R
Reporter Antigen Popliteal Lymph Node Assay
recognition of “infectious non self” and initiation of
events that induce adaptive immune responses. Rather,
special emphasis will be placed here on the unique
humoral immune
characteristics of the rabbit
system. The cells and molecules that contribute to
cell-mediated immunity, though less well characterized than those of mice and humans, appear to be
comparable.
3
RA
3
3
RA-PLNA
Reporter Antigen Popliteal Lymph Node Assay
3
Rabbit
Characteristics
An extensive review of the rabbit immune system has
been published previously (1). Many key ideas about
the immune system were first developed through studies of the rabbit model. The rabbit is rich in genetic
variants ( allotypes) that provided markers used for
documenting allelic exclusion, cis expression of linked
genes, and germline recombination within the heavy
chain locus. Figure 1 is a stick diagram of a rabbit
immunoglobulin G molecule, showing some structural
features and the locations of just a few of the many
markers that distinguish inheritable sequence differences of heavy and light chains.
Although a normal IgG molecule would have two
identical light chains, in this illustration the upper
light chain depicts an unusual inter-domain disulfide
bond that connects the variable and constant domains
of most kappa chains of Cκ1 type (allotypes b4, b5,
b6, and some light chains from b9 type). The lower
3
Rabbit Immune System
3
Rabbit Immune System
Rose G Mage
Molecular Immunogenetics Section Laboratory of
Immunology
NIAID Building 10 11 N 311, MSC 1892 NIH, 10
Center Drive
Bethesda, MD
USA
Synonyms
Rabbit, Oryctolagus cuniculus.
Definition
The rabbit immune system consists of the organs, tissues, cells, and molecules that interact to contribute to
specific responses to foreign antigens, infectious
agents, or—in autoimmune conditions—to self antigens. Included among the important molecules are
the genes and gene products that are necessary for
the development and proper functioning of the immune system including antigen-specific T cell and
B cell receptors and B cell-secreted immunoglobulins.
The innate immune system that constitutes the first
line of defense will not be considered here, although
it is now clearly recognized as important for early
Rabbit Immune System. Figure 1 Stick diagram of
rabbit immunoglobulin G showing locations of disulfide
bonds and some allelic types (allotypes) of heavy and
light chains.
546
Rabbit Immune System
light chain is drawn with only the intra-domain disulfide bonds typically found in other species, as well as
in rabbit kappa 2 and lambda light chains. I have
speculated that the great stability and long “shelf
life” of rabbit antibodies may in part result from stabilization of kappa 1 light chain structures by the unusual inter-domain disulfide bond. Conversely, the Cys
at position 80 in most rabbit Vκ genes presents a problem in generating recombinant chimeric Fab molecules with human Cκ because the Cys 80 in Vκ leads
to an unpaired thiol group. Compared to b4 rabbits,
those of the rare b9 and mutant bas types express a
higher proportion of Vκ that lack the Cys 80. When
rabbits of these types were immunized and recombinant rabbit-human Fab generated by phage display,
yields of distinct and specific high affinity Fab increased (2).
A summary of some of the rabbit kappa and lambda
genetic types and the organization of the kappa and
lambda light chain loci is shown in Figure 2. The
rabbit has an unusual duplication of the kappa light
chain locus (Cκ1 and Cκ2 in Figure 2).
The allelic forms of the Cκ1 genes b4, b5, b6 and b9
differ by multiple amino acids in their constant regions
and seem to have somewhat different sets of associated Vκ genes. There are more than 100 different
Vκ genes but they are not fully mapped and sequenced. Some of Vκ genes and Cκ2 are located
about 2 Mb away from Cκ1 in the duplicated rabbit
kappa locus. In wild-type rabbits, kappa 1 light chains
are the major expressed isotype along with 10–30%
of lambda light chains. However, in the mutant Basilea strain (bas) which has a defective acceptor site for
splicing Jκ to Cκ in mRNA for kappa 1 light chains,
there is elevated expression of both kappa 2 and lambda light chains. The allelic forms of kappa 2 chains are
the result of a single amino acid replacement change in
the Cκ2 sequences.
Figure 3 shows a schematic diagram of the heavy
chain locus with VH (the first few of more than 100
VH genes are shown), DH and JH genes, and the genes
that encode the constant regions of IgM, IgG, IgE and
IgA (μ, γ, ε and α). Rabbits are again unusual in
having only one γ gene but 13 α genes. A gene encoding a rabbit homologue of IgD has not been identified in the region downstream of rabbit μ where the δ
gene is found in some species. Perhaps most unusual
of all are the inherited forms of heavy chain variable
regions that are detectable using anti-allotype antisera
raised by immunization of rabbits of one type with
IgG of another type. The reason why allelic forms of
rabbit heavy chain variable regions are detectable became clear when it was found that in most rabbit
B lymphocytes, the first gene in the locus, VH1 is
rearranged; the different allelic forms have amino
acid differences encoded by the VH1 alleles in framework regions 1 and 3. This VH1 gene can rearrange to
one of several DH genes and one of three functional JH
genes, to form VHDHJH. As shown in Figure 3, VH1 is
usually rearranged. In a mutant strain named Alicia
(ali), the VH1a2 gene is deleted and the first gene
that is functional, VH4, is frequently found rearranged
along with a few other upstream genes.
Rabbit Immune System. Figure 2 Diagram (not to
scale) of the rabbit light chain kappa and lambda loci.
Rabbit Immune System. Figure 3 Diagram (not to
scale) of the rabbit heavy chain locus.
Rabbit H- and L-Chain Diversity is Generated by
Rearrangements, Somatic Hypermutation and Gene
Conversion
Today the rabbit remains a major source of polyclonal
antibodies found in catalogs of commercial suppliers.
There are some unique characteristics of the immune
system of rabbits that contribute to their special capability to produce diverse highly specific high-affinity
polyclonal antibodies. These include use of both gene
conversion (GC) and somatic hypermutation (SHM) to
alter the sequences of rearranged antibody heavy and
light chain genes; selection of favorable amino acid
replacements during clonal expansion of antigen-specific B lymphocytes in germinal centers; great diver-
3
3
Rabbit Immune System
sity of kappa light chain variable region genes; and
unusual germline Vκ-encoded variability of the length
of complementarity-determining region 3 (LCDR3).
Compensation for limited heavy chain VHDHJH by
diverse light chain VκJκ occurs even before the start
of somatic diversification processes. Despite this, gene
conversion further diversifies rearranged VκJκ both in
the appendix of young non-immunized rabbits and in
the spleens and lymph nodes of immunized rabbits.
