.
be an important mechanism of peripheral tolerance,
maintaining a high activation threshold of T cells
which can only be overcome by foreign antigen.
Drugs could potentially provide a “danger” signal to
the immune system resulting in upregulation of costimulatory molecules and activation of self-reactive
T cells. However, clearly not all drugs inducing cell
stress or cell death result in an activation of the im-
mune system. Evidence is emerging though that some
compounds can alter dendritic cells resulting in upregulation of the costimulatory molecule CD86 or provoking migration of dendritic cells by upregulating the
CCR7 receptor.
Failure to Encounter Self Antigens (Immune privilege)
Under normal conditions, the cells in nonlymphoid
Tolerance
organs throughout the body are not in contact with
T cells and are thus sequestered from the immune
system. This lowers the probability of a low affinity
self-reactive T cell encountering a specific self antigen.
Only in the presence of “danger” signals such as provided by bacteria can T cells enter non-lymphoid organs. Certain tissues are particularly protected from
the entry of T cells, such as the interior of the eye,
brain and testes. Constitutive expression of immunosuppressive receptors and cytokines ensures protection
of these organs from immune-mediated damage.
Receipt of Death Signals
An important mechanism of maintaining immune homeostasis is the downregulation of the immune response after activation. Activation of antigen-presenting cells by bacteria or viruses results in upregulation
of costimulatory molecules and production of proinflammatory cytokines such as tumor necrosis factor
(TNF)-α. Persistence of an inflammatory environment
increases the risk of activating T cells by self peptides
by providing costimulation to these cells. Also, there is
a risk of cross-reactivity of T cells activated by pathogens with self antigen, because activated T cells require less costimulation. Therefore, most activated
T cells ultimately undergo a process of programmed
death or apoptosis. Apoptosis of activated cells (activation-induced cell death, AICD) occurs by cytokine
withdrawal and by induction through fatty acid synthetase (FAS) and TNF-α. FAS acts on FAS ligand
expressed on activated T cells. These cells therefore
can kill themselves as well as activated B cells and
macrophages. Also, expression of other receptors
mediating immune suppression play an important
role in downregulating the immune response as discussed in the following section.
Immunosuppressive Receptors
The immune response can be terminated by upregulation of the T cell surface molecule CTLA-4. While
CTLA-4 is present at very low levels on resting
T cells, it is markedly upregulated after T cell activation. Similar to the positive costimulatory molecule
CD28, CTLA-4 binds to B7.1 and B7.2. Due to its
substantially higher affinity for these molecules,
CTLA-4 outcompetes CD28, thereby transducing inhibitory signals to the activated T cell. More inhibitory
molecules have recently been identified. PD-1 is expressed on activated T cells, B cells and myeloid cells,
and engagement by its ligands PD-L1 and PDL-2 inhibits T cell proliferation and cytokine production. Expression of immunosuppressive receptors on nonlymphoid organs is another safeguard mechanism against
self attack of T cells.
649
Regulatory Cells and Cytokine Milieu
A minor population of T cells known as regulatory
T cells (Treg) suppresses the proliferative response
and production of inflammatory cytokines of other
T cells. They may constitute a specialized T cell subset
to reduce the activity of autoreactive T cells. Treg constitutively express CTLA-4 and secrete transforming
growth factor(TGF)-β and interleukin(IL)-10. Mechanisms of action are still under debate, but they
seem to require direct cell-cell contact.
In addition to regulatory T cells, dendritic cells and
macrophages play a major role in immune tolerance.
The functional activities of dendritic cells are mainly
dependent on their state of activation and differentiation. Terminally differentiated mature dendritic cells
can efficiently induce the development of T effector
cells, whereas immature dendritic cells are involved in
maintenance of peripheral tolerance. The means by
which immature dendritic cells maintain peripheral
tolerance are not entirely clear, however, their functions include the induction of anergic T cells, T cells
with regulatory properties as well as the generation of
T cells that secrete immunomodulatory cytokines. Depending on the cytokines produced by the macrophage/dendritic cell, the immune response can be
steered towards a Th1 or Th2 response. Th1 cells produce IFN-γ, IL-2 and TNF-α and regulate classical
delayed (type IV) hypersensitivity. Th2 cells secrete
IL-4, IL-5, IL-6 and IL-10 and participate in immediate (type I) hypersensitivity reactions and B cell antibody-mediated immunity. The effect of drugs on cytokine production and the importance of the cytokine
milieu resulting in drug-induced autoimmunity are
being studied extensively.
Characteristics: B cell Tolerance
Similar to T cells, B cells are constantly being tolerized to self antigens. For a thorough discussion of
B cell tolerance the reader might refer to Jacquemin
et al. (3).
Central Mechanisms
B cells mature and undergo selection on self peptides
in the bone marrow. A large population of B cells with
different specificities is created by genetic recombination within the immunoglobulin locus generating a
broad range of heavy- and light-chain sequences that
rearrange to form a B cell receptor (BCR). If the immature B cell encounters extracellular antigen capable
of crosslinking its BCR, a signal is created that will
block further development of this autoreactive cell.
The B cell will initiate the receptor editing process
to produce BCR with new antigen specificities. If it
cannot alter its BCR effectively, the immature B cell
will be deleted by apoptosis. Some autoreactive
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Tolerance
B cells escape deletion and enter the peripheral circulation in an anergic state.
Peripheral Mechanisms
After recognition and uptake of antigen in the periphery, these partially activated B cells migrate through
the lymphoid tissue. If an activated B cell encounters a
T cell that has been activated by the same antigen,
antibodies against that antigen are produced. B cells
cannot respond to most antigens without receiving
help from T helper cells. Therefore, ensuring self tolerance of T cells is an important mechanism of keeping B cells from producing autoantibodies. However,
drugs affecting B cell tolerance can ultimately result in
autoimmunity when the individual has other predisposing factors, as might be the case for pristane.
Additional Mechanism for Drugs to Break
Immune Tolerance
The most common hypothesis of how drugs result in
immune stimulation is the formation of drug-protein
conjugates by reactive drug metabolites with self antigens. The resulting haptens might be recognized as
foreign by the immune system. Although formation
of haptens has been demonstrated for a number of
drugs associated with idiosyncratic immune adverse
reactions (e.g. phenytoin, carbamazepine, halothane,
tielinic acid, procainamide and diclofenac) these adducts are not a predictive factor for adverse immune
reactions indicating that additional factors are required
to induce the immune response. It has been demonstrated that binding of halothane to CF3CO proteins
mimics very closely the structure of the E2 subunit
proteins of the 2-oxoacid dehydrogenase complexes
and protein X—autoantigens associated with halothane hepatitis. Furthermore, binding of drugs to protein can alter their cleavage and presentation after cell
death. Exposure of macrophages to mercuric chloride
has been show to alter fibrillarin processing, resulting
in the appearance of self epitopes not normally encountered by the immune system.
In addition to covalent drug binding to proteins, noncovalent interactions of drugs such as sulfamethoxazole with MHC-peptide complexes have been implicated in immunological adverse reactions.
While disruption of immune tolerance by classical
chemical drugs leaves many unanswered questions,
immune responses after administration of bioengineered drugs is far more straightforward. The importance of antibodies in therapeutics gains increasing
recognition. Often, these antibodies are of mouse origin and certain residues are recognized as foreign by
the human immune system. Engineering methods
known as “humanization” and pegylation decrease
the risk of an immune response against the therapeutic.
Preclinical Relevance
Adverse drug reactions affecting immune tolerance are
difficult to address in the preclinical setting. However,
a number of assays have been developed to address the
potential of drugs to sensitize the immune system,
such as the popliteal lymph node assay that assesses
the effects of drugs on macrophages, or assays looking
for altered cytokine profiles. Few animal models demonstrating chemically induced autoimmunity are
available, but are specific for the compound used.
As far as immunogenicity of biotherapeutics is concerned, some animal models have proved to be useful.
For example, transgenic mice were developed to produce and secrete human tissue plasminogen activator
to which they developed immune tolerance. These
mice were capable of producing antibodies to a form
of human tissue plasminogen activator that had been
modified by a single amino acid substitution. Furthermore, nonhuman primates have been used successfully
in predicting the relative immunogenicity of different
forms of human growth hormone. Also, computer
modeling methods are used in predicting the immunogenicity of proteins.
Relevance to Humans
Adverse drug reactions account for 2%–5% of all hospital admissions, a portion of which is based on immune-mediated reactions. With more than 80 recombinant proteins in clinical use and more than 400 therapeutic antibodies in clinical trials, immune tolerance
to these proteins is a major issue and predicting immunogenicity is crucial (4).
Regulatory Environment
Regulatory issues for drug-induced autoimmunity and
allergy are covered in their respective chapters. For
clinical trials of recombinant proteins, patients are
screened for the development of antidrug antibodies.
References
1. Grossman Z, Singer A (1996) Tuning of activation
thresholds explains flexibility in the selection and development of T cells in the thymus. Proc Natl Acad Sci USA
93:14747–14752
2. Sharpe AH, Freeman GJ (2002) The B7-CD28 superfamily. Nat Rev Immunol 2:116–126
3. Jacquemin MG, Vanzieleghem B, Saint-Remy JM (2001)
Mechanisms of B-cell tolerance. Adv Exp Med Biol
489:99–108
4. Pendley C, Schantz A, Wagner C (2003) Immunogenicity
of therapeutic monoclonal antibodies. Curr Opin Mol
Ther 2:172–179
Toxicogenomics (Microarray Technology)
An illness associated with the ingestion of adulterated
rapeseed oil in Spain in 1981. The most distinctive
lesion is a non-necrotizing vasculitis involving different types and sizes of blood vessels in every organ.
Systemic Autoimmunity
Toxicogenetics
The genetic basis for individual differences in susceptibility to toxicity, with single nucleotide polymorphisms (SNPs) being the prime source of variability in
the genome.
Toxicogenomics (Microarray Technology)
3
Unresponsiveness to antigenic stimulation that is
either mediated by genetics, or is acquired by special
conditions of antigenic exposure. The immune system
has established several mechanisms that prevent immune reactions against self antigens. Of central importance is the tolerance of the immune regulatory helper
T cells. Activation of helper T cells can be controlled
by tolerance induction in the thymus, by sequestration
of antigens in immune privileged sites (brain, testis,
cornea) and by active suppression of immune responses by regulatory T cells.
Antigen Presentation via MHC Class II Molecules
Graft-Versus-Host Reaction
Autoantigens
Autoimmune Disease, Animal Models
Antinuclear Antibodies
Lymphocytes
Transforming Growth Factor β1; Control of T cell
Responses to Antigens
Toxic Oil Syndrome (TOS)
3
Tolerance and the Immune System
651
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3
3
3
3
Toll-Like Receptors
Toxicogenomic Studies
Studies in toxicology which screen for global changes
in gene expression following exposure to a toxicological agent.
Thymus: A Mediator of T Cell Development and
Potential Target of Toxicological Agents
3
A family of receptors expressed by cells of the innate
immune system and directed against conserved structures present on many micro-organisms. Ten members
of this receptor family are present in humans (e.g.
TLR4 specific for lipopolysaccharide; TLR2 for peptidoglycan; TLR5 for flagellin). They are named after
the Drosophila protein Toll which is involved in the
antibacterial defense of the fruit fly.
B Cell Maturation and Immunological Memory
Toxicogenomics
The measurement of altered gene expression upon exposure to a compound or drug, thereby identifying the
toxicant and characterising its mechanism of action.
Toxicogenomics (Microarray Technology)
3
3
Toxic Epidermal Necrolysis (TEN)
Toxic epidermal necrolysis (TEN) represents the most
serious extreme of the febrile mucocutaneous syndrome in which there is a full-thickness sloughing of
the epidermis. According to the criteria, TEN is defined as detachment affecting about 30% of the body
surface area. Stevens-Johnson syndrome is similar to
TEN in terms of the histopathology and the responsible drugs, indicating that these two conditions are part
of the same spectrum. Fas-Fas L interactions appear to
be involved in the epidermal necrolysis.
Drugs, Allergy to
Toxicogenomics (Microarray
Technology)
Rob J Vandebriel
Laboratory for Toxicology, Pathology and Genetics
National Institute for Public Health and the
Environment
3720 BA Bilthoven
The Netherlands
Synonyms
Gene profiling, expression profiling, global gene expression analysis
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Toxicogenomics (Microarray Technology)
Definition
Microarray technology is the simultaneous individual measurement of the mRNA expression level of
thousands of genes in a given sample by means of
hybridization. Toxicogenomics is the measurement
of altered gene expression upon exposure to a compound or drug, thereby identifying the toxicant and
characterizing its mechanism of action.
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3
Characteristics
Although individual differences exist, the basic principle of microarray technology is the same for different platforms (1). The term platforms means types of
arrays or array suppliers, in the latter case combined
with dedicated hardware and software. First, per gene,
a single probe or a few different probes are generated,
using either polymerase chain reaction(PCR)-amplified complementary DNA (cDNA), or synthetic
DNA segments (oligonucleotides or oligos) devised
on the basis of these cDNA sequences. Usually they
are spotted onto a glass surface in a regular array. This
process is called spotting or arraying, and requires
dedicated machinery. Some companies manufacture
oligos in situ, either using photolithography (Affymetrix) or chemical coupling (Agilent).
Several options exist to obtain arrays:
* ready-made arrays (e.g. Affymetrix, Agilent)
* custom-made arrays (e.g. Affymetrix, Agilent)
* in-house spotting of a PCR-amplified clone collection (e.g. Invitrogen) or of an oligo collection
(MWG, Operon, Sigma).
Other manufacturers of ready-made arrays include Operon, MWG, and Phase-1, but this list is by no means
exhaustive.
A clone collection is a collection of bacteria, each
containing a plasmid consisting of a different cDNA
insert. Care has to be taken that the individual clones
indeed contain the correct insert; verifying clone sets
by sequencing the inserts is not uncommon. Second,
RNA or mRNA is isolated from cells or tissues and
cDNA is synthesized. This cDNA is labeled using a
fluorescent label, either during or after synthesis. The
labeled cDNAs are then hybridized to the array. The
Affymetrix platform uses a single labeled cDNA (Cy3)
per hybridization, whereas other platforms rely on two
labeled cDNAs (Cy3 and Cy5; most often test and
control). The array is then read using a scanner (with
fitted laser (s)) that measures for each spot the fluorescence intensity. These data are then transferred to a
personal computer. This process is outlined in Figure 1.