The rabbit VHa allotypes are encoded by the 3’ VH1
gene which rearranges in most B cells. Some diversity
is generated by the choice of one of several DH and JH
genes. Even before diversification by GC and SHM,
there is diversity generated at the sites of VH to DH and
DH to JH DNA recombination by insertions and deletions of bases at the sites of joining. At the points of
joining, the additions and deletions of bases that occur
lead to great variability in the sequences of the heavy
chain third complementarity determining region
(HCDR3). In most rabbit B cells, only one chromosome of the allelic pair undergoes complete rearrangement, and the order of arrangement may also be VH to
DH followed by VHDH to JH; this differs from the order
DH to JH followed by VH to DHJH shown in most
textbooks. The sequence of the rearranged VHDHJH
gene is further diversified by gene-conversion-like
changes. Sequence blocks that vary in length are acquired from upstream donor VH genes. This was first
described as the mechanism of VH-gene diversification
in the chicken, where it occurs in specialized gut-associated lymphoid tissue (GALT), the bursa of Fabricius of embryos and young chicks, and later in life in
splenic germinal centers. In young rabbits, these
changes also take place in specialized GALT sites
such as the appendix (3,4), and in older rabbits in
germinal centers of spleens and lymph nodes in response to foreign antigens. Comparisons of the chicken bursa and rabbit appendix were first published in
the 1960s and suggested that the rabbit appendix
might be a homologue of the chicken bursa, based
on similarities in follicle development and the finding
that neonatal thymectomy of rabbits had no effect on
appendix development. The independence of appendix
cell development from the thymus, as well as remarkable histological resemblance, suggested that rabbit
appendix may be a central lymphoid tissue analogous
to chicken bursa. Subsequently it was shown that removal of appendix and Peyer’s patches resulted in
severe depletion of B cells and blunted immune responses. Once gene conversion was discovered to contribute to sequence diversification in both chicken
bursa and rabbit appendix, it was also shown that
removal of rabbit GALT structures limited—but did
not eliminate—diversification of rearranged heavy
chain sequences. There are similarities and differences
between development and diversification of B cells in
547
the two species, some of which are summarized in
Table 1.
Rabbit Central and Peripheral B cell Development and
V Gene Diversification
In rabbit appendix, development of the primary preimmune antibody repertoire requires endogenous gut
flora. The gut flora may primarily provide B cell survival and proliferation signals, either directly or indirectly through interactions that activate the innate immune system. The rearranged VHDHJH and VLJL in
appendix B cells diversify by gene conversion and
somatic hypermutation but the receptors may not be
specific for a provoking antigen (4). The sequences of
rearranged heavy and light chains within a single expanding clone are strikingly diverse in CDR3. This led
to the view that cells that diversify within individual
clones in appendix may not develop receptors specific
for a single antigenic epitope; the clonal diversification
contrasts with the response to specific antigens in peripheral lymphoid tissues such as the spleen, lymph
nodes, and Peyer’s patches where germinal centers
develop. There, B cells also diversify rearranged
heavy and light chain sequences by somatic hypermutation and gene conversion. This antigen-driven diversification leads to increased affinity of the receptors on
some B cells. Selection for cells with good affinity for
the immunizing antigen occurs via interactions with
antigen on the surface of specialized follicular dendritic cells (FDC). The cells with high affinity may process antigen picked up from FDC and present processed antigen to germinal center T cells which then
release stimuli for proliferation, class switching and
development into plasma cells or memory B cells.
Gene conversion and somatic mutation may also decrease the affinity of antigen receptors. Cells with decreased affinity may die by apoptosis or possibly undergo further rounds of mutation and selection. We
have also speculated that in adults peripheral germinal
centers may have a secondary role comparable to the
role of appendix in young rabbits. For example if some
cells with decreased affinity survive and exit as antigen-responsive cells, the germinal centers could be a
source of new repertoire in adults.
Rabbit Leukocyte Markers, Chemokines and Cytokines
Tables of rabbit leukocyte antigens, T cell receptors
and associated proteins and accessory molecules involved in signaling, leukocyte and endothelial adhesion molecules and some chemotactic molecules described in rabbits for which probes and/or monoclonal
antibodies are available can be found in reference (1).
Data on cytokines and chemokines summarized at the
time of this publication were limited. Although some
progress has been made in this area (5), no commercial
kits are available for rabbit. Some reagents specific for
R
548
Rabbit Immune System
Rabbit Immune System. Table 1 Similarities and differences between chicken bursa and rabbit appendix B cell
development
Chicken Bursa
Rabbit Appendix
VDJ and VLJL rearrangements in spleen, yolk sac
Generally, rearrangement on only one chromosome
VDJ and VLJL rearrangements in bone marrow, fetal
omentum, fetal liver, young spleen
Generally, rearrangement on only one chromosome.
Migration to embryonic bursa
Migration to newborn appendix
Rapid B cell expansion in bursal follicles even before
exposure to exogenous (foreign) antigens
Endogenous stimuli not characterized
Diversification by gene conversion and SHM pre-and
post-hatching to develop preimmune repertoire
Rapid B cell expansion requires presence of gut flora
(exogenous)
There may be some effects of endogenous stimuli such
as CD5
Diversification by gene conversion and somatic hypermutation (SHM) after about 2 weeks of age to develop
preimmune repertoire
Emigration from bursa to periphery
Bursa involutes by sexual maturity
Emigration from appendix to periphery
Appendix changes in appearance and possibly function
but does not involute
Emigrants represent the chicken’s preimmune repertoire
Further diversification by gene conversion and hypermutation occurs in germinal centers of spleen after
immunization
Emigrants thought to represent rabbit’s preimmune
repertoire
However, the diversification seen in spleen after immunization suggests some new B cells may also seed adult
spleen and initiate germinal centers
human markers (produced in species other than rabbit)
cross-react with homologous rabbit proteins.
Preclinical Relevance
The special characteristics of the rabbit immune
system that lead to high affinity and specificity of
antibodies described above make the rabbit a major
source of polyclonal antibodies used in diagnostics
and immunopathology. Although rabbits have been
used in toxicology for tests of eye irritation potential
(Draize rabbit eye irritancy test), as well as for tests of
dermal toxicity, many members of the scientific community and animal welfare organizations have criticized the tests as subjective and inhumane. In the United States, the validation status of in vitro screening
assays for ocular irritation is currently being evaluated.
stat.com), is widely used for the treatment of renal
transplant acute rejection, in conjunction with concomitant immunosuppression. However, such a therapeutic cannot be used to treat patients who are not
immunosuppressed because they would mount immune responses to the foreign rabbit immunoglobulin.
Attempts are currently under way to genetically engineer rabbits that will produce therapeutic human polyclonals (http://www.polyclonals.com). The technology
for production of rabbit monoclonal antibodies has
also developed to the point that highly specific highaffinity rabbit monoclonal antibodies may find use in
drug discovery, diagnostics and possibly as the starting
point for development of humanized therapeutic
monoclonals (http://www.epitomics.com/technology/
tech.html).
Relevance to Humans
References
Rabbit models for diseases of immunological relevance include various infectious diseases such as anthrax, syphilis, tuberculosis, virus-induced papilloma,
and HTLV1: a rabbit model of hemolytic disease of
newborns; complement deficiencies; and a variety of
autoimmune diseases. Rabbits have been used as the
starting source of potential humanized therapeutic
monoclonal antibodies because they produce highly
specific antibodies with high affinities (2). A polyclonal rabbit anti-human thymocyte globulin (Thymoglobulin), approved by the United States Food and Drug
Administration in December 1998 (http://www.sang-
1. Mage RG (1998) Immunology of lagomorphs. In:
Pastoret PP, Bazin H, Griebel HP, Govaerts H (eds)
Handbook of Vertebrate Immunology. Academic Press,
London, pp 223–260
2. Popkov M, Mage RG, Alexander CB, Thundivalappil S,
Barbas CF, Rader C (2003) Rabbit immune repertoires as
sources for therapeutic monoclonal antibodies: the impact
of kappa allotype-correlated variation in cysteine content
on antibody libraries selected by phage display. J Molec
Biol 325:325–335
3. Pospisil R, Mage RG (1998) Rabbit appendix: A site of
development and selection of the B cell repertoire. In:
Kelsoe G, Flajnik M (eds) Current Topics in Microbiology and Immunology ,Vol 229: Somatic Diversification
Real-Time Polymerase Chain Reaction
Ras
Ras is a small-molecular-weight G protein responsible
for regulating the MAP kinase cascades, which lead to
activation of transcription factors.
Signal Transduction During Lymphocyte Activation
3
of Immune Responses. Springer-Verlag, Heidelberg,
pp 59–70
4. Seghal D, Obiakor H, Mage RG (2002) Distinct clonal Ig
diversification patterns in young appendix compared to
antigen-specific splenic clones. J Immunol 168:5424–
5433
5. Perkins HD, van Leeuwen BH, Hardy CM, Kerr PJ
(1999) The complete cDNA sequences of IL-2, IL-4, IL-6
and IL-10 from the European rabbit (Oryctolagus cuniculus). Cytokine 12:555–565
549
Rat Immune System
3
Radiation Mucositis
Rodent Immune System, Development of the
Oral Mucositis and Immunotoxicology
3
Reaction
Radioimmunoassay (RIA)
3
An immunoassay that is based on the use of radioactivity (e.g. 125Iodine-labeled antigens) to generate
counts per minute upon the binding of a radiolabeled
antigen with its antibody.
Immunoassays
Delayed-Type Hypersensitivity
Reactive Oxygen Intermediate (ROI)
3
3
An experimental design utilizing several homogeneous groups of subjects. There are as many subjects
in a block as there are treatment conditions, and within
each block subjects are randomly assigned to treatment conditions.
Statistics in Immunotoxicology
3
Randomized Complete Blocks Design
Products, like hydrogen peroxide and superoxide
anion, of the oxidative burst that occurs in neutrophils,
macrophages, and other cells in response to phagocytosis or other forms of receptor stimulation. These
reactive intermediates can be released into the phagosome, where they can attack ingested microbes, or are
secreted outside the cell where they might attack extracellular pathogens, or contribute to inflammation
and local tissue damage.
Opsonization and Phagocytosis
Antibody-Dependent Cellular Cytotoxicity
3
R
Polymerase Chain Reaction (PCR)
Real-Time Polymerase Chain Reaction
A system that detects and quantifies gene expression
or concentration of a pathogen. PCR product is monitored cycle-by-cycle by combining thermal cycling,
fluorescence detection, and application-specific software.
Polymerase Chain Reaction (PCR)
3
RANTES (regulated on activation normal T cell expressed and secreted; CCL5) is a member of the C-C
subgroup of chemokines. RANTES is secreted by circulating T cells and is chemotactic for T cells, eosinophils, and basophils and plays an active role in recruiting leukocytes into inflammatory sites. It increases the
adherence of monocytes to endothelial cells, selectively supports the migration of leukocytes, and causes
the release of histamines. RANTES binds to CCR5
which is an HIV co-receptor.
Cancer and the Immune System
Chemokines
Interferon-γ
Real-Time and Quantitative PCR
3
RANTES
3
3
3
Real-Time Reverse Transcription PCR
Polymerase Chain Reaction (PCR)
3
Rearrangement
During B cell development in the bone marrow a rearrangement of the genomic DNA takes place. The
gene encoding the variable domain of the light chain
is generated by the stepwise recombination of two
gene elements, the VL gene and the JL gene. The
gene encoding the variable domain of the heavy
chain is generated by the stepwise recombination of
three gene elements, the VH gene, the DH element and
the JH gene.
B Cell Maturation and Immunological Memory
Recombinant
Transgenic Animals
Recombinant Antibodies
Antibody molecules produced in prokaryotic and eukaryotic cells in culture or whole animals and plants
using genetic engineering methods.
Antibodies, Antigenicity of
3
Real-Time Reverse Transcription PCR
3
550
Reconstructed Human Skin/Epidermis
3
Three-Dimensional Human Skin/Epidermal Models
and Organotypic Human and Murine Skin Explant
Systems
3
Recall Antigens
Antigens, usually of microbial origin, such as tetanus
toxoid or pneumococcal antigens, to which the organism has been previously exposed to and to which the
organism has developed a memory capacity.
Primate Immune System (Nonhuman) and Environmental Contaminants
Red Pulp
Part of the spleen comprising venous sinuses filled
with blood and splenic cords. Main function is phagocytosis of particulate material and removal of aged
erythrocytes from blood. In some species, the red
pulp is a site of hematopoiesis.
Spleen
3
3
Receptor Shedding
Regenerative Anemia
Anemia characterized by the presence of increased reticulocyte count or increased polychromasia, indicative of adequate bone-marrow response.
Antiglobulin (Coombs) Test
3
Some transmembrane cytokine receptors can be released from the surface by proteolytic cleavage
through ektoproteases. Receptor shedding has two effects: it rapidly deprives the target cell of functional
receptors on the cell surface and thus interrupts or
terminates cytokine signaling. It also provides soluble
cytokine receptors which may have agonist properties,
e.g. by protecting the circulating cytokine from proteolytic degradation, or may have antagonistic effects by
scavenging and neutralizing cytokines.
Cytokine Receptors
3
Regression Analysis
Cytokine Receptors
A statistical technique in which the relationship between the dependent variable and an independent variable or variables is fit using linear or nonlinear equations. Often used for deriving a prediction equation.
Statistics in Immunotoxicology
3
Receptors for Mediators of the
Immune System
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Regulatory Guidance in Immunotoxicology
Regulated on Activation, T Cell
Expressed and Secreted (RANTES)
RANTES is a chemokine involved in intracellular signaling including stimulation of G protein-coupled receptor activity, and tyrosine phosphorylation of multiple proteins.
Interleukin-1β (IL-1β)
3
Regulatory Cells
Specialized populations cells that modulate the function of other immune cells to prevent uncontrolled or
prolonged responses.
Autoimmunity, Autoimmune Diseases
3
Regulatory Environment
Regulatory Guidance in Immunotoxicology
3
Regulatory Guidance in Immunotoxicology
Robert V House
DynPort Vaccine Company LLC
64 Thomas Johnson Drive
Frederick, MD 21702
USA
Synonyms
Regulatory environment, guidelines in immunotoxicology.
Definition
From its inception in the late 1970s, immunotoxicology has developed from an essentially academic discipline to an important tool for assessing the risk of
human exposure to various xenobiotics. From its early
days, immunotoxicology has been virtually synonymous with immunosuppression; this is perhaps due
to the dual influences of early assays used to assess
immunotoxicity and the more immediately obvious
sequelae of decreased host resistance in comparison
to, for example, autoimmunity. However, it is increasingly recognized that any perturbation of the immune
response from its tightly regulated normal range can
have serious adverse consequences on health. In recognition of this, most of the regulatory guidance spe-
551
cific for immunotoxicology emphasizes individual
evaluation of an agent based on prior information
and its expected/intended molecular mechanism of action. In this review regulatory guidance is divided into
generalized chemical class, with the understanding
that overlap is inevitable.
Characteristics
Industrial and Environmental Chemicals
Some of the earliest codified immunotoxicology test
guidelines were developed to augment toxicological
assessment of chemicals with some of the greatest potential for large-scale human exposure, namely pesticides. In 1996 the Office of Prevention, Pesticides and
Toxic Substances (OPPTS) of the US Environmental
Protection Agency (EPA) published guidelines entitled
Biochemicals Test Guidelines: OPPTS 880.3550 Immunotoxicity (1), which described the preferred study
design for evaluating potential immunotoxicity in biochemical pest control agents. The panel of tests included in this guideline is exceptionally thorough, including standard toxicology tests as well as many of
the standard functional tests being employed at that
time, including both humoral and cell-mediated immune function (the exceptions being primarily cytokine quantification and flow cytometry). Although this
document explains the “how” of testing, it is lean on
the “why”. To address this deficiency, a second document was published concurrently, entitled Biochemicals Test Guidelines: OPPTS 880.3800 Immune Response (2). This companion document provides a
good rationale for why pesticides must be tested for
immunotoxicity, together with more detailed explanations for testing strategies, and additional details on
advanced (mechanistic) tests including host resistance
and bone marrow function.