During and after this process a number of controls
have to be performed to assure that the results obtained
are correct. For the arrays these controls include the
shape of the spots and the amount of DNA spotted (e.
g. by hybridization of labeled random hexamers).
After hybridization these controls include a similar
average staining intensity over the entire array, and
plotting the intensity ratio of both labels against the
intensity of the label for the control sample. This ratio
should be independent of the intensity for most of the
genes interrogated. To exclude artifacts caused by differential incorporation of the two labels into the
cDNAs a dye swab is useful. If replicate samples are
tested, statistics can be performed. Ratios of test vs
control of > 2 are generally considered significant. If
several time points, dose groups, or organs are analyzed, more advanced statistics can be done, such as
cluster analysis and/or principal component analysis
(2). To this end several algorithms have been written,
most of them being freely available on the internet.
Commercial software packages have the advantage
of easier data handling, compared to the tedious process of uploading data-sets to algorithms on the web
(see Baxevanis and Francis Ouelette for a primer on
the subject) (3).
The number of genes to be analyzed is of interest.
Obviously, for mechanistic studies as well as for seeding databases that are ultimately aimed at identifying
toxic profiles of compounds, the number of genes
should be maximal, nowadays meaning virtually all
genes. With statistics aiding in the process of gene
selection, signatures of toxicity (such as peroxisome
proliferators) or pathology (such as liver necrosis) may
eventually be addressed by interrogating a small number of genes.
Preclinical Relevance
A first important issue of toxicogenomics is to establish specific types of toxicity, or even compounds on
the basis of signature expression profiles. A proof-ofprinciple approach to obtain such signature profiles
proved to be successful (4,5). A first step towards
preclinical relevance is to obtain a database consisting
of gene profiles for a range of model compounds.
Since studies aimed at seeding such a database are
usually divided between different laboratories and
the outcome has to be useful also for laboratories outside the study group, care has to be taken that results
from these laboratories can be compared, or used back
and forth. With the current state of technology, various
methodologies and platforms exist for assessing gene
expression, making it difficult to compare and compile
data across laboratories.
An important initiative in this respect is the “minimum
information about a microarray experiment”
(MIAME) document (6), produced by the microarray
gene expression database (MGED) society (http://
www.mged.org). This set of guidelines is in the process of extension for toxicogenomics (MIAME/Tox),
aiming to define the core that is common to most
Toxicogenomics (Microarray Technology)
653
Toxicogenomics (Microarray Technology). Figure 1 Schematic illustration of microarray analysis. In this
particular example PCR amplified cDNAs are dotted.
toxicogenomic experiments. The major objective of
MIAME/Tox is to guide the development of toxicogenomics databases and data management software. The
draft document can be found at http://hesi.ilsi.org. Efforts to build international public toxicogenomics
databases are underway at the National Center for
Toxicogenomics, National Institute of Environmental
Health Sciences, USA (http://www.niehs.nih.gov/nct)
and at the EMBL European Bioinformatics Institute
(http://www.ebi.ac.uk/microarray/index.html) in conjunction with the International Life Sciences Institute
Health and Environmental Sciences Institute (http://
hesi.ilsi.org). This database will be made public in
late 2003 or early 2004.
A provisional conclusion from experiments conducted
so far is that multiple sources of variability exist, including expected sources of biological variability, isolation and labeling of mRNA samples, hardware and
software settings, microarray lot numbers and gene
coverage, and annotation. Nevertheless, the gene expression profiles relating to biological pathways are
robust enough to allow insight into mechanism, strong
information on topographic specificity is provided,
dose-dependent changes are observed, and concerns
of over sensitivity may be unfounded (http://hesi.ilsi.
org).
Relevance to Humans
A second important issue of toxicogenomics is the
genetic basis for individual differences in susceptibil-
ity to toxicity. Much of the variability in the genome
stems from single nucleotide polymorphisms or SNPs,
that occur roughly every 1000 nucleotides. A map
describing over 1.4 million SNPs (7) is available
(http://snp.cshl.org). The next step is then to find an
association of a particular SNP and a disease trait.
Generally, two approaches can be taken to find such
associations: one is a candidate gene approach, where
genes in key biochemical pathways are investigated
for SNPs, and in the second approach SNPs and thereby target genes are identified by whole genome approaches. Mixed approaches can of course also be
taken. An example of a successful candidate gene approach is the SNP mapping of the hypersensitivity response (HSR) to the drug abacavir. Over 100 SNPs
were tested on the basis of candidate genes. Polymorphisms from two of the candidate genes (tumor
necrosis factor(TNF)-α and human leukocyte antigen
(HLA)-B57) were found to be highly associated with
the hypersensitivity response to abacavir (8).
Similar to gene profiling, creating a database that describes associations between SNPs and disease is an
important goal. Using high-density SNP mapping it
should be feasible to study the genetic basis for several
common diseases simultaneously. For drug adverse
effects this will surely be more difficult since only
few patients with a certain drug prescribed will show
adverse effects.
A recent development comes from the finding that the
human genome can be parsed into haplotype blocks,
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Regulatory Environment
Regulations that rely on genomics are not yet in place
but there is little doubt that within the next 5–10 years
gene expression profiles will be used for safety as well
as efficacy assessment. This requires a firm database
of expression profiles that can be directly related to
well characterized toxicological and pathological endpoints.
Second, risk assessment has traditionally been performed across whole populations with widely varying
responses. The goal is that by genetically identifying
sensitive subpopulations, the accuracy of risk assessment can be improved. Possibly, this may eventually
lead to personalized risk profiles.
References
1. Duggan DJ, Bittner M, Chen Y, Meltzer P, Trent JM
(1999) Expression profiling using cDNA microarrays.
Nature Genet 21S:10–14
2. Eisen MB, Spellman PT, Brown PO, Botstein D (1998)
Cluster analysis and display of genome-wide expression
analysis. Proc Natl Acad Sci USA 95:14863–14868
3. Baxevanis AD, Francis Ouelette BF (eds) (2001)
Bioinformatics. John Wiley & Sons, New York
4. Hamadeh HK, Bushel PB, Jayadev S et al. (2002) Gene
expression analysis reveals chemical-specific profiles.
Tox Sci 67:219–231
5. Hamadeh HK, Bushel PB, Jayadev S et al. (2002)
Prediction of compound signature using high density
gene expression profiling. Tox Sci 67:232–240
6. Brazma A, Hingamp P, Quackenbush J et al. (2001)
Minimum information about a microarray experiment
(MIAME)—toward standards for microarray data. Nature
Genet 29:365–371
7. Sachidanandam R, Weissman D, Schmidt SC et al. (2001)
A map of human genome sequence variation containing
1.42 million single nucleotide polymorphisms. Nature
409:928–933
8. Roses AD (2002) Genome-based pharmacogenetics and
the pharmaceutical industry. Nature Rev Drug Disc
1:541–549
9. Gabriel SB, Schaffner SF, Nguyen H et al. (2002) The
structure of haplotype blocks in the human genome.
Science 296:2225–2229
TR1 Cells
Suppressor Cells
Trace Metals
Those metals commonly found in minute amounts in
the organism.
Trace Metals and the Immune System
Trace Metals and the Immune System
Judith T Zelikoff
Depart. of Environmental Medicine
New York University School of Medicine
57 Old Forge Road
Tuxedo, NY 10987-5007
USA
Synonyms
CD4+, T helper lymphocyte, CD4+/CD8−, T helper
lymphocyte, CD8+, T suppressor lymphocyte,
COPD, chronic obstructive pulmonary disease, asthma, bronchitis, emphysema.
Definition
Trace metals are normally present in minute quantities
in the body. Many of them are also transition elements,
essential for life due to their ability to control metabolic and signaling functions, such as zinc (Zn), manganese (Mn), and copper (copper). However, these
same essential metals can also be toxic because of
their ability to evade established controls for cellular
uptake, transport, and compartmentalization. Aluminum (Al) is a toxic trace element, unavoidable by
the general population because of its widespread environmental distribution. The immunotoxicity of trace
metals other than Al, copper , Mn, and zinc can be
found in a number of review articles (1–3).
Molecular Characteristics
Aluminum
Aluminum is the third most prevalent element in the
Earth's crust. It is an A-type metal, or hard acid, that
strongly prefers oxygen-donor ligands; hydroxide, citrate, phosphate, and nucleoside phosphate groups are
probably the most important low-molecular-mass bioligands for the predominant trivalent cation (Al3+). It
also binds readily to the two high-affinity iron-binding
sites of the serum transport protein, transferrin (TF).
There is a wide variation in the ability of different
ligands to solubilize and transport the Al3+ ion to critical target sites.
3
being regions over which there is little evidence for
historical recombination and within which only a few
haplotypes are observed (9). Markers for these haplotype blocks are now available, which makes it possible
to identify the genetic control of responses to toxicants
without the necessity to identify the specific SNP responsible.
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Copper
Copper is a Group II (or IB) element, the third most
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Trace Metals and the Immune System
Manganese
Manganese is the only Group VIIB element commonly found in biological environments. Although
the inorganic chemistry of manganese displays a
range of stable oxidation states, its biological chemistry is dominated by the divalent form (Mn2+). Because
Mn2+ is very similar in size and charge density to
magnesium (Mg2+) and Zn2+ and also prefers to assume thetrahedryl and octahedral geometric structures,
Mn2+ can replace Mg2+ in the enzyme pyruvate carboxylase and Zn2+ in superoxide dismutase (SOD)
with only negligible effects on enzyme activities.
Zinc
Zinc is found in large quantities in the vertebrate body
(second only to iron); it is the first member of
Group IIB elements and forms stable complexes
with sulfur, phosphate, and carbon atoms. Biological
complexes contain zinc only in the divalent oxidation
state (Zn2+). Since Zn2+ is the only stable oxidation
state of the metal, it does not play a redox-active role
in biological processes. However, Zn2+ can actively
participate in enzymatic reactions as a Lewis acid or
as a structural cofactor. Zinc is part of, or a cofactor
for,
such enzymes as carbonic anhydrase, carboxypeptidase, SOD, lactate dehydrogenase, phosphatase, and
glutamate dehydrogenase. Zinc also displays a structural role in biological systems, as exemplified by its
role in maintaining the integrity of zinc finger transcription factors that bind to DNA and regulate the
transcription of genetic information.
Relevance to Humans
Aluminum
While some daily exposure to aluminum is unavoid-
able, inhalation by the general population is usually
considered negligible (i.e. 0.14 mg aluminum dust per
day). However, smelters, miners, welders and other
workers involved in various metal industries are
often acutely exposed to localized atmospheres containing 2–4 mg/m3 of aluminum, resulting in timeweighted-average (TWA) intakes of > 23 mg per 8hour shift. Increases in pneumonia, bronchitis, asthma,
pneumoconiosis, lung cancers, and pulmonary fibrosis
have been described in occupationally exposed workers. In addition, there is little doubt that aluminum can
cause encephalopathy, osteopathies, and anemia in
kidney dialysis patients. Although early studies set
100 μg/l plasma as the level of aluminum below
which neurotoxicity failed to occur, recent studies
have demonstrated subtle neurocognitive and/ or psychomotor effects, as well as EEG abnormalities in dialysis patients expressed at levels well below this
limit. Infants are a particularly susceptible subgroup
for aluminum toxicity partly due to their rapidly growing and immature brain and skeleton and their developing blood-brain barrier; preterm infants are generally recognized to be at risk for aluminum loading due
to their immature kidney function. While the reference
range for blood aluminum levels in healthy individuals
is < 10 µg/l, studies in infants have demonstrated plasma aluminum levels > 50 μg/l after oral intake of
aluminum-containing antacids.
Copper
As an essential element, copper promotes iron absorption from the gastrointestinal system, it is involved
in the transport of iron from tissues into plasma, it
helps maintain myelin in the nervous system, it is
necessary for hemoglobin synthesis, and it is important in the formation of bone and brain tissue. Apart
from occupational exposure, daily copper intake
averages ∼ 0.02 mg. The fine balance required for
copper in humans is evident in genetically inherited
inborn errors of copper metabolism. For example, in
Wilson's disease there is failure to excrete copper from
the liver to the bile, resulting in copper overload in the
liver, brain, kidneys, and cornea; and in Menkes disease, which is characterized by severe copper deficiency due to an error in copper transport from the intestines. Copper, usually in the form of cuprous oxide
and cupric hydroxide (which converts to cupric
oxide), is generally encountered in high concentrations
in the air of metallurgical processing plants, iron and
steel mills, and around coal-burning power plants. In
contrast to airborne copper concentrations in rural/suburban areas that average 0.01–0.26 μg/m3, particulate
copper levels in workplace sites be 50–900 μg/m3.
Inhalation of such levels can result in an immunologically-based condition called “copper fever”.
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abundant transition metal found in living things. It
exists in one of two stable oxidation states: as cuprous
(Cu1+) and cupric (Cu2+) ions. Consequently, its biological chemistry is dominated by participation in
redox reactions. Copper is necessary in the diet for
iron utilization and as a cofactor in enzymes associated
with oxidative metabolism. It is transported in serum
bound initially to albumin and later more firmly to αceruloplasmin where it is exchanged in the cupric
form; normal copper serum level is 120–145 μg/l.
At elevated levels, copper is toxic to cells, presumably
by binding indiscriminately to thiol moieties or by
catalyzing a Fenton-type reaction to produce reactive
hydroxyl radicals. Binding of copper by biological
ligands such as small peptides, large proteins, and enzymes is required to minimize potential deleterious
effects. Most stored copper is usually bound to metallotheinein (MT), a ubiquitous class of proteins that is
well suited to the role of metal sequestration.