Whereas immunotoxicity evaluation encompassed by
the 880 series of guidelines would arguably detect any
type of immunotoxicity, its breadth would probably
render it tremendously expensive and time consuming.
In 1998, the EPA followed up with Health Effects Test
Guidelines: OPPTS 870.7800 Immunotoxicity (3)
which described immunotoxicology testing for nonbiochemical agents that would be regulated by EPA.
This document provides descriptions of both why and
how, with a far more abbreviated panel of testing to be
performed. While the 880 series of immunotoxicology
guidelines are probably excessive, the testing approach
mandated by 870.7800 has stood up well in intervening years and reflects the more limited, case-by-case
approach currently favored. Most notably, the functional assessment is pared down to T-dependent antibody formation (plaque assay), natural killer (NK) cell
function, and quantitation of T cells and B cells; this
combination is derived from the early work of Luster
and colleagues which demonstrates the greatest pre-
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Regulatory Guidance in Immunotoxicology
dictivity of known immunotoxicants using these three
assays. This study design described in this document is
amenable for testing a wide range of industrial and
environmental chemicals.
In Europe, the Organisation for European Cooperation
and Development (OECD) regulates testing of chemicals for toxicity. The OECD Guideline 407 entitled
Repeated Dose 28-day Oral Toxicity Study in Rodents
(4),while not specific for immunotoxicology, includes
a variety of toxicological endpoints that can provide
early evidence of immune system alterations. Missing,
however, are any functional assays to directly measure
any immune deficit. Although meetings have been
held to suggest the addition of functional assays (e.g.
Immunology Work Group Meeting, 11–12 December
1996), at present the 407 guideline does not include
such assays.
Food Additives
After industrial and environmental chemicals, food
additives may have the greatest potential for human
exposure. In the USA these chemicals are regulated by
the Food and Drug Administration’s (FDA) Center for
Food Safety and Applied Nutrition. In March 1993 the
FDA published the Draft Redbook II, which recommended safety testing practices for food additives.
This document contained an extensive description of
immunotoxicology testing; although Redbook II was
never finalized, the approach was described in some
detail in a number of publications (5,6). In general, the
Redbook guidelines resembled the “tier” approach that
was used with such success in the early development
and qualification studies performed under the aegis of
the National Toxicology Program. However, Redbook
emphasized a step-wise approach, beginning with “retrospective level I” (expanded) studies utilizing data
obtained in standard toxicology testing as an initial
indicator of potential immunomodulation. Subsequent
stages included enhanced (expanded) level I, level II,
and enhanced (expanded) level II testing designs. This
approach was very much case-by-case, with each level
predicated on positive findings in its predecessor.
In 2001, the FDA began offering an electronic version
of Redbook, entitled Toxicological Principles for the
Safety of Food Ingredients (Redbook 2000) (7). As of
the writing of this review, the guidelines for immunotoxicity studies exist only in outline form in Redbook
2000.
Pharmaceuticals
In the USA, safety testing of small molecule pharmaceuticals is the purview of the US FDA Center for
Drug Evaluation and Research (FDA CDER). In October of 2002, the CDER released a long-awaited
document entitled Guidance for Industry: Immunotoxicology Evaluation of Investigational New Drugs (8).
This document is arguably the most comprehensive of
any published guidance, describing a diversity of adverse events including immunosuppression, immunogenicity, hypersensitivity, autoimmunity, and adverse
immunostimulation. The document describes each of
these types of immunotoxicity (more accurately, immunomodulation) in detail, and provides not only approaches but also suggests methodology for evaluating
each type. Like the document produced by the Committee for Proprietary Medicinal Products (CMPM) (as
described below) the FDA CDER guidance advocates
the use of information derived from standard repeatdose toxicity studies to provide early evidence of immunotoxicity, with subsequent evaluations to be rationally designed to use a minimum of animals and
resources while deriving the maximum amount of information. Subsequent to the publication of the
FDA CDERthe primary purpose of this particular
document was to describe an overall approach to safety testing of pharmaceuticals, it was important as the
first guidance document mandating specific immunotoxicology screening for pharmaceuticals. An appendix of this document describes a staged evaluation,
emphasizing that information gained in standard toxicology evaluation can be useful as a primary indicator
for immunotoxicity. Functional tests may be incorporated to gain additional information, first as an initial
screen and then progressing to extended studies as
indicated. The choice of assays to be used includes
combinations of functional tests known to be predictive of immunotoxicity, as described in the early National Toxicology Program publications.
As the first published document requiring immunotoxicology evaluation, CPMP/SWP/1042/99 predictably
was met with a combination of resistance and confusion. Much of this was allayed in a workshop held in
Noordwijk in the Netherlands in November of 2001,
sponsored by the Drug Information Association
(DIA). At this meeting the intent of the guideline
was clarified. A summary of the workshop has been
published (11).
A second CPMP document that includes reference to
immunotoxicity assessment is Note for Guidance on
the Quality, Preclinical and Clinical Aspects of Gene
Transfer Medicinal Products (CPMP/BWP/3088/99)
(12) currently in draft form. This document recognizes
the possibility of adverse immunological events as a
consequence of gene transfer therapy, although it
makes no specific recommendations for testing.
Japanese regulatory agencies have been cautious in
promulgating immunotoxicology guidelines. In 1999,
the Japanese Pharmaceutical Manufacturers Association (JPMA) published two documents, International
Trends in Immunotoxicity Studies of Medicinal Products (13) and Survey on Antigenicity and Immunotoxicity Studies of Medicinal Products (14). These
Regulatory Guidance in Immunotoxicology
comprehensive documents provided a survey of immunotoxicologic methods and study designs in use
in Japan and elsewhere, without advocating or requiring any studies per se.
At the DIA meeting in Noordwijk (11) a representative
from the Japanese Pharmaceutical Manufacturers’ Associated presented an Interim Draft Guidance for Immunotoxicity Testing (15), which describes the current
thinking on such testing. As of the preparation of this
review, this draft guidance document has not been
published and is not readily available for review.
Thus, as of 2004, there are no published Japanese
guidance documents specifically regulating immunotoxicology evaluation.
Biologicals
Biologicals (for the purposes of this review defined as
therapeutics derived by biotechnology) present a unique challenge for immunotoxicity assessment for two
primary reasons. First, many of these agents (e.g. cytokines and other immunomodulatory molecules) are
intended to modulate therapeutically the immune response. Therefore, it can be difficult to differentiate
between the agent’s efficacy and a truly adverse reaction. Second, because many of these agents are proteins or peptides, their introduction into a host often
triggers an immune response directed against the molecule itself. This can lead to alterations in pharmacodynamics, or to other adverse reactions. Thus, development of appropriate guidance on testing these agents
is problematic. One approach is promulgated by the
International Conference on Harmonisation (ICH) in
the document Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals S6 (16). This
document includes sections on immunogenicity (as
described above) as well as a brief mention of immunotoxicity studies. In short, the S6 document recognizes the inappropriateness of a structured tier approach, opting instead for careful design of screening
studies, followed by mechanistic studies to clarify any
potential evidence of immunotoxicity. Specific techniques and approaches are not described in the S6 document.
Safety evaluation of biological drugs is regulated in
the USA by the FDA Center for Biologics Evaluation
and Research (CBER). To date, the CBER has not
promulgated any written guidance on immunotoxicology. The reason for this lack of written guidance is the
extreme diversity of biological therapeutics, which
makes it difficult to design a standardized testing approach. Rather, the approach of the CBER to addressing potential immunotoxicology has always been
case-by-case, generally following suggestions provided in the ICH S6 document.