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Trace Metals and the Immune System
Zinc
Zinc is ubiquitous in the environment and present in
most foodstuffs, water, and air. It is a nutritionally
essential element that serves as a cofactor for more
than 70 metalloenzymes. Daily dietary intake of zinc
is usually 12–15 mg/day and ~ 20%–30% of ingested
zinc is absorbed; zinc deficiency results in a wide
spectrum of clinical effects depending upon age,
stage of development and deficiencies of related metals (i.e. zinc deficiency can exacerbate impaired copper nutrition and exacerbate cadmium and lead toxicity). Airborne concentrations of zinc are usually < 1
μg/m3, with the majority of zinc being derived from
automobile exhaust, soil erosion, and local commercial, industrial or construction activities. In urban
areas, atmospheric zinc concentrations are in the
range 0.02–0.50 μg/m3; rural air contains 0.01–0.06
μg/m3. Because zinc also contaminates certain workplace environments, national guidelines of 1.0 mg, 5–
10 mg, and 0.1 mg/m3 have been established for soluble zinc, insoluble zinc oxide, and carcinogenic zinc
chromate, respectively.
Putative Interactions with the Immune System
Aluminum
Although limited in number, immunotoxicologic studies using a variety of animal models have demonstrated that injection of soluble aluminum compounds increases mononuclear cell mitotic index; injection of
the metal or insoluble aluminum agents alter monocyte/macrophage numbers and immune function (1).
Dietary exposure of rodents to soluble aluminum reduces cytokine production, T helper (Th) and
T suppressor (Ts) cell numbers, and host resistance
to Listeria monocytogenes infection. Repeated inhalation exposure of rabbits and hamsters to soluble aluminum increases lung immune cell numbers; similar
effects were not seen in aluminum-exposed workers.
Effects of inhaled aluminum on host resistance are
inconsistent, showing decreased resistance to subsequent bacterial challenge in some studies and no effect
in others. Differences between the studies are thought
to be due to intratracheal versus inhalation exposure
routes. In vitro studies employing soluble aluminum
salts demonstrate a range of effects on immune cells
derived from a variety of animal species including
humans. For example, aluminum chloride treatment
of rat alveolar macrophage reduced reactive oxygen
intermediate production.
Copper
Much the same as for manganese and zinc, studies of
copper immunotoxicity are complicated by the fact
that copper is essential to maintenance of immunocompetence and, thus, most immunotoxicity occurs
as a result of copper insufficiency. Splenomegaly
and thymic atrophy are consistent findings in copper-deficient mice. Alterations in antibody response
and B-lymphocyte function are also well documented
with experimentally induced copper deficiency. In
contrast, serum antibody levels in humans with nutritional or genetic copper-deficiency are reported to be
normal. B-lymphocytes are increased in number in
copper-deficient animals, but they respond poorly to
mitogen stimulation. Although the effects of copper
deficiency on T-lymphocyte populations are well characterized, the overall effect on cell-mediated immunity
is unclear. Except in female rats, CD4+ and CD8+
subsets are decreased in the peripheral blood and
spleen of copper-deficient rodents. Though no gender
effect has been observed in mice, the immune system
of male rats appears more susceptible to copper deficiency than that of females. Clinical studies involving
healthy men on low copper diets fail to support the
animal studies with respect to circulating T-lympho3
Manganese
Manganese, an essential trace element for all living
organisms, is necessary for bone formation, cholesterol and fatty acids synthesis, and as a dissociable cofactor for several enzymes including SOD. Despite its
essentialness, the toxic effects of manganese are well
known, particularly those associated with the nervous
system (4). Manganese is widely employed in many
industries: in alloy steel manufacture for deoxidation
and to promote hardenability; in the electric industry
for production of dry cells; in the chemical industry,
where they are used as oxidants, for the manufacture
of fertilizers, paints and varnishes, and in the production of glass and glazes (3). Apart from the direct
release of manganese into the air by several types of
mining industries and alloy and steel production facilities, manganese is introduced into the ambient environment by the combustion of manganese-containing
fossil fuels (used as anti-knock additives and combustion improvers). Manganese (whose toxicity in many
cases depends upon compound solubility) has been
found at measurable levels in the majority of suspended particulate matter (including coal flyash) in
urban environments. While air levels of manganese
in many metropolitan areas containing steel or alloy
plants can range from 0.5–3.3 μg/m3, the majority
have levels ≤ 0.1 μg/m3; average air levels in the absence of any contributing point sources are in the
range 0.03–0.07 μg/m3. Alternatively, occupational
airborne levels of manganese are usually in the range
1–≥ 100 μg/m3 (although levels as high as 1 mg/m3
have been measured); workplace permissible exposure
limits (PEL) of 300 (TWA) and 500 μg/m3 have been
recommended by the World Health Organization and
OSHA (Occupation Safety and Health Association),
respectively.
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Transendothelial Migration
cytes and CD4+ and CD8+ subsets. While effects of
copper-deficiency on innate immunity are inconclusive in animal studies, reduced neutrophil numbers
and functionality are well defined in clinical studies.
The most consistent immune defect associated with
copper deficiency in epidemiologic, clinical, and toxicological studies is impaired host resistance due primarily to suppressed antibody-mediated responses and
phagocyte antimicrobicidal activities (2).
Manganese
While relatively few studies have investigated the effects of manganese on the immune system, immunotoxicity appears dependent (like many of the other
metals discussed herein) upon compound solubility.
Immune responses of the lung appear particularly sensitive to the immunomodulating effects of manganese.
Inhalation of insoluble manganese compounds reduces
the ability of the lungs to resist and clear subsequent
bacterial/viral infections and exacerbates ongoing viral
infections (3). Inhalation studies examining the effects
of soluble manganese reveal little effect on lung immune cell-related functionality. In contrast, studies
wherein rabbit alveolar macrophages were exposed
in vitro to manganese chloride demonstrated decreased
cell viability and number, increased incidence of cell
lysis, and reduced phagocytic activity.
657
cific immune cell types, effects of zinc on innate immunity are conflicting.
References
1. Zelikoff JT, Cohen MD (1997) Metal immunotoxicology.
In: Massaro EJ (ed) Handbook of Human Toxicology.
CRC Press, New York, pp 811–852
2. Omara FO, Brousseau P, Blakley BR, Fournier M (1998)
Iron, zinc, and copper. In: Zelikoff JT, Thomas PT (eds)
Immunotoxicology of Environmental and Occupational
Metals. Taylor and Francis, London, pp 231–262
3. Cohen MD (2000) Other metals: aluminum, copper,
manganese, selenium, vanadium, and zinc. In: Cohen M,
Zelikoff JT, Schlesinger RB (eds) Pulmonary Immunotoxicology. Kluwer, Boston, pp 267–299
4. Inoue N, Makita Y (1996) Neurological aspects of human
exposures to manganese. In: Chang LW (ed) Toxicology
of Metals. CRC Lewis, New York, pp 415–421
5. Zelikoff JT, Chen LC, Cohen MD et al. (2003) Effects of
inhaled ambient particulate matter (PM) on pulmonary
anti-microbial immune defense. Inhal Toxicol 15:101–
120
Trans-Signaling
3
Transcription Factors
Proteins (enzymes) that bind to regulatory sequences
(response elements) in the promoter region of a gene,
forming a complex to which RNA polymerase binds.
The process of transcription converts the genetic information contained in DNA into an RNA message for
synthesis of a specific protein.
Glucocorticoids
Signal Transduction During Lymphocyte Activation
3
3
Zinc deficiency (like copper) can impair humoral and
cell-mediated host immunocompetence. Zinc-deficient
children and laboratory animals consistently present
with thymic hypoplasia; oral administration of zinc
supplements appear to reverse this effect. In zinc-deficient animals, secondary antibody responses to T-dependent antigens are suppressed in conjunction with
accelerated thymic hypoplasia and a decreased number
of CD4+ cells. Administration of zinc to immunosuppressed human populations appears to increase numbers of CD4+ and CD8+ thymocytes which, in turn,
give rise to increased numbers of Th-cells important
for the activation of cytotoxic T- and B-lymphocytes.
In contrast, zinc suppresses concanavalin A-induced
T lymphocyte proliferation by in vitro-exposed
human immune cells (2) and compromises pulmonary
host resistance against bacterial infection (5); suppressive effects of inhaled zinc on pulmonary antimicrobial activity are most likely due to zinc-induced reductions in macrophage phagocytic activity. While the
main immunological effect of occupational zinc exposure is metal fume fever, inhalation of particulate zinc
by occupationally exposed workers also alters pyrogenic, chemotactic, and anti-inflammatory cytokines.
While zinc appears to play an important regulatory
role in membrane-associated events of certain nonspe-
3
Zinc
The soluble Interleukin-6 receptor α-chain binds interleukin-6 and can then interact with the transmembrane
receptor subunit glycoprotein 130 (gp130) and induce
signal transduction. Thus a cell lacking an endogenous
binding subunit of the interleukin-6 receptor can respond to Interleukin-6 in the presence of the soluble
receptor-derived from distant producer cells, hence
trans-signaling.
Cytokine Receptors
Transendothelial Migration
This is the exit of circulating leukocytes from blood
into tissue by means of traversing the microvascular
endothelium. This process involves loose interactions
of blood leukocytes with the luminal side of blood
T
3
Transferrin
vessels (mostly mediated by selectins), and this is followed by firm adhesion and leukocyte transmigration.
This latter step critically depends on the rapid and
transient modulation of integrin function, which itself
is controlled by chemokine receptor signaling. Only
those leukocytes firmly arrest that bear the appropriate
set of chemokine receptors and, therefore, the chemokines present on the luminal side of microvessels are
viewed as key controllers of leukocyte extravasation.
Immune Cells, Recruitment and Localization of
3
Transferrin
A protein that combines with and competes for iron
with bacteria.
Trace Metals and the Immune System
3
Transferrin Receptor
These are cell membrane receptors for transferrin.
They play a role in iron uptake by the cell, and are
highly expressed in proliferating cells.
Interferon-γ
3
Transforming Growth Factor β1;
Control of T Cell Responses to
Antigens
Susan C McKarns
Laboratory of Cellular and Molecular Immunology
NIAID/NIH
Building 4, Room 111, MSC 0420, 4 Center Drive
Bethesda, MD 20892
USA
Synonyms
TGF-β1(the nomenclature is used worldwide with the
number designating the isoform)
Definition
The transforming growth factor-β (TGF-β) superfamily consists of more than 40 structurally related secreted proteins (1). Three members (TGF-β1, 2, 3)
are expressed in mammals; despite a 70%–76% sequence homology, these isoforms have expression pattern and functional differences. Whereas, TGF-β2 and
TGF-β3 are important for cellular differentiation, development, and embryogenesis, the effects of TGF-β1
are predominantly—albeit not exclusively—immunologic. Lymphoid cells selectively produce TGF-β1.
The name 'transforming' is something of a misnomer
because this factor is not always associated with oncogenesis. TGF-β1 is possibly the most pleotropic of
all the cytokines and growth factors, and its activity
is cell-type- and context-dependent. The ability of
TGF-β1 to suppress cell growth distinguishes it
from most other cytokines/growth factors. Mechanistically, it converts receptor ligation at the cell surface
into an enzymatic signaling cascade within the cell to
change the level of expression of target genes. In this
manner, it is able to target a vast array of immune cell
lineages to modulate their ability to proliferate, differentiate, survive, perform effector functions, and migrate to sites of antigen presentation and/or inflammation. These events are vital to the initiation, progression, and resolution of inflammatory responses. Dysregulated expression or function of TGF-β1 is implicated in autoimmune disease, chronic inflammation,
and tumor progression. The driving force behind
TGF-β1 seems to be maintaining homeostasis of controlled immune responses, and it achieves its goal by
orchestrating a network of intracellular signaling
crosstalk that enables cells to rapidly respond to
changes in their environment.
3
658
Characteristics
Cellular sources
TGF-β1 expression is present at the four-cell embryo
stage and persists, in most tissues, during morphogenesis and into adulthood. Most all mature cells have
been shown to produce this factor. Likewise, nearly
all cell types have functional TGF-β1 receptors.
Although controversial, it has been postulated that
the primary effector function of a small cohort of regulatory T cells (e.g. Th3 and CD4+CD25+) is to secrete
TGF-β1.
Regulation of activity
It is well established that the bioavailability and activity of TGF-β1 are influenced by the environment
(Table 2), and it generally is accepted that some of
Transforming Growth Factor β1; Control of T Cell
Responses to Antigens. Table 1 GenBank
accession numbers for transforming growth factor-β1
(TGF-β1)
Accession numbers
(partial listing only)
Species
Gene
Protein
Human (Homo sapiens) J04431, J05114 PO1137
Mouse (Mus musculus) AH003562
P04202
Transforming Growth Factor β1; Control of T Cell Responses to Antigens
TGF-β1 signaling
The predominant mechanism by which TGF-β1 elicits
its activity is through modulation of gene transcription.
TGF-β1 mediates the association of transmembrane
type II (TβRII) and type I (TβRI) receptors. Ligand
binding propagates signaling through phosphorylation
of multiple effector proteins. The only known direct
TGF-β1 signaling effectors are a class of structurally
similar Smad proteins (2). Once the ligand has
bound to the serine-threonine kinase receptor a signaling complex is formed, leading to the phosphorylation
3
Transforming Growth Factor β1; Control of T Cell
Responses to Antigens. Table 2 Factors that
modulate bioactivity of transforming growth factor-β1
(TGF-β1)
Enhance TGF-β synthesis and secretion
Liver hepatotoxicants (carbon tetrachloride, acetaminophen, alcohol)
Tissue injury (liver, renal, and lung)
Hypoxia
Stress
Viral infection
Parasitic infection
Steroid hormones (retinoids, vitamin D, and
tamoxifen)
Activate extracellular latent TGF-β1
Mannose 6-phosphate/insulin-like growth factor 2
receptor (M6P/IGF2R)
of Smad 2 and Smad 3 and their subsequent trafficking
to the nucleus, where they bind well-defined Smad
response elements and function as transcriptional
modulators to regulate transcription of TGF-β1 target
genes (Fig. 1).
Smads can also positively regulate gene expression by
recruiting coactivators such as CBP/p300 or negatively by forming complexes with histone deacetylases
(HDACs) or corepressors (such as c-ski and SnoN)
which themselves associate with HDACs. One key
negative regulation of Smad signaling is the expression of the inhibitory Smad, Smad7. Smad7 blocks
TGF-β signaling by competing with R-Smads for association with TβRI, or by targeting receptors for ubiquitin-mediated degradation. Potent inducers of
Smad7 are interferon-γ (IFN-γ), tumor necrosis factor
(TNF)-α, and interleukins IL-1β, and IL-7. Induction
of Smad7 represents an important regulatory interplay
between TGF-β1 and cytokines in immune cell function. It is noteworthy that type TβRI and TβRII receptors distinguish themselves from other cytokine/
growth factor receptors by their specificity for serine/
threonine, rather than tyrosine kinase, activity.