Currently there are institutional changes underway
within FDA that would put therapeutic proteins now
553
regulated by CBER under the regulatory authority of
CDER; therefore the CDER guidance document could
apply to these products
Vaccines
Along with certain biologicals, vaccines present a
challenge for immunotoxicological evaluation since
they are specifically designed to induce an immune
response—a situation deemed undesirable (or potentially so) for most of the other agents described in this
review. Since methodology is well established to evaluate the desirable immunomodulation produced by
vaccine, the concern of regulatory agencies is the propensity of these therapeutics to produced undesired or
deleterious effects on the immune system.
European regulation of vaccines is described in Note
for Guidance on Preclinical Pharmacological and
Toxicological Testing of Vaccines (17) by the CPMP.
In this document, immunotoxicology is to be considered during toxicology testing. In particular, vaccines
should be considered for their immunological effect on
toxicity, such as antibody complex formation, release
of cytokines, induction of hypersensitivity reactions
(either directly or indirectly), and association with autoimmunity. No specifics are described for methods or
approaches; rather, each vaccine is to be evaluated on
a case-by-case basis.
The FDA CBER is tasked with regulating vaccines in
the US. One of the primary documents describing vaccine studies is Guidance for Industry for the Evaluation of Combination Vaccines for Preventable Diseases: Production, Testing and Clinical Studies (U.S.
Department of Health and Human Services, 1997).
Animal immunogenicity is covered in detail in the
document, although immunotoxicity is not specified
as an area of concern. On the other hand, the
CBER’s Considerations for Reproductive Toxicity
Studies for Preventive Vaccines for Infectious Disease
Indications (18). Although this is intended primarily to
assess effects of vaccination on reproductive function
(including generalized toxicity such as fetal malformations), it acknowledges the potential immunological
reactions resulting from the vaccination process to
exert unintended consequences. No specific guidance
is provided on methods or approaches to be used in
this evaluation.
Devices and Radiological Agents
It has been recognized by the FDA that immunotoxicity may result not only from chemical or biological
agents that dynamically interact with the physiology of
humans (such as small molecule drugs or biologicals),
but also from medical devices that contact the body
externally (via skin or mucosa), or internally (implantable devices), or by external communication to the
blood or tissue.
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554
Regulatory Guidance in Immunotoxicology
Thus, FDA Center for Devices and Radiological
Health published the guidance entitled Guidance for
Industry and FDA Reviewers: Immunotoxicology Testing Guidance (19) in May 1999 that addresses testing
for medical devices. This guidance is based on General Program Memorandum G95-1, an FDA-modified
version of International Standard ISO-10993, Biological Evaluation of Medical Devices—Part 1: Evaluation and Testing. Immunotoxicology Testing Guidance
provides detailed guidance for determining when immunotoxicity testing should be performed (including a
flowchart and numerous tables), but does not provide
details on which methods should be employed, or for
overall study design.
Some additional details on the use of this guidance
were published by Anderson and Langone in 1999
(20). This manuscript, similar to the guidance, provides little information on which specific assays to
use. It is, however, a useful adjunct to the guidance
document.
American Society for Testing and Materials
The American Society for Testing and Materials
(ASTM) is a not-for-profit organization promoting
the development of voluntary standards for materials,
products, systems and services. ASTM develops documents that serve as a basis for manufacturing, procurement, and regulatory activities. Since the ASTM standards are voluntary, they are included in this review
only for the sake of completeness. The two relevant
documents are F1905-98 (Standard Practice for Selecting Tests for Determining the Propensity of Materials to Cause Immunotoxicity) (21) and F1906-98
(Standard Practice for Evaluation of Immune Responses in Biocompatibility Testing Using ELISA
Tests, Lymphocyte Proliferation, and Cell Migration)
(22).
Hypersensitivity
Although much attention is paid to immunosuppression (low immune response) in the majority of guidance documents, it is hypersensitivity (hyperactive immune response) that is the most common type of immunomodulation resulting from exposure to xenobiotics. Due to the acknowledged frequency of this occurrence, as well as the multiplicity of testing methods
that have been developed, a complete coverage of this
condition will not be included here. However, one
method for assessing hypersensitivity has taken priority in assessing contact hypersensitivity, namely the
murine local lymph node assay (LLNA). Detailed explanations of this assay and its use are covered in the
OECD 429 guideline, entitled Skin Sensitisation:
Local Lymph Node Assay (23); the US EPA document
OPPTS 870.2600 Skin Sensitization (24), and the
ASTM document Standard Practice for Evaluation
of Delayed Contact Hypersensitivity Using the Murine
Local Lymph Node Assay (LLNA) (25).
Regulatory Environment
The extended bibliography below includes guidelines
and guideline drafts which are to be considered for the
special aspects of immunotoxicologic screenings mentioned here.
Acknowledgement
This article was prepared under the Immunotoxicology
Workgroup supported by the EPA Office of Research
and Development (National Center for Environmental
Assessment), the EPA Office of Children’s Health Protection, National Institute of Environmental Health
Sciences (National Toxicology Program) and National
Institute for Occupational Safety and Health (Health
Effects Laboratory Division). Members of the workgroup not included as authors are Laura Blanciforti
(NIOSH), David Chen (EPA/OCPH), Dori Germolec
(NIEHS, NTP), Michael Kashon (NIOSH), Marquea
King (EPA/ORD/NCEA), Robert Luebke (EPA/ORD/
HERL) Michael Luster (NIOSH) Christine Parks
(NIEHS) and Yung Yang (EPA, OPPTS). Special
thanks to Bob Sonawane (EPA/ORD/NCEA) for helping to organize this effort.
References
1. Biochemicals Test Guidelines: OPPTS 880.3550 Immunotoxicity. United States Environmental Protection
Agency, February 1996
2. Biochemicals Test Guidelines: OPPTS 880.3800 Immune Response. United States Environmental Protection
Agency, February 1996
3. Health Effects Test Guidelines: OPPTS 870.7800
Immunotoxicity. United States Environmental Protection
Agency, August 1998
4. OECD Guideline for the Testing of Chemicals 407:
Repeated Dose 28-day Oral Toxicity Study in Rodents.
Adopted 27 July 1995
5. Hinton DM (1995) Immunotoxicity testing applied to
direct food and colour additives: US FDA ‘Redbook II”
Guidelines. Hum Exp Toxicol 14:143–145
6. Hinton DM (2000) US FDA “Redbook II” immunotoxicity testing guidelines and research in immunotoxicity
evaluation of food chemicals and new food proteins.
Toxicol Pathol 28:467–478
7. Toxicological Principles for the Safety of Food Ingredients: Redbook 2000. Draft. Food and Drug Administration
8. Guidance for Industry: Immunotoxicology Evaluation of
Investigational New Drugs. US Department of Health
and Human Services, Food and Drug Administration
Center for Drug Evaluation and Research (CDER).
October 2002
9. Hastings KL (2002) Implications of the new FDA/
CDER Immunotoxicology guidance for drugs. Int
Immunopharmacol 2:1613–1618
Relative Risk
Regulatory T Cells
A specific T cell subset controlling the response of
other T cells by cell-cell contact and secretion of cytokines.