Smad-independent signaling pathways also regulate
TGF-β1 signaling. For instance, TGF-β1 activates mitogen-activated protein (MAP) kinases including the
extracellular regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 kinases. TGF-β1 has also
been shown to activate Rho-like GTPases and phosphatidulinostiol-3-kinase (P13K) and signal through
protein phosphatase 2A (PP2A). One key point of
cross-talk among signaling intermediates is MAP kinase activation that occurs downstream of growth factors, integrins, and chemokine receptors. In most
cases, activated MAP kinases promote the actions
of TGF-β1 to enhance cell migration. Thus, activation
of growth factor receptor and the pattern of cytokine/
chemokine signaling have a tremendous impact on the
response of cells to TGF-β1 (Fig. 1). Finally, TGF-β1
3
these changes increase human susceptibility to immunologic-related diseases. TGF-β1 predominantly is secreted as a biologically inert complex consisting of
mature TGF-β1, latency associate protein (LAP),
and latent TGF-β1-binding protein (LTBP). Prior to
binding to TGF-β receptors, the latent complex must
be cleaved into the 25 kDa active TGF-β1 homodimer.
659
Transglutaminase
T
Plasmin/plasminogen activator
Apoptotic T cells
Reactive oxygen species
αvβ6 Integrin receptor
Suppress activation of extracellular latent TGF-β1
α2-Macroglobulin
Decorin
Endoglobin
Mucosal mast cell protease (MMCP)
Antagonize TGF-β1 signaling
Cytokines: tumor necrosis factor-α, interferon-γ, interleukins IL-1β, IL-6, IL-2
Transforming Growth Factor β1; Control of T Cell
Responses to Antigens. Figure 1
660
Transforming Growth Factor β1; Control of T Cell Responses to Antigens
signaling can also modulate protein stability. For example, it enhances degradation of TβRI.
Immunological activities
A loss-of-function mutation in TGF-β1 results in the
rapid onset of lethal multiorgan inflammation and autoimmune phenotype. These transgenic mouse models
clearly establish the critical role of this factor in maintaining immune homeostasis for the prevention of disease and chronic inflammation (Fig. 2).
A role for TGF-β1 has been implicated, in several
different mouse models, including tolerance, and
particularly in mucosal immunity. While these studies
clearly demonstrate the onset of inflammation in the
absence of TGF-β1 signaling, more recent data suggest that the role for TGF-β1 in controlling T cell
homeostasis may be restricted to preventing inappropriate responses to self- or environmental antigens,
rather than regulating T cell responses to low-avidity
self-ligands (3). In addition to its immunosuppressive
and anti-inflammatory properties, TGF-β1 is capable
of promoting inflammation (4). For example, at the
early stages of inflammation, it enhances lymphoid,
neutrophil, monocyte, and macrophage migration, presumably to enhance the localization of these cells at
the site inflammation. Probably TGF3 also prolongs
the inflammation associated with numerous autoimmune disorders by actively sequestering activated
T cells at the site of inflammation. TGF-β1 also exerts
numerous suppressive effects on T and B lymphoid
effector and antigen-presenting cells and many of
these effects are summarized in Table 3.
3
Preclinical Relevance
Implications for disease
Dysregulated expression of TGF-β1 or response of
immune cells to TGF-β1 signaling have been implicated in the pathogenesis of many human diseases,
including hypersensitivity reactions such as asthma
and food allergies, as well as autoimmune disorders,
including encephalomyelitis, arthritis, systemic lupus
erythematosus, and allograft rejection. While it is com-
Transforming Growth Factor β1; Control of T Cell
Responses to Antigens. Figure 2
monly accepted that both environmental and genetic
factors contribute to the incidence of these immune
responses, it remains unclear why some individuals
are susceptible to these disorders while others are
not. Appropriate levels of TGF-β1 have been shown
to be essential for maintaining immunologic balance,
to prevent the pathogenesis of hypersensitivity reactions/chronic inflammation and autoimmune disorders.
Perhaps a better mechanistic understanding of how it
modulates cellular and molecular pathways will provide important insights that will enhance our understanding of susceptibility to these diseases. Outlined
below are three prevalent immune disorders that occur
in response to common environmental exposure;
which, TGF-β1 is key to regulation of the ensuing
pathological immune responses.
Asthma
The development of asthma in response to environmental antigens affects up to 20% of the population
in developed countries. Asthma is a chronic inflammatory disease of the airways that is characterized by
mononuclear infiltration, eosinophil degranulation,
and bronchoconstriction. TGF-β1 is constitutively expressed by airway epithelial cells, eosinophils,
T lymphocytes, macrophages, and fibroblasts, and
stored in the extracellular matrix of the airways. Rodent models of asthma suggest that it mediates both
anti-inflammatory and profibrotic effects (5). Prior to
allergen exposure, it is thought to play a critical protective role against the onset of asthma by suppressing
airway inflammation and hyper-responsiveness
through the suppression of T lymphocytes, dendritic
cells, eosinophils, mast cells, and IgE production. Notably, mononuclear cell infiltration into the lungs is
prevalent in TGF-β1 null mice. Additionally,
TGF-β1 may further suppress airway CD4+ T cell allergen exposure by enhancing activity of T regulatory
cells. However, repeated long injury is accompanied
by a profound TGF-β1-mediated recruitment of fibroblasts into the airways, a progressive deposition of
extracellular matrix, and subsequent fibrosis and bronchoconstriction.
Food allergy
Food allergy is characterized by an adverse hypersensitive response to food consumption. A normal healthy
gastrointestinal immune response must discriminate
between harmful pathogens and harmless dietary antigens and commensal bacterial flora. The mucosal immune system has generated two adaptive immune responses to meet this challenge: induction of a local
secretory IgA response, which is propagated in the
absence of a measurable systemic immune response,
to clear potentially dangerous antigens; and induction
of oral tolerance, a state of non-responsiveness or
Transforming Growth Factor β1; Control of T Cell Responses to Antigens
661
Transforming Growth Factor β1; Control of T Cell Responses to Antigens. Table 3 Biological activities of
transforming growth factor-β1 on the immune system
Parameter
TGF-β1-mediated effect
T lymphocytes
TCR-induced CD4+ and CD8+ T cell Suppress; memory CD4+ resistant to G1 cell cycle arrest
proliferation
IL-2-induced CD4+ and CD8+ T cell
proliferation
Suppress, dependent upon IL-2 concentration
Th1 differentiation
Suppress, but dependent upon strength of T cell stimulation; inhibits T-bet
and INF-γ expression
Th2 differentiation
Suppress; inhibits GATA-3 and IL-4 expression
Th1 effector function
Suppress; inhibits INF-γ and IL-2 production; inhibits IL-12 signaling
Th2 effector function
Suppress; inhibits IL-4 and IL-5 production
CD8+ cytotoxic T cell effector function
Suppress
CD4+ and CD8+ migration/adhesion Enhance; increases CXCR4 and α4β7 expression
CD4+CD25+ regulatory T cell function
Enhance; increases Foxp3, GITR, CD103, CTLA-4 expression
IL-12 signaling
Suppress; downregulates IL-12 receptor β2 chain
T cell apoptosis
Suppresse or enhance, dependent on microenvironment
B lymphocytes
Proliferation
Suppress
Effector function
Enhance IgA and IgG2b and suppressed most other isotopes
Antigen-presenting cells
MHC class I and class II molecules
Suppress
Monocytes and macrophages
Monocyte chemotaxis
Enhance or suppress
Macrophage chemotaxis
Suppress
Neutrophils
Neutrophil chemotaxis
Enhance or suppress
Chemokine and receptor expression Suppress or enhance: chemokine, receptor, and cell type-dependent
CTLA=cytotoxic T-lymphocyte-associated protein 4; GITR= glucocorticoid induced TNF receptor; Ig=immunoglobulin;
IL=interleukin; INF=interferon; MHC=major histocompatibility complex; TCR=T cell receptor; TGF=transforming growth factor;
Th=T helper cell.
hypo-responsiveness which minimizes unnecessary
immune reactions against harmless antigens. A failure
to induce or an inability to maintain oral tolerance may
leads to a food allergy. TGF-β1 is abundant throughout the mucosa and has been shown in several experimental models to play a profound role in maintaining
oral tolerance (5). It is well documented that a population of TGF-β1-secreting T helper cells is generated
when low doses of antigen are consumed. These
TGF-β1-secreting regulatory T cells also produce various amounts of IL-4 and/or IL-10. Secreted TGF-β1
suppresses T cell proliferation and promotes class
switching of B cell IgA isotypes to modulate the adaptive immune response. Moreover, it also enhances the
preservation of the epithelial barrier between environmental antigens in the gut flora and T lymphocytes in
the mucosa to add yet another level of regulatory control over adaptive T cell responses.
Childhood food allergies have been associated with a
reduction in the number of mucosal TGF-β1-producing lymphocytes. Aberrant levels of mucosal TGF-β1
and associated dysregulated responses to the normal
gut flora have also been implicated in the pathogenesis
of inflammatory bowel disease. Collectively, these
data implicate a role for TGF-β1 to maintain oral tolerance in humans.
T
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Transforming Growth Factor β1; Control of T Cell Responses to Antigens
Autoimmunity
Discordance in incidence of autoimmune disease in
monozygotic twins demonstrates a role for environmental exposure in regulating immune homeostasis.
Although numerous environmental factors have been
implicated, the underlying mechanisms remain relatively undefined. The autoimmune phenotype of the
TGF-β1 knockout mouse, characterized by circulating
antinuclear antibodies and glomerular deposit s of immune complexes, probably best defines the role of this
factor in the disease process. 100% of TGF-β1 knockout mice succumb to a massive multiorgan inflammation involving the heart, lung, liver, gut, salivary
glands, eyes, brains, and other tissues. The inflammatory infiltrates are predominantly perivascular and
vary from neutrophilic in the stomach to lymphocytic
in the brain. In agreement, systemic administration of
exogenous TGF-β1 or adoptive transfer of TGF-β1
−producing T cells protect against autoimmune diseases in several experimental models, including diabetes, encephalomyelitis, inflammatory bowel disease,
arthritis, systemic lupus erythematosus, and allograft
rejection. Targeted deletion of TGF-β signaling in
T cells alone has been demonstrated to be sufficient
to induce an autoimmune phenotype. It remains to be
determined whether other non-T cell TGF-β1-producing cells (e.g. macrophages) contribute to the disease
process as well. The precise mechanisms of actions
underlying the ability of this factor to regulate autoimmune disorders remains speculative. Recent evidence implicates a significant role for regulatory
T cells. CD4+ CD25+ regulatory T cells, also called
suppressor T cells, can be delineated into two subsets
of CD4+ CD25+ T cells with inherent activity to suppress autoreactive T cells: these are ‘natural’ regulatory CD4+ CD25+ T cells that emerge from the thymus, and adaptive regulatory CD4+ CD25+ T cells that
are induced in the periphery. TGF-β1 has been shown
to positively regulate both subsets, and TGF-β1mediated expansion of CD4+ CD25+ T cells protects
against autoimmune diabetes (7). However, in view of
the diversity of pathology associated with autoimmune
disorders, it is highly likely that TGF-β1 also utilizes
other critical mechanisms of action. For instance,
modulation of Th2/Th1 cytokine balance, cell survival, migration, effector function, and Th3-mediated
tolerance represent likely alternative mechanistic
routes (8).
Relevance to Humans
The phenotype of the TGF-β1 mouse resembles
human SLE, Sjögren syndrome, graft-versus-host disease, and polymyositis, suggesting that TGF-β1 may
play a similar regulatory role in human immunologic
disorders. The levels of TGF-β1in serum and of its
mRNA in tissue can be measured and have been used
as diagnostic or prognostic markers for other human
diseases. For example, high levels of the factor in
RNA in tissues are associated with gastric cancer.
High serum levels also correlate with the development
of fibrosis in patients with breast cancer who have
received radiation therapy. Understanding the mechanisms of action of environment-induced immune disorders in experimental models will potentiate the development of better predictive risk assessment assays to
prevent disease as well as more specific therapeutic
regimens aimed at increasing effectiveness and diminishing deleterious side effects.
Regulatory Environment
Although interaction of chemicals with cytokines or
chemokines may have an important impact on the
function and regulation of the immune system they
are not regulated by any specific immunotoxicity
guideline. The cytokine network is mentioned in different guidelines or guideline drafts but exclusively in
connection with extended 'case-by-case' investigations.
The regulation of the immune system is complex, and
identification of the mechanism, of action of chemicalinduced immune toxicity is critical for the understanding of the disease process. The regulatory cytokine
TGF-β1 may be of special interest for such investigations. A better understanding of the disease process
will provide the basis for the development of more
sensitive and predictable assays for risk assessment.
Chemical-induced immunotoxicity may be indirectly
mediated via the soluble potent immune modulator,
TGF-β1. TGF-β1 may be a useful biomarker of chemical-induced and/or environmental-induced immunotoxicity. A critical challenge is to determine the appropriate therapeutic level of active TGF-β1 or signaling
pathways that positively influence cell responsiveness
to ameliorate disease while minimizing deleterious
side effects.
References
1. Flanders CF, Roberts AB (2000) TGF-β. In: Oppenheim
JJ, Feldman M, Durum SK, Hirano T, Vilcek J, Nicola
NA (eds) Cytokine reference: A compendium of
cytokines and other mediators of host defense. Academic
Press, New York, pp 719–746
2. Shi Y, Massague J (2003) Mechanisms of TGF-β
signaling from cell membrane to the nucleus. Cell
13;113:685–700
3. Gorelik L, Flavell RA (2002) Transforming growth
factor-β in T-cell biology. Nat Rev Immunol 2:46–53
4. McCartney-Francis NL, Frazier-Jessen M, Wahl SM
(1998) TGF-β: A balancing act. Int Rev Immunol
16:553–580
5. Duvernelle C, Freund V, Frossard N (2003) Transforming
growth factor-β and its role in asthma. Pulmon Pharmacol Ther 16:181–196
Transgenic Animals
6. Weiner HL (2001) Oral tolerance: immune mechanisms
and the generation of Th3-type TGF-β-secreting regulatory cells. Microbes Infect 11:947–954
7. Peng Y, Laouar Y, Li MO, Green EA, Flavell RA (2004)
TGF-β regulates in vivo expansion of Foxp3-expressing
CD4+CD25+ regulatory T cells responsible for protection
against diabetes. Proc Natl Acad Sci USA 101:4572–
4577
8. Prud'homme GJ, Piccirillo CA (2000) The inhibitory
effects of transforming growth factor-beta-1 (TGF-β1) in
autoimmune diseases. J Autoimmun 1:23–24
663
neoplastic and cardiovascular disease), in immunology, mutagenesis and carcinogenesis research, and in
novel target evaluation and drug discovery. In immunotoxicology, although the potential usefulness of
transgenic mice is widely recognized, practical application has been limited.
Characteristics
Generation of Transgenic Animals (1)
There are two principal methods by which transgenic
mice are created: microinjection of genetic material
into the pronucleus of a fertilized ova; or gene transfection of embryonic stem cells (ES cells) cells followed by injection of the transgenic cells into a blastocyst. In either case, the resulting transgenic embryo
is implanted into a recipient female prepared for pregnancy. The principal difference between the techniques is that, when successful, microinjection of the
pronucleus results in homozygous offspring, while
transfection of ES cells results in chimerae (see chimera) that must be selectively bred to yield homozygous animals.
There are two principal types of transgenic events,
those with one or more random insertions of the transgene and those where homologous recombination
results in targeted insertion. Either type of transgenic
event can result in a knock-in (KI) that will express the
coding sequences of the transgene. Although random
insertion will by definition result in a mutation in the
recipient genome, as most DNA is noncoding these
mutations are generally silent. Homologous recombination is used to selectively disrupt expression of the
homologous gene, resulting in a knock-out (KO). This
requires design of the inserted DNA so as to contain
sequences homologous to the desired host species’
gene.
A third type of transgenic animal—the knock-inknock-out (KI-KO) mouse—has also been created. If
the inserted DNA has a sequence homologous to a
murine gene and also codes for a foreign protein, a
KI-KO mouse can be created in a single step. Alternatively, these two types of transgenic events can be
combined in a two-step process to result in a KI-KO
strain.
3
3
3
Peter J Bugelski
Experimental Pathology
Centocor, Inc.
R-4-2, 200 Great Valley, Parkway
Malvern, PA 19355
USA
Synonyms
Knock-out, knock-in, genetically modified, recombinant
Definition
Animals whose genome has been modified using recombinant DNA technology so as to have a foreign
gene expressed (knock-in) or a native gene suppressed
(knock-out) in a heritable fashion. A number of transgenic species have been created: mice, rats, pigs,
goats, cattle, sheep and fish. Currently, the vast majority of transgenic animals are mice. Transgenic mice
have applications in numerous areas of biomedical
research (e.g. neurologic, inflammatory, autoimmune,
Transgene Expression
In some cases, expression of the transgene by the host
is not important. For example in mutagenesis research
the endpoint can be a mutation in the transgene that
will be expressed and detected ex vivo. In most cases
however, expression of the transgene is desired. Insertion of multiple copies of the gene and linkage to a
potent promoter (e.g. simian virus 40 promoter, will
likely ensure widespread and high-level gene expression. Depending on the experiments to be conducted, however, it may be important that the site (s)
3
3
Transgenic Animals
3
TGF-β1 is the prototype for a superfamily of secreted
proteins that control many aspects of growth and development. It was named transforming growth factor
because upon its discovery it was shown to induce a
transformed or tumor cell phenotype in normal cells.
TGF-β1 is now known to regulate a diverse array of
cellular functions unrelated to cell transformation.
Within the immune system, TGF-β1 is critical for
cell growth, differentiation, effector cell function, survival, and migration.
Transforming Growth Factor β1; Control of T cell
Responses to Antigens
Mucosa-Associated Lymphoid Tissue
3
Transforming Growth Factor β1
(TGF-β1)
T
Transgenic Animals
of expression, the magnitude of expression and timing
of expression be controlled. This can be accomplished,
but is no means guaranteed by selecting the gene promoter sequences included in the transgene. Techniques
are now reasonably well established for controlling the
sites and magnitude of expression and “conditional”
gene expression in transgenic animals is an active field
of research.
3
Preclinical Relevance
Transgenes
Transgenic mice have been created which express a
wide and ever increasing range of genes (as of June
2003 the database maintained by BioMed Net lists
2300 transgenic mice (2)). These genes include reporter constructs (e.g. β-galactosidase or green fluorescent protein (GFP)) and viruses (e.g. hepatitis C),
and a wide variety of human proteins.
Of greatest relevance to immunotoxicology is the expression of human cytokines, cell surface markers and
immunoglobulins. Mice transgenic for mutant reporter
genes have application in genotoxicity, and mice transgenic for mutant oncogenes have application as shortterm replacements for 2-year cancer bioassays.
In efforts to facilitate xenotransplantation, transgenic
pigs have been created that express a small number of
human genes. There have also been reports on a model
of colitis in rats expressing a human major histocompatibility antigen (HLA)-B27.
Function
Expression, however, is not sufficient for the transgenic strain to have preclinical relevance. The transgene
gene product (i.e. the protein) must be functionally
active in the transgenic animal. In the case of immunotoxicology, this will generally require that the
human protein binds, and will result in signal transduction in its respective murine receptor (e.g. CCR
and FcR or binding proteins such as major histocompatability complex (MHC) class II).
Genetic Background
Two strains of inbred mice are widely used for creating transgenic mice; 129 and C57 black. Once a transgenic strain has been created however, it may be possible to “move” the transgene into an alternate genetic
background (e.g. BALB/c, by selective breeding. This
can be of critical importance in application of transgenic mice in immuntoxicology where the genetic
background of the mice can have a significant impact
on the experiment (e.g. delayed-type hypersensitivity,
transplantation, immunogenicity and host defense
against infection or neoplasia).
Fecundity
Fecundity can also be an important factor in determin-
ing the success of application of transgenic mice in
toxicology. Many strains of transgenic mice show
low fecundity as determined by fertility and number
of offspring. As we must have sufficient numbers of
animals for study, low fecundity can have a serious
impact on our ability to conduct a given experiment.
Simple animal husbandry (e.g. selection of proven
breeders) may be sufficient to solve this issue. Maintaining the breeding colony as heterozygotes may be
required. However, as the offspring of heterozygotes
will be a mix of transgenic and nontransgenic, the
offspring must be genotyped or phenotyped prior to
enrolment in studies.
Application of Transgenic Rodents in Immunotoxicology
Transgenic mice have been used extensively for studying the immune system. As of June 2003 the National
Institutes of Health Medline lists over 2800 papers
describing the use of transgenic mice to study immunology. Obviously, there are far too many examples to
list here. However, applications of direct relevance to
immunotoxicology are much rarer. Some selected examples are listed in Table 1.
Relevance to Humans
As with any animal system, the relevance of transgenic animals to humans is somewhat limited. Some of
the factors which lead to this limited relevance are
listed in Table 2. One must also keep in mind that in
most cases while the transgenic animal may be transgenic for one human protein (and therefore immunotolerant to that human protein) it will likely not be
inherently tolerant to any administered human therapeutic protein. With these caveats in mind, a priori,
transgenic mice should have a much relevance to humans as any murine system.
Regulatory Environment
Use of transgenic animals for demonstrating pharmacologic activity and safety are gaining increasing acceptance by regulatory authorities. They are specifically dealt with in the following guidance documents:
* FDA Guidance for Industry. Clinical Development
Programs for Drugs, Devices, and Biological Products for the Treatment of Rheumatoid Arthritis
(RA) http://www.fda.gov/cder/guidance/1208fnl.
pdf
* FDA Guidance for Industry. Immunotoxicology
Evaluation of Investigational New Drugs. http://
www.fda.gov/cder/guidance/4945fnl.doc
* ICH Guidance for Industry. S1B Testing for Carcinogenicity of Pharmaceuticals http://www.fda.gov/
cder/guidance/1854fnl.pdf
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664
Animal Models of Immunodeficiency
Transgenic Animals
665
Transgenic Animals. Table 1 Examples of application of transgenic rodents in immunotoxicology (KI, knock-in;
KO, knock-out)
Transgenic system
Application
Reference
Various cytokine KI and
KO mice
Drug hypersensitivity
3
TNF-α receptor KO
Mechanism of toluene diisocyanate asthma
4
Human CD4 KI-murine CD General and immunotoxicity of a chimeric antihuman CD4 monoclonal 5
KO
antibody
Human CD4 KI-murine CD Embryo–fetal and immunotoxicologic development study of a chimeric 6
KO
antihuman CD4 monoclonal antibody
Human growth hormone
KI rats
Immunogenicity
7
Human interferon-α KI
Breaking immune tolerance to interferon-α
Human carcinoembryonic Safety of human carcinoembryonic antigen tumor vaccine
antigen KI mice
8
9
Transgenic Animals. Table 2 Examples of sources of limitation of the relevance of transgenic mice to human
immunotoxicity
Physiology
Kinetics
Generally more rapid clearance of xenobiotics and therapeutic proteins in mice
Metabolism
Differences between murine and human P450 usage and inducibility, substrate
specificity and metabolite profile
Immunology
Ontogeny
Differences in timing of cytogenesis, histogenesis and organogenesis of the immune
system
Receptors
Differences in binding affinity and signal transduction of human proteins for murine
receptors and binding proteins
Immunogenicity and
tolerance
Differences between human and murine antigen processing and MHC restrictions
T cells
Differences in T helper 1 and 2 usage and switching
B cells
Differences in immunoglobulin class switching
Macrophages
Differences in Fc receptor utilization
References
1. Hofker MH, Van Deursen J (eds) (2002) Transgenic
Mouse: Methods and Protocols. Methods Molecular
Biology, Vol. 209. Humana Press, Clifton NJ
2. BioMed Net. http://www.biomednet.com/db/mkmd (accessed June 2003)
3. Moser R, Quesniaux V, Ryffel B (2001) Use of transgenic
animals to investigate drug hypersensitivity. Toxicology
158:75–83
4. Matheson JM, Lemus R, Lange RW, Karol MH, Luster
MI (2002) Role of tumor necrosis factor in toluene
diisocyanate asthma. Am J Respir Cell Mol Biol 27:396–
405
5. Bugelski PJ, Herzyk DJ, Rehm S et al. (2000) Preclinical
development of keliximab, a Primatized anti-CD4 monoclonal antibody, in human CD4 transgenic mice:
characterization of the model and safety studies. Hum
Exp Toxicol 19:230–243
6. Herzyk DJ, Bugelski PJ, Hart TK, Wier PJ (2002)
Practical aspects of including functional endpoints in
developmental toxicity studies. Case study: immune
function in HuCD4 transgenic mice exposed to antiCD4 MAb in utero. Hum Exp Toxicol 21:507–512
7. Takahashi R, Ueda M (2001) The milk protein promoter
is a useful tool for developing a rat with tolerance to a
human protein. Transgenic Res 10:571–575
8. Braun A, Kwee L, Labow MA, Alsenz J (1997) Protein
aggregates seem to play a key role among the parameters
influencing the antigenicity of interferon alpha (IFNalpha) in normal and transgenic mice. Pharm Res
14:1472–1478
9. Francini G, Scardino A, Kosmatopoulos K et al. (2002)
High-affinity HLA-A(*)02.01 peptides from parathyroid
hormone-related protein generate in vitro and in vivo
antitumor CTL response without autoimmune side
effects. J Immunol 169:4840–4849
T
Transgenic Mouse
Transgenic Mouse
Transgenic mice are genetically engineered mice that
over-express foreign DNA and are typically referred to
as transgenic, while those in which foreign DNA has
replaced an endogenous gene are termed gene targeted
(or knockout). In the strict sense, however, both these
procedures yield a transgenic mouse (i.e. one with
added genetic material).
Knockout, Genetic
Trichinella spiralis
A helminthic parasite, invading the gut mucosa and
residing as larvae in striated muscle tissues.
Host Resistance Assays
3
666
Triglycerides
3
Tricglycerides are molecules that consist of a glycerol
backbone esterified to three fatty acids.
Fatty Acids and the Immune System
3
Transglutaminase
Trivalent Chromium
The ionic form of chromium when three outer shell
electrons (one from 4s and two from 3d orbitals) have
been shed, thereby giving the atom an overall charge
of +3.
Chromium and the Immune System
3
The epidermal keratinocyte transglutaminase I is a calcium-dependent enzyme that plays a central role in
keratinocyte cornification. It catalyzes the cross-linking between glutamine and lysine residues of isopeptides at the inner surface of keratinocyte cell membranes, which is an essential step for the stabilization
of their cornified cell envelope (CCE).
Three-Dimensional Human Skin/Epidermal Models
and Organotypic Human and Murine Skin Explant
Systems
3
Transition Element
Elements that occupy the middle portions (the dblock) of the periodic table, have valence electrons
in two or more shells instead of only one, and are
characterized in most cases by variable oxidation
states and magnetic properties.
Chromium and the Immune System
Vanadium and the Immune System
Trypanosomes, Infection and
Immunity
Ronald Kaminsky
Centre de Recherche Santé Animale
Novartis
CH-1566 St-Aubin
Switzerland
Synonyms
hemoflagellates
3
3
Definition
Transporter Associated with Antigen
Processing (TAP)
TAP is composed of two subunits, TAP1 and TAP2.
This heterodimer, which belongs to the ABC (ATPbinding cassette) transporter family is responsible for
the shuttling of peptides from the cytosol into the
lumen of the endoplasmic reticulum.
MHC Class I Antigen Presentation
Trypanosomes are protozoan parasites of the family of
Trypanosomatidae, belonging to the order of Kinetoplastida of the class of Zoomastigopohora.
Three species are pathogenic to man—Trypanosoma
brucei gambiense and T brucei rhodesiense cause
African human sleeping sickness in sub-Saharan Africa, while T. cruzi causes Chagas disease in South
America (Table 1).