Tolerance
Suppressor Cells
Hapten and Carrier
3
3
3
10. Committee for Proprietary Medicinal Products (CPMP).
Note for Guidance on Repeated Dose Toxicity (CPMP/
SWP/1042/99). October 2000
11. Putman E, van Loveren H, Bode G et al. (2002)
Assessment of the immunotoxic potential of human
pharmaceuticals: a workshop report. Drug Info J 36:
417–427
12. Committee for Proprietary Medicinal Products (CPMP).
Note for Guidance on the Quality, Preclinical and
Clinical Aspects of Gene Transfer Medicinal Products
(CPMP/BWP/3088/99). Draft version
13. International Trends in Immunotoxicity Studies of
Medicinal Products. JPMA Drug Evaluation Committee
Fundamental Research Group, Data 92. April 1999
14. Survey on Antigenicity and Immunotoxicity Studies of
Medicinal Products. JPMA Drug Evaluation Committee
Fundamental Research Group, Data 93. April 1999
15. Interim Draft Guidance for Immunotoxicity Testing.
MHLW/JPMA, 2001 (unpublished)
16. ICH Topic S6: Preclinical Safety Evaluation of Biotechnology Derived Pharmaceuticals (CPMP/ICH/302/95).
March 1998
17. Committee for Proprietary Medicinal Products (CPMP).
Note for Guidance on Preclinical Pharmacological and
Toxicological
Testing
of
Vaccines
(CPMP/
SWP/4654/95). June 1998
18. Guidance for Industry: Considerations for Reproductive
Toxicity Studies for Preventive Vaccines for Infectious
Disease Indications. US Department of Health and
Human Services, Food and Drug Administration Center
for Biologics Evaluation and Research. Draft version,
August 2000
19. Guidance for Industry and FDA Reviewers: Immunotoxicology Testing Guidance. US Department of Health
and Human Services, Food and Drug Administration
Center for Devices and Radiological Health. 6 May 1999
20. Anderson JM, Langone JJ (1999) Issues and perspectives on the biocompatibility and immunotoxicity
evaluation of implanted controlled release systems. J
Control Rel 57:107–113
21. American Society for Testing and Materials: Standard
Practice for Selecting Tests for Determining the
Propensity of Materials to Cause Immunotoxicity.
F1905–1998
22. American Society for Testing and Materials: Standard
Practice for Evaluation of Immune Responses in
Biocompatibility Testing Using ELISA Tests, Lymphocyte Proliferation, and Cell Migration. F1906–1998
23. OECD Guideline for the Testing of Chemicals 429: Skin
Sensitisation: Local Lymph Node Assay. Adopted 24
April 2002
24. Health Effects Test Guidelines: OPPTS 870.2600 Skin
Sensitization. US Environmental Protection Agency,
March 2003
25. American Society for Testing and Materials: Standard
Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay
(LLNA). F 2148–21401
555
Relative Risk
Stephen B Pruett
Department of Cellular Biology and Anatomy
Louisiana State University
Health Sciences Center
Shreveport, Louisiana 71130
USA
Synonyms
None (in specific situations odds ratios can be numerically similar to relative risk but they are calculated
differently).
Definition
Relative risk is the probability of an outcome in individuals exposed to a particular factor or condition
divided by the probability of that outcome in individuals not exposed (1).
Characteristics
The characteristics of relative risk can best be understood by considering an example, as in Table 1.
The relative risk of cancer for persons exposed to this
toxicant is (25 ÷ 5025) ÷ (5 ÷ 5005)
=0.004975 ÷ 0.000999=4.98. Thus, the risk of developing cancer is 4.98 times greater for the group exposed to the toxicant than for the nonexposed control
group. Statistical analysis using a chi-square test or
Fisher’s exact test is done to determine the statistical
significance of this difference and to determine the
confidence intervals. In this example, the P value is
< 0.005 and the 95% confidence interval is 1.9–13.0.
The fact that this interval excludes 1.0 (the value expected if there is no difference in risk between the
exposed and unexposed groups) can also be used to
demonstrate that the relative risk noted in this case is
significant.
Relative risk analysis is most often used with prospective studies (either cohort studies or randomized clinical trials). It is not suitable for case-control studies
because these involve selection of cases on the basis
of outcome, not exposure (1). The odds ratio can be
R
556
RELISPOT
Relative Risk. Table 1 An example of the characteristics of relative risk
Cancer
No cancer
Total
Toxicant exposure
25
5 000
5 025
No exposure
5
5 000
5 005
Total exposure
30
10 000
10 030
used for case-control studies. Interpretation of the biological relevance of relative risk data depends in part
on an awareness of the distinction between relative
risk and absolute risk. Increases in relative risk are
more meaningful when the underlying absolute risks
are relatively large than when they are small. Thus, a
relative risk of 2.0 for an exposed population may
cause relatively little concern if the frequency of the
adverse outcome in the control population is
1:10 000 000, whereas more concern would be raised
if the frequency of the adverse outcome is 1:100 in the
control population.
human populations) is still required for most toxicants
in the risk assessment and regulatory process.
References
1. Rosner B (2000) Fundamental Biostatistics. Duxbury
Thompson Learning, Pacific Grove, CA, pp 54–58
2. Immune Deficiency Foundation. The Clinical Presentation of the Primary Immunodeficiency Diseases. A
Primer for Physicians. The Laboratory Diagnosis of
Immunodeficiency. http://www.primaryimmune.org
Preclinical Relevance
Relative risk analyses from human studies can be useful in risk assessment, particularly when toxicant exposure is associated with increased relative risk for a
detrimental outcome. However, it should be noted that
population sizes in published studies are generally not
sufficient to permit small (but potentially meaningful)
increases in risk to be demonstrated. For example, an
increase from 2 cases of a particular outcome per
100 000 people to 4 cases per 100 000 people represents a relative risk of 2.0, but the 95% confidence
interval is 0.37–10.9. Therefore, this two-fold change
in risk is not statistically significant. An additional
difficulty in using relative risk from human studies
in the risk assessment process is that exposure assessment has generally received little attention, so that it is
not usually possible to quantify the amount of exposure required to produce an adverse effect. Thus, extrapolation from animal studies (with uncertainty factors added for cross-species extrapolation and sensitive
An experimental design in which each subject is measured at multiple time points. Measures over time
within a subject will be correlated with one another
and it is necessary to incorporate this into the analysis.
Statistics in Immunotoxicology
Replicate Cultures
For many assays in cell biology (e.g. limiting dilution
analysis), parallel cultures are set up under identical
conditions to increase the precision of quantitative
measurements.
Limiting Dilution Analysis
Reporter Antigen
3
Regulatory Environment
Repeated Measures Design
3
Relative risk is commonly used in epidemiological
studies to determine if exposure to toxicants is significantly associated with adverse health effects or changes in values for standard clinical tests (2).
Enzyme-Linked Immunospot Assay
3
Relevance to Humans
RELISPOT
3
Relative risk values could be calculated for most preclinical toxicology studies. However, this is usually
not done, because other methods (e.g. analysis of variance with a post hoc test to compare means of multiple
groups) are generally more appropriate and have greater statistical power.