Characteristics
Characteristics of the parasites
The prominent morphological feature of the unicellular protozoan parasites is the kinetoplast, an organelle
which contains about 15% of the cells DNA. The kinetoplast can be visualized by Giemsa staining or flu-
3
Trypanosomes, Infection and Immunity
667
Trypanosomes, Infection and Immunity. Table 1 Characteristics of human pathogenic trypanosomes
Trypanosoma
species
Disease
T brucei gambiense
Sleeping
sickness
T. brucei rhodesiense
T. cruzi
Transmission
Mode of
transmission
Animal reservoirs
Geographic
distribution
Tsetse flies
(Glossina spp.)
Bite
Mainly dogs, pigs, and
certain game animals
West and Central Africa
Sleeping
sickness
Tsetse flies
(Glossina spp.)
Bite
All major domestic animals and various game
animals
East Africa
Chagas
disease
Reduviid bugs
(Triatoma spp.,
Rhodnius spp.,
Panstrongylus
spp.)
Contamination
by bug feces
Southern and
Domestic (dogs, cats,
guinea-pigs), rodents and Central
wild animals (opossums America
etc.)
vector
orescent dies like DAPI. Movement of trypanosomes
is via a flagellum which originates at the basal body
near the kinetoplast and which is attached to the body
of the parasite by an undulating membrane.
The African trypanosomes are extracellular parasites
(16–30 μm long) which move within the blood (hence
their designation as hemoflagellates) or within the cerebral spinal fluid. T. cruzi occurs in man in both as
extracellular and intracellular form. After introduction
into the blood T. cruzi invades various cell types including macrophages and muscle cells. The intracellular form (3 μm in diameter) is much smaller than the
extracellular form and does not posses a flagellum, but
still contains the kinetoplast.
All bloodstream forms of African trypanosomes are
coated with variable surface glycoproteins (VSGs).
The VSGs are anchored through a glycosyl phosphatidyl inositol lipid to the body of the parasite. These
highly immunogenic VSGs have, at any one point of
time, the same structure resulting in a specific variant
antigen type (VAT). However, the VSGs are periodically removed and replaced with the result that the
parasite population bearing one VSG are killed by
an antibody response and are replaced by a new population with another variant antigen type. This antigenic variation is a mechanism that plays a key role in
the escape of trypanosomes from total destruction by
the immune response of their mammalian hosts (1).
'kissing' bugs, the family of Reduviidae (Table 1),
not by direct inoculation when the vector is feeding
but by contamination through parasites in feces. Tri-
Trypanosomes, Infection and Immunity.
Figure 1 Trypanosoma brucei brucei bloodstream
forms.
3
T
Cyclical transmission
African trypanosomes are cyclically transmitted by
tsetse flies (various Glossina species) (Table 1).
After a fly has taken a blood meal from an infected
host, the trypanosomes undergo various changes and
multiplication within the fly. They finally mature in
the salivary glands of the tsetse fly, to infectious
metacyclic forms which are transmitted to a naive
host.
The American T. cruzi is transmitted cyclically by
3
Trypanosomes, Infection and Immunity.
Figure 2 Trypanosoma cruzi: in vitro cultured amastigote forms in mammalian feeder cells.
Trypanosomes, Infection and Immunity
atoma infestans is the major transmitting species, but
various others species including Triatoma spp., Rhodnius spp., and Panstrongylus spp. are capable of transmitting T. cruzi.
Characteristics of the diseases
Sleeping sickness is 100% fatal if left untreated.
There are two disease stages for human African trypanosomiasis. A chancre, a primary lesion at the site
of the bite, it is not observed frequently. The first
stage, when trypanosomes are in the blood, is characterized by non-specific symptoms, such as fever, severe headache, joint or muscle aches. The second stage
of the disease (also called late stage) starts with the
invasion of the central nervous system by trypanosomes, which cross the blood-brain barrier 3–
6 month post infectionem. It is in the late stage of
the disease that the characteristic symptoms of sleeping sickness occur, such as sleep disturbances, alteration of mental state, muscle tone disorders, abnormal
movements, and sensory and coordination disorders,
up to a final general apathy.
The acute form of Chagas disease is characterized by
general malaise with a variety of clinical manifestations. Symptoms can be very mild and atypical. At the
site of entry of T. cruzi a local inflammation called a
chagoma may develop; this is known as a Romana
sign if it occurs at the eyelid. The acute form is followed by a period of an indeterminate form without
any clinical symptoms. It is estimated that 20%–50%
of persons with the indeterminate form of the infections will suffer from cardiac, digestive, or neurological damage 10–20 years after infection (2).
3
Preclinical Relevance
African and American trypanosomes can be manipulated in vitro and in various animal models. T. brucei
gambiense appears to be the most difficult species for
laboratory work. In vitro assays and in vivo models are
being used to identify new active compounds, nonvariant vaccine targets, and to monitor drug resistance.
Some forms play an important role in host resistance
models for immunotoxicity screenings.
Relevance to Humans
Infection
Sixty Million people in 36 countries of sub-Saharan
Africa live at risk of acquiring sleeping sickness. In
1999 around 45 000 cases were reported, but the number of people thought to have the disease at any one
time is between 300 000 and 500 000. Chagas disease
affects 16–18 million people, and about 100 million
(25% of the population of Latin America) are at risk of
acquiring Chagas disease. Due to the chronic character
(indeterminate stage) of Chagas disease, transmission
occurs not only via insect vectors, but also by congen-
ital transmission, and from transfusions with contaminated blood, and organ transplantations.
Immunity
Due to antigenic variation of the African trypanosomes, which can express approximately 1000 different variant antigen types (1), immunity to the parasites
develops only to specific VATs but does not provide
protection against infection.
Treatment
Two drugs, pentamidine and suramin, are used in the
first stage of sleeping sickness prior to CNS involvement. The first-line treatment for late-stage cases,
when trypanosomes are established in the CNS, is
the arsenic-based drug melarsoprol (3). The drug has
been in use since 1949. However, up to 5% of treated
patients may die because of lethal encephalopathy due
to the drug. Recently a new treatment schedule (4) was
designed, but the number of patients with encephalopathy syndromes was the same as before. Nevertheless,
the new 10-day schedule is a useful alternative to the
present standard 26-day treatment schedule. Eflornithine (DFMO) is used mainly as a back-up in instances of melarsoprol-refractory T. brucei gambiense.
Its efficacy against East African sleeping sickness is
limited due to an innate lack of susceptibility of
T. brucei rhodesiense based on higher ornithine decarboxylase turnover.
The unsatisfactory treatment situation for sleeping
sickness is hampered further by the occurrence of melarsoprol-resistant trypanosomes (5) in several regions
of sub-Saharan Africa. A molecular mechanism in the
resistant isolates was identified: the majority of individual resistant isolates from geographically distant
localities contained the same set of point mutations
in their TbAT1 genes (6), which codes for an adenosine transporter (7).
The drug of choice for treatment of Chagas disease is
nifurtimox, with benznidazole as a back-up. However,
these drugs are associated with side effects (2). Nifurtimox and benznidazoles were introduced at the beginning of the 1970s. Treatment success varies according
to the phase of Chagas disease, the period of treatment,
and the dose, the age, and geographical origin of the
patients. Good results have been achieved in the acute
phase, in recent chronic infection, and congenital infections. However, there is still controversy about their
use in chronic cases (2).
3
668
Regulatory Environment
At present there is only one new antitrypanosomal
drug on clinical trial in Africa—the diamidine derivative DB289. Identification of novel compounds and
their development to drugs is pursued by various private-public initiatives. Registration of new drugs
Tumor-Associated Antigens
TSK
An acronym for tight skin which is associated with
thickened skin and fibrosis due to mutations in the
fibrillin gene.
Systemic Autoimmunity
3
might be facilitated when these drugs are classified as
orphan drugs. As mentioned above, trypanosomes are
indirectly regulated by different immunotoxicology
guidelines by the recommendation for infection models using these parasites in host-resistance assays.
More detailed information is given in the relevant entries in this book.
669
References
3
Tsetse Fly
Tsetse flies (Glossinidae; more than 30 species) are
sub-Saharan bloodsucking flies (Diptera). The females
do not lay eggs but give birth to living larvae. Both
sexes feed on the blood of humans, livestock, and wild
animals. Tsetse flies transmit human and animal pathogenic trypanosomes. Ingested trypanosomes of an infested host undergo a development cycle in the tsetse
fly to mature to metacyclic forms which are infective
for the next host.
Trypanosomes, Infection and Immunity
Tuberculin-Type Reaction
A classical example of a delayed-type hypersensitivity
(DTH) is the tuberculin-type reaction. In sensitized
individuals, it is induced by an intradermal injection
of tuberculin, an extract of Mycobacterium tubercolosis. This particular example of DTH was first described by R. Koch. He who observed that patients
with tubercolosis reacted with fever and shock after
the subcutaneous injection of tuberculin. Typically, the
T cell mediated local immune reaction appears one or
two days after the application.
The tuberculin test, however, is not an allergic reaction. It is a diagnostic proof for the previous infection
with M. tubercolosis and also other pathogens such as
M. leprae or Leishmania tropica.
Delayed-Type Hypersensitivity
Tumor Antigen
Any molecule leading to immune recognition of
tumor. A generic term that encompasses tumor-specific antigens, antigens shared by normal and neoplastic
cells, and specificities recognized by xenogeneic antibodies (e.g. human molecules bound by mouse monoclonal antibodies) that can be non-antigenic in the species of origin.
Tumor, Immune Response to
3
α-Amino-β-indole-propionic acid; a component of
proteins; it is chromogenic, producing a violet color
with chlorine or bromine solution.
Serotonin
Mixture of antigens obtained from the culture of Mycobacterium tuberculosis.
Mitogen-Stimulated Lymphocyte Response
3
Tryptophan
Tuberculin
3
1. Borst P (2002) Antigenic variation and allelic exclusion.
Cell 109:5–8
2. Coura JR, de Castro SL (2002) A critical review on
Chagas disease chemotherapy. Mem Inst Oswaldo Cruz
97:3–24
3. Legros D, Ollivier G, Gastellu-Etchegorry M et al. (2002)
Treatment of human African trypanosomiasis—present
situation and needs for research and development. Lancet
Infect Dis 2:437–440
4. Burri C, Nkunku S, Merolle A, Smith T, Blum J, Brun R
(2000) Efficacy of new, concise schedule for melarsoprol
in treatment of sleeping sickness caused by Trypanonosoma brucei gambiense: a randomized trial. Lancet
355:1419–1425
5. Kaminsky R, Mäser P (2000) Drug resistance in African
trypanosomes. Curr Opin Anti-infect Invest Drugs 2:76–
82
6. Matovu E, Geiser F, Schneider V et al. (2001) Genetic
variants of the TbAT1 adenosine transporter from African
trypanosomes in relapse infections following melarsoprol
therapy. Molec Biochem Parasitol 117:71–81
7. Mäser P, Sütterlin C, Kralli A, Kaminsky R (1999) A
nucleoside transporter from Trypansoma brucei involved
in drug resistance. Science 285:242–244
Tumor-Associated Antigens
Tumor-associated antigens (TAA) are tumor-specific
proteins that can be recognized by immune effector
cells of the host. To date, a variety of TAA are
T
3
Tumor, Immune Response to
known. These are derivatives of either (i) physiological self-antigens or tissue specific differentiation antigens that are dramatically overexpressed by tumor
cells in comparison to other cells, (ii) mutated selfproteins or specific oncogenic antigens inappropriately
expressed by tumor cells, or (iii) those derived from
virally encoded antigens. The recognition pattern induced by TAA allows the immune system to distinguish the transformed neoplastic cells from surrounding normal tissue cells and triggers the immune cascade against them.
Cancer and the Immune System
3
Tumor, Immune Response to
Pier-Luigi Lollini
Cancer Research Section
Department of Experimental Pathalogy, University of
Bologna
Viale Filopanti 22
I-40126 Bologna
Italy
Synonyms
Immune response to cancer, anti-tumor immunity.
Definition
The immune system of the host responds to tumor
growth as it does to infectious agents, with specific
(e.g. T cells and antibodies) and non-specific (e.g. natural killer cells and cytokines) effector and regulatory
mechanisms. The immune response reduces the number of tumors arising in the host, but is no longer
effective against established tumors. Tumor immunotherapy is the attempt to elicit a therapeutic immune
response in cancer patients.
Characteristics
The immune response against tumors was formally
demonstrated in the late 1940s and early 1950s using
transplantable tumors induced with chemical carcinogens or retroviruses in inbred mice (1). The experiments showed that mice vaccinated with a given
tumor reject a subsequent challenge with the same
tumor (immune memory), but fail to reject an unrelated tumor (specificity).
The immunization-challenge system was extensively
used to characterize the effector and regulatory mechanisms of the immune response against tumors
using two strategies:
* cellular and molecular analysis of local and systemic components elicited by immunization and/or involved in rejection
*
use of mice with selective immune deficiencies of
genetic origin (spontaneous mutation or genetically
modified mice) or induced by exogenous treatments
like monoclonal antibodies or drugs.
Specific immune responses against tumors are mainly
due to T cells. Cytotoxic T cells (CTL) expressing the
CD8 surface molecule are the final effectors capable of
tumor cell lysis. Helper T cells (Th) expressing CD4
play a fundamental positive or negative regulatory
role. Tumor immunologists tend to downplay the importance of B cells, antibodies and complement because solid tumors are resistant to complementmediated cytotoxicity (tumor cells express complement inhibitors like CD55 and CD59) and in immunization-challenge systems B cells can even favor tumor
growth (“enhancement”).
Most cells of the innate (also called natural or nonadaptive) immune system directly affect tumor growth,
and are required for the generation of T cell immunity.
Professional and non-professional phagocytes destroy
tumor cells and generate antigenic material that is subsequently picked up by antigen-presenting cells (APC)
like dendritic cells, that are indispensable to activate
T cell responses. Natural killer (NK) cells can kill
tumor cells in tissues and in the bloodstream, thus
are important in the control of systemic metastatic
spread. In the course of the immune response many
cytokines released by various cell types have regulatory and effector activities. Interferons IFN-α, IFN-β,
and IFN-γ and tumor necrosis factors TNF-α and
TNF-β, in addition to their roles as internal mediators
of the immune system, directly inhibit tumor cell proliferation, trigger apoptosis, and induce the secretion
of anti-angiogenic chemokines like MIG and IP-10
(2).