Reporter Antigen Popliteal Lymph Node Assay
Reporter Antigen Popliteal Lymph Node Assay
Reporter Antigen Popliteal Lymph
Node Assay
Raymond Pieters
Head Immunotoxicology
Institute for Risk Assessment Sciences (IRAS)
Yalelaan 2
P.O. Box 80.176
3508 TD Utrecht
The Netherlands
Synonyms
Popliteal lymph node assay, PLNA, reporter antigen,
RA, RA-PLNA, ELISPOT
Short Description
Abbreviations
RA-PLNA:
RA-PLNA=reporter antigen popliteal lymph node
assay
TNP-OVA=trinitrophenyl-ovalbumin
TNP-Ficoll=trinitrophenyl-Ficoll
ASC=antibody secreting cells
The reporter antigen-popliteal lymph node assay (RAPLNA) is a modification of the PLNA to determine
compound-induced specific antibody responses (i.e.
number of antibody secreting cells (ASC) by ELISPOT) to selected bystander antigens (1). This approach allows assessment of the nature and type of
the immune response induced by chemicals in a
straightforward and simple manner. The T-cell independent type 2 antigen TNP-Ficoll, which is susceptible to noncognate T cell help, is used as a reporter
antigen (RA) that indicates or reports whether a chemical can induce neoantigen specific T cell help. By
using the T cell-dependent antigen TNP-OVA (in a
nonsensitizing concentration) as RA one can assess
whether a chemical has adjuvant or sensitizing potential. By using the RA approach, characteristics of the
chemically induced immune response (T cell dependency, adjuvant potential, type of immune response)
can be determined without the need to know the
neoantigens that are elicited by a compound and without the need to isolate or synthesize these neoantigens
for assessment of anamnestic immune reactivity. So, as
for the primary PLNA, the RA-PLNA allows fast
screening of compounds for immunopotentiating effect, but in addition it enables discrimination between
immunosensitizing and mere adjuvant or irritant potential of compounds.
investigation; and the TNP-specific antibody secretion
is determined in addition to cell numbers of the draining lymph nodes. Briefly, chemicals and RA (fixed
final dose of 10 µg per mouse) are mixed in solution
and injected into the hind footpad of a mouse. After
7 days the PLN is excised and used to prepare singlecell suspensions. PLN cells are counted and amounts
of TNP-specific ASC in PLN suspensions are determined by ELISPOT.
TNP-OVA and TNP-Ficoll are chosen because of their
specific immunogenic properties. TNP-Ficoll is a
T cell-independent type 2 antigen, that cannot be recognized by T cells, but that is very well capable of
triggering B cells to produce immunoglobulin(Ig)M.
Once triggered by TNP-Ficoll, B cells become susceptible to noncognate T cell help, meaning that such
B cells will also produce switched isotypes such as
IgG1 when soluble T cell help is available. Thus an
IgG1 response to TNP-Ficoll indicates that T cells are
activated (and possibly sensitized) and that these
T cells recognize neoantigens induced by the chemical. These can be hapten-carrier complexes or other
neoantigens (e.g. previously cryptic epitopes or hidden
autoantigens).
TNP-OVA is a protein antigen that can be recognized
by T cells as well as B cells. The dose of TNP-OVA
(10 µg per animal) that is used in the RA-PLNA is
unable to elicit a specific immune response by itself.
For a measurable immune response to TNP-OVA,
extra or adjuvant signals are necessary. These adjuvant
signals can be provided by any chemical that has some
irritant or proinflammatory effect. In other words, a
specific response against TNP-OVA indicates that
the chemical is at least able to elicit an adjuvant signal.
A specific antibody response against TNP-OVA does
not, however, exclude sensitizing potential. But if an
IgG1 response is elicited against TNP-OVA—but not
against TNP-Ficoll—it is highly probable that the
compound itself is not a sensitizer but that it has
mere adjuvant potential. A chemical that does not eli-
Characteristics
The RA-PLNA differs from the PLNA in two ways:
the RA are injected together with the chemical under
557
Reporter Antigen Popliteal Lymph Node Assay.
Figure 1 Reporter antigens.
R
558
Reporter Antigen Popliteal Lymph Node Assay
cit an IgG response against any of the two RA can be
considered as non-immunopotentiating. So, by combining the outcomes of the antibody (IgG1 or other
switched isotypes) responses against TNP-Ficoll and
TNP-OVA one can assess whether a chemical is a
sensitizer (IgG1 to TNP-Ficoll and TNP-OVA) or a
mere adjuvant (IgG1 to TNP-OVA but not to TNPFicoll), or is unable to elicit an immune response at
all (no IgG1 to any of the RA) (see Figure 1).
Interestingly, by changing the isotype specificity of the
detection antibody in the ELISPOT assay (i.e. by using
anti-IgG2a, anti-IgG2b and anti-IgE in addition to antiIgM and anti-IgG1) it is possible to determine the type
(type 1 vs type 2) of the immune response. This can be
done particularly well with TNP-OVA. This RA is also
suitable to follow the immune response over a longer
period of time (at least 4–5 weeks) and the type of
memory response can be evaluated again without the
need to know the relevant antigen induced by chemical exposure (2).
Recent studies have been published in which the RAPLNA with TNP-Ficoll was combined with oral exposures to the drug diclofenac (3). Twenty days after
single oral exposure to diclofenac, TNP-specific IgG1
responses were observed in the PLN upon footpad
injection of a subsensitizing dose of diclofenac together with TNP-Ficoll. Hence, it appears possible to detect compound-specific anamnestic responses by using
TNP-Ficoll which is susceptible to non-cognate neoantigen specific T cell help.
It is important to note that coinjection of TNP-OVA or
TNP-Ficoll did not appear to interfere with the type of
immune response raised by the chemical.
Pros and Cons
The evident advantage of the RA-PLNA over the primary PLNA is that by the use of RA nonsensitizers
can in principle be distinguished from nonsensitizing
irritants. Moreover, the RA-PLNA provides a more
robust indicator of immunostimulation (for chemicals
the cell number varies from 1–2 × 106 to around 107
cells per PLN, whereas the number of IgG1 AFC varies from 0–10/106 to around 1300/106 PLN cells) and
immunologically relevant information can be obtained
in particular with respect to the type of immune response that is elicited. The kinetics of the immune
response to TNP-OVA initiated by a certain chemical
can be easily followed over a certain period of time.
This allows easy verification of the adjuvant potential
of a chemical by detecting memory responses to the
RA.
Apart from this, the RA-PLNA has pros and cons
resembling those of the primary PLNA: it is a simple,
straightforward, objective, and cheap test (despite the
fact that it requires detection of ASC by ELISPOT)
that allows fast screening of the immunostimulatory
potential of compounds. Moreover, the outcome of
the response depends likewise on the genetic background of the strain of mice used. Although false-positive chemicals (if they are so by irritancy) can be
distinguished, false-negative pro-haptens remain undetectable without a metabolizing system.
The RA-PLNA is also limited by the irrelevance of the
route of exposure. Interestingly, however, the RA technique can be used in combination with exposure to
compounds via the oral route.
Predictivity
The RA-PLNA was developed in 1996 and has been
used primarily for fundamental research into immunomodulating capacity of low-molecular-weight chemicals. Hence, the number of compounds tested in the
RA-PLNA is limited to around 20–30. Although not
formally evaluated or validated, based on a comparison between two independent laboratories, the chemicals tested in the two laboratories showed similar outcomes (1,2,4). The RA-PLNA seems to be more robust then the primary PLNA (because of the use of an
immunological parameter) so the selectivity of the
assay may be improved by the use of RA.
Relevance to Humans
As for the primary PLNA, the RA-PLNA should be
regarded as a screening test for the possibility of a
compound to cause immunosensitization, and as a
first step to evaluate whether the compound has also
potential to stimulate the immune system via the relevant route of exposure. The relevance of the outcomes
of the RA-PLNA might be higher because it gives
substantially more information about the possible effect of the chemical exposure.
Regulatory Environment
The RA-PLNA was developed only 5–6 years ago and
was mainly used to perform mechanistic studies. The
RA-PLNA is regarded as a modification of the PLNA
that has improved predictivity; it is therefore included
in the ILSI-HESI initiative to be evaluated as a predictive test.
References
1. Albers R, Broeders A, van der Pijl A, Seinen W, Pieters R
(1997) The use of reporter antigens in the popliteal lymph
node assay to assess immunomodulation by chemicals.