Immune Surveillance
The immune surveillance hypothesis, originally proposed in the late 1950s, postulates that the immune
system protects the host not only from infectious
agents, but also from tumor onset (1). Two predictions
can be derived from the theory:
* tumors that grow despite the immune system have
found a way to escape surveillance, thus must be
poorly immunogenic
* tumor incidence should be higher in immunodepressed than in immunocompetent individuals.
3
670
The low immunogenicity of spontaneous (as opposed
to carcinogen-induced or viral-induced) tumors in
mice was easily verified, and is also a property of
human tumors. Demonstration of the second prediction has been more controversial, because the degree
and duration of immunodepression in experimental
systems and in human conditions is highly variable
Tumor, Immune Response to
3
Tumor Antigens
The search for tumor antigens in human tumors was
conducted for many years by means of antisera and
monoclonal antibodies obtained after immunization of
rodents with human cells or tissues. This endeavor led
to the discovery of a wealth of molecules expressed by
human tumors that are recognized by xenogeneic antibodies. However some molecules detected by rodent
antibodies display little or no antigenicity in the
human species, or data on recognition by the human
immune system are not available. Application of the
term “tumor antigens” to molecules that are not recognized as such in the species of origin is inappropriate,
whereas “tumor markers” is more appropriate. Even
though the immunological import of tumor markers is
dubious, they have a great clinical relevance in tumor
diagnosis, prognosis, and follow-up. Some examples
are lactate dehydrogenase (used to monitor treatment
of testicular cancer, Ewing’s sarcoma and other human
tumors), neuron-specific enolase (neuroblastoma and
small cell lung cancer), and DU-PAN-2 (pancreatic
carcinoma).
To distinguish “true” tumor antigens, that can induce a
specific immune response in the species of origin,
leading to tumor rejection, the terms “tumor rejection
antigens” or “tumor specific transplantation antigens”
are sometimes used. Having clearly established the
distinction between tumor markers and tumor antigens, here we will simply use the latter term. Molecular cloning of tumor antigens became possible in the
1980s thanks to technologies based on T cell recognition under syngeneic or autologous conditions. Identification of tumor antigens and measure of specific
responses are currently based on T cell clones with
helper or cytotoxic activity in vitro, identification of
peptides bound to major histocompatibility complex
(MHC) molecules on the surface of tumor cells, molecular cloning of T cell receptor (TCR) genes from
tumor-infiltrating lymphocytes (TIL), soluble
MHC tetramers produced in vitro and bound to synthetic peptides, and screening of DNA libraries with
patient’s sera ( SEREX) (2). It can be noted that
SEREX is antibody-based, however it makes use of
high affinity human IgGs that derive from a Th-induced immunoglobulin class switch, thus SEREX
can be viewed as a T-B hybrid technology.
The main groups of tumor antigens are shown in
Table 1 (3). One major fact is that most tumor antigens
are not tumor specific. The protein expressed by tumor
cells, and the antigenic peptides derived from it are
identical to those of normal cells, thus leading to the
conclusion that the immune response to tumors is actually an autoimmune response. Experimental and
clinical proofs of the autoimmune nature of antitumor immune responses were obtained in melanoma-bearing individuals, who develop autoimmune vitiligo as a consequence of vaccination with tumor antigens (2). The autologous nature of many tumor antigens is one of the reasons why tumors are poorly immunogenic, suggesting that a break of immune tolerance is a prerequisite to an effective anti-tumor immune response. In a few cases immune tolerance
does not operate, either because normal cells expressing the antigen are in immunologically privileged
sites (e.g. cancer-testis antigens), or because the antigen is involved in a physiological network of immune responses (idiotypes of T and B cell neoplasms).
The only truly tumor-specific antigens are those that
derive from mutations of oncogenes (RAS, CDK4) or
tumor suppressor genes (p53), from chimeric proteins
encoded by chromosomal translocations (BCR-ABL),
or from tumor-specific alternative splicing (MUC-1,
possibly HER-2). Experimental evidence shows that,
even when tumor antigens are not shared by normal
cells and are tumor-specific, spontaneous immune responses are quite low and ineffective in the tumorbearing host.
3
and rarely complete. Only recently, with the advent of
knockout mice, has it been clearly demonstrated that
aging immunodepressed mice develop significantly
more tumors than immunocompetent mice (1). Tumors
arising in such immunodepressed mice are more immunogenic than tumors of immunocompetent mice,
thus providing a further demonstration of the hypothesis. In long-term immunodepressed adult humans (e.
g. transplant recipients or HIV-infected patients) the
incidence of virus-induced tumors (such as Kaposi
sarcoma or cervical carcinoma) is increased, but
many other tumor types display an incidence similar
to that of the immunocompetent population.
671
Low Immunogenicity of Tumors
A complete understanding of the reasons why tumors
are poorly immunogenic is of paramount importance
to devise immunotherapeutic strategies to induce a
protective response (1). Basically tumors are tolerated
by the immune system because their antigenic profile
is almost identical to that of normal cells. In addition,
genetic instability of tumor cells generates a large
array of phenotypes that can escape immune recognition using a variety of passive and active strategies.
Down-regulation of antigen expression is an obvious
alternative that has been incompletely investigated.
The most common defect in human tumors (80%–
90% of all solid tumors) is a partial down-regulation
of MHC class I molecules required for peptide binding
and T cell recognition (4). Active strategies may include the induction of regulatory (i.e. suppressive)
cells of myeloid (CD11b+/Gr1+) or lymphoid (CD4+/
CD25+) origin, the secretion of suppressive cytokines
like TGF-β or IL-10, or the expression of pro-apopto-
T
3
3
3
3
672
Tumor, Immune Response to
Tumor, Immune Response to. Table 1 Examples of tumor antigens
Tumor antigen group
Examples*
Cancer-testis antigens
MAGE-A1–A12, B1–B4, C1, C2
BAGE
GAGE-1–8
NY-ESO-1
Differentiation or lineage-specific tumor antigens
gp100
Melan-A (MART-1)
Prostate specific antigen (PSA)
Tyrosinase
Tyrosinase-related proteins (TRP)
Shared tumor antigens
Carcinoembryonic antigen (CEA)
HER-2/neu
MUC-1
Telomerase catalytic unit (TERT)
Mutated antigens
RAS
β-catenin
Cyclin-dependent kinase 4 (CDK4)
MUM-1
p53
Fusion proteins
BCR-ABL
PML-RARα
PAX3-FKHR
SYT-SSX1/2
EWS-WT1, EWS-FLI1
* Complete listing is given in reference 3.
3
From Tumor Immunology to Immunotherapy
Spontaneous immune responses are incapable of eradicating established tumors (spontaneous regression
has been rarely described in human melanoma and
renal cell carcinoma). Preclinical evidence demonstrates that the immune response, if properly activated,
can cure tumors. Analogous conclusions can be drawn
from some successful clinical approaches. A convincing clinical example is the ability of allogeneic T cell
transplants to reduce the risk of leukemic relapse by
30%–40%, a phenomenon known as “graft versus leukemia” (GvL). The main strategies to induce a therapeutic immune response in human patients are based
on the administration of preformed immunologic
“drugs” ( passive immunotherapy) or of therapeutic
vaccines ( active immunotherapy).
Passive immunotherapy is currently the most successful way to target human tumors (2). A small number of
monoclonal antibodies with significant activity against
human tumors emerged from clinical trials and is approved for clinical use. The best known examples are
trastuzumab (Herceptin), a humanized monoclonal an-
tibody against HER-2 for breast cancer, and rituximab,
a monoclonal antibody against CD20 for non-Hodgkin’s lymphoma (NHL). Several other monoclonal
antibodies against similar, or different, target antigens
are being developed. It is interesting to note that the
therapeutic activity of monoclonal antibodies is only
partly mediated by classical immune functions such as
complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). Therapeutic effect is also attributable to the activity of
monoclonal antibodies as “receptor antagonists”,
through the inhibition of receptor dimerization and
signaling, and the induction of receptor internalization
and degradation.
Immunotherapy with cytokines (2) received a considerable attention throughout the 1980s and 1990s. The
major clinical drawback has been the high toxicity of
cytokines. Immune cytokines physiologically reach
high local concentrations, but high systemic dosages
are usually associated with severe toxicity. Toxicity
hampered the clinical development of promising molecules such as IL-2, TNF-α, and IL-12. IFN-α is a
good example of a cytokine that can be administered
systemically at active dosages to cancer patients with
tolerable toxicity. IFN-α initially showed therapeutic
activity against hairy cell leukemia, and has signifi3
tic surface molecules (1). On top of all immune regulations, an expanding tumor could overcome the immune response by sheer cell kinetics.
3
3
Tumor, Immune Response to
cantly prolonged survival in chronic myeloid leukemia
(CML). It is also used for some solid tumors, such as
melanoma, with a significantly lower activity than
against hematologic malignancies. As previously
noted for therapeutic monoclonal antibodies, IFN-α
owes its anti-tumor activity to a combination of immune and non-immune effects. The latter include inhibition of tumor cell proliferation, induction of cell
differentiation, and inhibition of neo-angiogenesis.
Molecular definition of tumor antigens prompted a
large number of vaccination trials based on a variety
of immunological approaches to cancer vaccines (2).
One possibility is to vaccinate with the DNA encoding
a tumor antigen that is picked up and translated by
host cells (DNA vaccination). Alternatively, vaccines
are made of whole cells, recombinant proteins or synthetic peptides admixed with adjuvants. Dendritic cellbased vaccines exploit the pivotal role of antigen presentation in the generation of T cell responses. Dendritic cells cultured in vitro are fed (“pulsed”) with
tumor antigens and then injected in vivo. Promising
results were obtained in small phase I/II clinical trials,
but definitive evidence of a marked clinical benefit
from therapeutic cancer vaccines is still lacking.
3
Preclinical Relevance
Study of the immune response to tumors was largely
conducted in preclinical model systems. The results
were mostly confirmed by human studies, when possible, thus it is generally assumed that preclinical evidence and features of the immune response to tumors
apply to human and clinical situations.
One area that requires caution is the toxicity of cytokines endowed with anti-tumor activity. Because of
species specificity, human cytokines are inactive, or
partially active in rodents, therefore mouse cytokines
must be used for mouse studies. This situation is quite
different from the development and testing of conventional anticancer drugs, in which the same molecule is
used in preclinical and in clinical studies. Some cytokines like TNF-α that display a potent anti-tumor activity in mice are clinically useless because in humans
the maximum tolerated dose is much lower than the
effective dose (2).
A major stumbling block for cancer vaccines, as well
as for other forms of tumor immunotherapy, is the fact
that most clinical trials recruit advanced patients that
are heavily immunosuppressed and poorly responsive
to vaccines, whereas preclinical data clearly show that
the ideal use of vaccines would be for cancer prevention in healthy individuals at risk, or for adjuvant therapy against micrometastatic foci, rather than for therapy of bulky and advanced lesions (5). In conclusion it
is conceivable that active immunotherapy will demonstrate its anti-tumor potential only when clinical stu-
673
dies will follow the path clearly marked by preclinical
data.
Relevance to Humans
Cancer patients treated with monoclonal antibodies
respond to therapy only if the tumor expresses high
levels of the target antigen, thus demonstration of high
antigen levels in tumor lesions is a prerequisite for
therapy. Some indications are available for specific
antigens (e.g. HER-2), for which clinical benefit of
antibody therapy at intermediate antigen levels is dubious.
Assessment of the anti-tumor immune response in cancer patients is not routinely performed outside clinical
trials of immunotherapy. Most immune tests applied in
patients receiving immunotherapy are not standardized, and in some instances are of questionable
value. For example it is not clear if tests performed
on peripheral blood lymphocytes (the easiest sampling
route) correlate with the immune response at tumor
sites. Correlation of positive or negative clinical results with the immune status of patients and with modifications of the immune response induced by immunotherapy is a major open issue (2).
Regulatory Environment
Preclinical data are required to design clinical trials,
but analysis of human immune responses to tumors is
confined to in vitro systems, thus there is no need for
guidelines concerning animal testing. Within clinical
trials, study of the immune response is highly dependent on treatment (e.g. type of vaccine or cytokines
used) and in most instances there are no gold standards
or guidelines pertinent to immune testing of cancer
patients for what concerns antibody responses, cytokine release or T cell cytotoxicity against autologous
or non-autologous tumor cells. Skin tests (cf. delayedtype hypersensitivity) are used to detect responses elicited by anti-tumor vaccines.
Immunotherapy trials are being conducted with a wide
range of approaches that span practically all classes of
therapeutic agents and therapies. The range of adverse
or unwanted effects that can affect patients is correspondingly wide. In addition to general toxicity of the
therapeutic agent, and to the presence of contaminants
in the preparation, a specific type of potential adverse
effect of cancer immunotherapy is the induction of
autoimmunity. In practice all treatments aimed at inducing an immune response against tumor antigens
shared by normal cells involve an autoimmune response. It must be underlined that regulatory requirements for prophylactic vaccines to be administered to
healthy individuals in the general population are quite
different from those applying to cancer patients. In fact
some relatively mild forms of autoimmunity induced
by immunotherapy in cancer patients, such as vitiligo
T
Tumor Immunology
*
*
*
*
*
*
*
*
European Agency for the Evaluation of Medicinal
Products (EMEA) http://www.emea.eu.int
European portal to the pharmaceutical regulatory
sector (EudraPORTAL) http://www.eudra.org
US Food and Drug Administration (FDA) http://
www.fda.gov
FDA Center for Biologics Evaluation and Research
(CBER) http://www.fda.gov/cber
FDA Center for Drug Evaluation and Research
(CDER) http://www.fda.gov/cder
Japanese Ministry for Health, Labour and Welfare
http://www.mhlw.go.jp
Organization for Economic Co-operation and Development (OECD) http://www.oecd.org
International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) http://www.ich.
org
References
1. Pardoll D (2003) Does the immune system see tumors as
foreign or self? Ann Rev Immunol 21:807–839
2. Rosenberg SA (2000) Principles and practice of the
biologic therapy of cancer. Lippincott Williams &
Wilkins, Philadelphia
3. Renkvist N, Castelli C, Robbins PF, Parmiani G (2001) A
listing of human tumor antigens recognized by T cells.
Cancer Immunology Immunotherapy 50:3–15
4. Garrido F, Algarra I (2001) MHC antigens and tumor
escape from immune surveillance. Adv Cancer Res
83:117–158
5. Lollini PL, Forni G (2002) Antitumor vaccines: is it
possible to prevent a tumor? Cancer Immunol Immunother 51:409–416
Cancer and the Immune System
Tumor-Infiltrating Lymphocytes
Lymphocytes (usually T cells) isolated from tumor
specimens. Tumor-infiltrating lymphocytes (TILs)
can be cultured in vitro to analyze their functional
and molecular features, and can be also injected in
vivo for therapeutic purposes.
Tumor, Immune Response to
Tumor Necrosis Factor (TNF)
TNF-α and TNF-β lymphotoxin are produced by
macrophages and T lymphocytes. First described as
cytotoxins for tumor cells, later as important cytokines
for the inflammatory response, cooperation with other
leukocytes, induction of fever and interference with fat
metabolism; therefore TNF is also named cachectin.
Cytokines
3
Regulatory Bodies and Agencies
Tumor Immunology
3
in melanoma, are regarded as surrogate markers of
anti-tumor immune response (2).
In various instances a single therapeutic rationale can
be implemented with different treatment modalities
that depend on different regulatory environments.
For example, to enhance tumor antigen recognition
cytokines can be administered systemically to the patient (drug therapy), or tumor cells can be genetically
modified to secrete the cytokine (gene therapy), or be
fused with autologous or allogeneic dendritic cells
(adoptive cell therapy). A complete listing of all the
guidances, guidelines, and regulations pertaining to
each and every immunotherapeutic approach goes beyond the scope of this article. The reader is referred to
the web sites of the regulatory bodies for comprehensive listings and full text of documents.
3
674
Tumor Necrosis Factor-α
Victor J Johnson
Toxicology and Molecular Biology Branch
National Institute for Occupational Safety and Health
1095 Willowdale Road
Morgantown, WV 26505
USA
Synonyms
tumor necrosis factor-α, lymphotoxin, cachectin,
TNF-α
Definition
Tumor necrosis factor-α (TNF-α) is a pleiotropic
proinflammatory cytokine that mediates key roles in
homeostasis, cell growth and proliferation, tissue damage, repair and chronic diseases. TNF-α production
is induced by a plethora of stimuli including bacterial
products, oxidative stress, other cytokines, and general
tissue damage. As such, this cytokine has a central role
in orchestrating many injury and disease states including immunotoxicity.
Tumor Necrosis Factor-α
Human TNF-α is synthesized as a 26-kDa pro cytokine destined for expression on the plasma membrane.
Proteolytic processing by members of the matrix metalloproteinase family of enzymes results in the extracellular release of the mature soluble 17-kDa form of
TNF-α. Both the membrane-bound and soluble forms
are biologically active and play important roles in
overlapping and distinct signaling processes. Signaling is achieved through ligation of two structurally
distinct receptor subtypes, TNF-receptor 1 (TNF-R1)
and TNF-R2. TNF-R1 is constitutively expressed on
most nucleated cells whereas TNF-R2 has a more restricted expression, mainly on cells of the immune
system and is inducible. A schematic of the major
signaling pathways and molecular mediators is
shown in Figure 1.
Membrane and soluble TNF-α form trimers that induce the trimerization of the TNF- receptor upon binding. Activation of the receptor initiates the formation
of unique signaling complexes that are distinct for
each receptor subtype. A death-inducing signaling
complex is formed at the intracellular domain of
TNF-R1 involving the recruitment and binding of a
number of accessory proteins, including TNF-R1-associated death-domain-containing factor (TRADD),
Fas-associated
death-domain-containing
protein
(FADD) and TNF-R-associated factor-2 (TRAF2).
Binding is achieved through mutual death domains
(DD) present on TNF-R1 and the accessory proteins,
and the DD sequence is unique to the intracellular
portion of TNF-R1. The resulting complex recruits
3
Molecular Characteristics and Mediators of
Signaling
675
T
Tumor Necrosis Factor-α. Figure 1 Death and survival pathways in tumor necrosis factor (TNF)-α signaling.
TNF-α exerts its biological effects through ligation of two distinct receptors, TNF-R1 and TNF-R2. Activation of
TNF-R1 results in the formation of a death-inducing signaling complex, consisting of TNF-R1 intracellular DD,
TRADD, FADD and TRAF2. This complex recruits other intracellular signaling molecules that activate pathways
culminating in cell death (caspase and JNK pathways) and cell survival (NFκB activation pathway). Additionally,
activation of MAP kinases and NFκB can result in upregulation of genes involved in inflammation, including TNF-α
itself. On the other hand, activation of TNF-R2 leads to the formation of a signaling complex via mutual TRAF
domains. This complex is known to lead to NFκB activation and anti-apoptotic signaling. Increasing evidence
suggests a role for TNF-R2 in apoptosis, possibly through potentiation of TNF-R1 pro-apoptotic signaling. Overall,
these signaling pathways can contribute to immunotoxicity by directly inducing cell death and tissue damage,
initiating and contributing to inflammation, and/or altering the proliferative capacity of cells and tissues.
676
Tumor Necrosis Factor-α
other proteins with enzymatic activity culminating in
the induction of several major signaling pathways, including the caspase pathway, mitogen-activated protein (MAP) kinase pathways and pathways that lead
to nuclear factor κB (NFκB) activation. The cell death
and tumor regression properties of TNF-α are attributed to TNF-R1-mediated activation of caspases and
c-Jun NH2-terminal kinase (JNK). JNK activation has
been shown to cleave the Bcl-2 interacting domain
(Bid) resulting in jBid translocation to the mitochondria. This induces the release of Smac/DIABLO from
the mitochondria, which then sequesters inhibitor of
apoptosis (cIAP) proteins leading to FADD-induced
activation of caspase 8. Caspase 8 activates effector
caspases 3 and 7 leading to the cleavage of several
intracellular proteins and apoptotic cell death. Apoptosis is not the predominant outcome in most cell types
in vivo and can be prevented through the activation of
the NFκB survival pathway. Activation of NFκB results from the coordinated action of receptor interacting protein (RIP), NFκB inducing kinase (NIK) and
inhibitor of κB (IκB) kinases. The outcome is phosphorylation-dependent ubiquitination and degradation
of IκB, which results in the release of NFκB into the
cytoplasm which then translocates to the nucleus via a
nuclear localization sequence. Heterodimers and
homodimers of the NFκB/Rel family drive the transcription of many survival and inflammation genes
containing NFκB response elements. Therefore, this
pathway functions to prevent cell death in normal
healthy cells and upregulates the production of proteins involved in inflammatory processes.
The intracellular domain of TNF-R2 lacks the DD and
instead has TRAF domains responsible for the recruitment of TRAF1, TRAF2, and TRAF3. This complex
recruits cIAP and NIK both responsible for NFκBmediated anti-apoptotic signaling. However, several
investigators have reported that TNF-R2 may be important in the regulation and potentiation of TNF-R1induced apoptosis. Several mechanisms have been
proposed, including TNF-R2, acting as a high-affinity
trap/ ligand passer and TNF-R2-induced upregulation of endogenous TNF-α production, both leading
to autocrine and paracrine activation of TNF-R1mediated apoptosis. A dual role in cell survival and
death has been shown such that in the absence of TNFR1 signaling, TNF-R2 promotes not only proliferation
of naïve T lymphocytes but also apoptosis in activated
CD8+ T lymphocytes. Additionally, the affinity of
TNF-R2 for membrane-bound TNF-α is much greater
than that for the soluble form (affinities are equal for
TNF-R1), suggesting that TNF-R2 is important in direct cell to cell regulation and localized immune responses. TNF-R2 can also be shed from the plasma
membrane resulting in soluble TNF-R and downregulation of TNF-α signaling.
Relevance to Immunotoxicity
TNF-α is produced by many cell types including immune cells (macrophages, monocytes, dendritic cells,
T lymphocytes, B lymphocytes), endothelial cells, epithelial cells, and fibroblasts following activation by
appropriate stimuli. Therefore, this cytokine can be
envisioned to play a role in immunotoxicity in many
target organs. Indeed, TNF-α, through its ability to
influence inflammatory processes, has been demonstrated in response to immuntoxicants targeting the
liver, kidney, lung, muscle, eye, skin and brain, to
name a few sites. Gene knockout of TNF-α, its receptors, or neutralizing-antibody studies have been used
to investigate the role of TNF-α in immunotoxicity.
For example, mice deficient in TNF-R1 and TNF-R2
show reduced lung inflammation and cytokine changes in response to toluene diisocyanante, an occupational asthmogen. Removal of TNF- signaling almost
completely abrogated inflammation and fibrosis in
the liver following treatment with the known immunotoxicant, carbon tertrachloride. TNF-α plays an important role in the immunotoxicity of many mycotoxins such as vomitoxin and fumonisin B1. Kidney
TNF-α levels increase in adriamycin-induced nephropathy and may play a direct role in the associated
proteinuria. Immediate production of TNF-α in the
skin is evident following exposure to agents that
cause allergic and irritant contact dermatitis and is
positively correlated with the inflammatory response.
Significantly, mice deficient in TNF-Rs show reduced
development of contact dermatitis. In addition to its
role in chemical-mediated immunotoxicity, TNF-α
also mediates critical events in the pathogenesis of
iodiopathic diseases involving the immune system, including bacterial infection and sepsis, chronic inflammatory lung diseases, cancer, and autoimmunity.
The vastness of TNF-α involvement in chemical and
idiopathic immunotoxicity stems from its central role
in many cytokine/chemokine networks. TNF-α signaling (see Figure 1) can modulate the expression of other
important mediators of inflammation required for the
recruitment and activation of effector cells (macrophages, lymphocytes, neutrophils, eosinophils) that
can contribute to tissue injury. Therefore, enhanced
synthesis and release of TNF-α following immunotoxicant exposure or during disease can initiate and exacerbate acute and chronic inflammation—known
contributors to tissue injury, repair and remodeling (fibrosis).
Relevance to Humans
Examining the association between genetics and disease prevalence and disease severity provides valuable
insight into the mechanisms of disease. As such, greater than 1000 scientific studies have been conducted
investigating the association between polymorphisms
3
Type 1 or Type 2 T Cell Responses
Tumor-Specific Antigen
Antigen expressed by tumor cells, but not by normal
cells. Truly specific tumor antigens are generated by
oncogenic genetic lesions, such as mutations in oncogenes and tumor suppressor genes, or chromosomal
rearrangements leading to the synthesis of fusion proteins. The idiotype of T and B cell receptor in lymphoid malignancies is considered a tumor-specific antigen (but there might be non-neoplastic clones sharing
the same idiotype). Cancer-testis antigens are also considered tumor specific because male germ line cells
(the only normal cell type sharing such antigens)
lack MHC expression, thus cannot present the antigen
to the immune system.
Tumor, Immune Response to
3
Type I Error
A decision error in which a true null hypothesis is
incorrectly rejected.
Statistics in Immunotoxicology
3
in the TNF family and diverse human diseases. Genetic variation in TNF-α has been associated with chemical toxicities and idiopathic diseases of the immune
system and diseases with immune involvement. Examples include occupational lung diseases, such as silicosis and coal workers' pneumoconiosis, chemotherapy-induced pulmonary fibrosis, adverse drug reactions, response to hepatitis B vaccination, asthma, diabetes, and rheumatoid arthritis. The strong association with human disease has prompted research into
potential therapies related to inhibition of TNF-α signaling. To date, several US FDA approved proteinbased injectable inhibitors that block TNF-TNF-R interactions have been used to successfully treat human
disease, including rheumatoid arthritis and juvenile
chronic arthritis. Ongoing clinical trials show promise
for these therapeutics in other diseases like psoriasis,
psoriatic arthritis, ankylosing spondylitis, and Crohn’s
disease. Second-generation small-molecule inhibitors
of TNF are now undergoing clinical trials and function
through blocking specific mediators in the TNF signaling cascade. Caution must be exercised in the use of
these treatments as side effects including potential exacerbation of congestive heart failure, activation of
latent tuberculosis infection, development of antinuclear antibodies, and systemic lupus erythematosis
have been reported. Nevertheless, TNF-α is a pinnacle
cytokine in acute and chronic inflammatory disease
and toxicity and represents a promising therapeutic
target.
677
Type I Reactions According to Gell
and Coombs
IgE-Mediated Allergies
3
References
Type I–IV Reactions
Gell and Coombs described antibody and T cellmediated reactions with distinct clinical pathology
and underlying pathomechanism. The type IV reactions can be subdivided in type IVa–IVd reactions,
which reflect the involvement of distinct effector cells.
Lymphocyte Transformation Test
Hypersensitivity Reactions
3
3
1. Luster MI, Simeonova PP, Gallucci R, Matheson J (1999)
Tumor necrosis factor alpha and toxicology. Crit Rev
Toxicol 29:491–511
2. Palladino MA, Bahjat FR, Theodorakis EA, Moldawer
LL (2003) Anti-TNF-α therapies: the next generation.
Nature Rev Drug Discov 2:736–746
3. Gupta S (2002) A decision between life and death during
TNF-α-induced signaling. J Clin Immunol 22:185–194
4. Chen G, Goeddel DV (2002) TNF-R1 signaling: a
beautiful pathway. Science 31:1634–1635
5. Lui Z-G (2004) Adding facets to TNF signaling: the JNK
angle. Mol Cell 12:795–796
6. Schook L, Laskin D (eds) (1994) Xenobiotics and
Inflammation. Academic Press, San Diego CA
Type 1 or Type 2 T Cell Responses
Tumor Necrosis Factor ReceptorAssociated Factor-6
TRAF-6 induces multiple signals from TOLL-like receptors that sense infection.
Interleukin-1β (IL-1β)
Subset of T lymphocytes called T helper (Th) cells can
respond to different stimuli by secreting different cytokine patterns. Two well characterized patterns are
categorized as type 1 (Th1) and type 2 (Th2) responses. Type 1 responses promote inflammation
and cell mediated immunity primarily through the production of IFN-γ. Type 2 responses promote allergies
T
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Type II Activation
and antibody mediated responses primarily through
production of IL-4 and IL-5. A type 1 response is
antagonistic to a type 2 response and vice versa.
Cytokine Assays
Type II Error
A decision error in which a false null hypothesis is not
rejected.
Statistics in Immunotoxicology
3
3
Type II Activation
Type II Interferon
3
Macrophage Activation
Interferon-γ
3