Toxicol Appl Pharmacol 143:102–109
2. Albers R, de Heer C, Bol M, Bleumink R, Seinen W,
Pieters R (1998) Selective immunomodulation by the
autoimmunity-inducing xenobiotics streptozotocin and
HgCl2. Eur J Immunol 28:1233–12342
3. Gutting BW, Updyke LW, Amacher DE (2002) BALB/c
mice orally pretreated with diclofenac have augmented
and accelerated PLNA responses to diclofenac. Toxicology 172:217–230
Respiratory Infections
4. Gutting BW, Schomaker SJ, Kaplan AH, Amacher DE
(1999) A comparison of the direct and reporter antigen
popliteal lymph node assay for the detection of immunomodulation by low molecular weight compounds.
Toxicol Sci 51:71–79
Resident Macrophages
Monocytes that migrate into normal tissues downregulate many activities and become resident macrophages
which have reduced phagocytic and killing capacities
but enhance signaling ability.
Macrophage Activation
559
tagion, epidemics, consumption, air pollution, host defense systems
Short Description
Humanity has always been vulnerable to microbes that
cause respiratory disease. Presently respiratory infection is the sixth leading cause of death in the USA—a
situation which may intensify in coming years. Being
aware of the health risk of respiratory infections has
become more critical for five reasons:
* the selection of resistant microbial flora due to the
multitude of antimicrobial drugs currently available
* the rapid international transport of microbes due to
population migration
*
immunodeficiency diseases
* increased mean life expectancy
* the development of (formerly unavailable) surgical
and systemic therapies for treating diseases.
3
3
Animal Models for Respiratory Hypersensitivity
The severity and risk of infection varies with the virulence, antigenicity ( immunogenicity) and viability of the invading organism, the number of viable
organisms at the target site, their ability to damage
host tissue by the production of toxins, and the function of the individual’s normal microbial defenses.
Microorganisms are highly versatile and widely distributed, occurring nearly everywhere in the environment. They are capable of replicating themselves or
merely surviving in habitats that are extremely diverse
and hostile. Ambient air that contains living organisms such as viruses, bacteria, fungi, protozoa, and
algae (as well as products of their metabolism or
their decomposition, such as toxins) is referred to as
bioaerosols. When bioaerosols are inhaled and deposited in the respiratory tract, a normal host defense
system exists to maintain health, and when this system
is impaired, an individual’s risk of respiratory disease
is increased.
3
3
3
Respiratory Hypersensitivity Test
Animal Models for Respiratory Hypersensitivity
3
Respiratory Infections
Donald E Gardner . Susan C Gardner
Inhalation Toxicology Associates Inc.
P.O. Box 97605
Raleigh, NC 27624
USA
Synonyms
Bioaerosols, pulmonary infections, pneumonia, influenza, bronchitis, common cold, SARS, airborne con-
3
The activation of oxidative metabolism of neutrophils,
which is manifested by the production of highly reactive oxygen species, such as superoxide, hydroxyl radical and hydrogen peroxide. Respiratory burst is based
on the activation of a multicomponent enzyme,
NADPH-oxidase, in the neutrophil plasma membrane
in response to various activators and during phagocytosis.
Chemotaxis of Neutrophils
3
Respiratory Burst
3
Respiratory Allergy Assay
Characteristics
In the natural environment, healthy people exist in
equilibrium with microorganisms. Microbes can be
classified as pathogens, opportunists, or nonpathogens. Opportunistic microbes are organisms that normally are not capable of causing disease in a healthy
immunocompetent person, but can cause disease in
those with impaired host defense. The respiratory
system is a most vulnerable target for such infectious
agents because it is directly exposed to the external
environment and has nearly four times the total surface
area (70m2) as the combined total surface areas of the
gastrointestinal tract and the skin. Although microbial
uptake through ingestion and through the skin is generally intermittent, inhalation provides a continuous
means of exposure. Thus, for airborne biological
R
Respiratory Infections
3
3
3
3
3
3
3
3
3
3
3
Environmental Factors Influencing Infectious Disease
The presence of microbes in humans may be considered as the normal state, and the process of disease is a
disturbance of the equilibrium between the host, the
parasite, and the environment. The process of respira-
tory infection and the subsequent disease involves the
interaction of a host, a microbe and the environment.
Thus, it becomes necessary that in addition to considering the virulence of the biological agent and the
susceptibility of the host, attention must be given to
a variety of environmental and physiological factors
that might influence the course and severity of the
disease. A person exposed to a combination of stresses, such as those of a physical or chemical nature,
may be more susceptible to certain biological agents
and thus may be at a greater risk of contacting a disease. A variety of gaseous and particulate airborne
pollutants may adversely affect the normal functioning
of the host’s respiratory defenses, which increases susceptibility to pulmonary infections. These include numerous inhaled metals (e.g. cadmium, lead, vanadium,
nickel, manganese), gaseous pollutants (e.g. ozone,
nitrogen dioxide, sulfur dioxide, phosgene, benzene,
toluene, HCHO), particles (e.g. sulfuric acid), and
complex mixtures (e.g. auto exhaust, tobacco smoke,
wood smoke, fly ash) (1,2).
Mechanisms by which these environmental factors
could exacerbate the incidence and severity of infectious respiratory disease in individuals with normal
immune function includes the following:
* enhancing deposition
* interfering with clearance and
bactericidal activity
* initiating inflammation leaving damaged epithelial
tissue vulnerable to microbial invasion
* by a mechanism that enhances delivery or infectivity of viruses, fungi and bacteria.
Relevance to Humans
The human relevance of understanding the source,
transmission, pathogenesis, and the need for testing
of bioaerosols is obvious. This has been clearly demonstrated with the recent epidemic of the new coronavirus that is most likely the cause of severe acute
respiratory syndrome (SARS). Within 4 weeks of the
first appearance of SARS, the microorganism infected
people around the world. Success in controlling such
epidiemics can be contributed to the worldwide global
network of laboratories that pool resources to collaborate on such outbreaks.
The 21st century faces a significant health threat from
of the use of biological agents as terrorist weapons.
The potential to cause large numbers of serious casualties among military forces and civilians provides an
excellent reminder to medical planners of the limits of
medicine. Biological weapons include any organism
or toxin found in nature that can be used to incapacitate, kill, or impede people. Examples of human respiratory diseases associated with biological agents include anthrax, meningitis, plague, tularemia, brucellosis, smallpox, viral encephalitis and hemorrhagic
3
agents the respiratory system is a major route of entry
into the body.
An important distinction must be made between infection and disease. Infection implies that a microorganism has taken up residence in a host and is multiplying
within that host—perhaps with no outward signs of
disease. Thus it is possible to be infected with an
agent, yet not have the disease (although disease
may develop at a latter time). In contrast, those who
appear “sick” are said to have a “disease”.
Humans are the primary host for many microorganisms. After the microorganism has been inhaled and
deposited in the respiratory tract, there are a number of
elaborate defense mechanisms by which the respiratory system can protect this large surface area, including
anatomical barriers (complicated shape of the nasal
passages), physical clearance mechanisms (sneezing,
coughing and ciliary activity), local antibody production (mainly immunoglobulin A, the preferred bacteriopsonin), production of
interferons,
proal
antiproteases, antioxidants,
fibronectin,
teases,
lactoferrin, phosholipids, phagocytic cells (alveolar macrophages and neutrophils) and immune effector cells (lymphocytes and natural killer cells). Together these defenses maintain the integrity of the respiratory system. Despite this protective system there
are a number of ways that the inhaled invading organism can circumvent these defenses and cause disease
either from deficiency in one or more of the mechanisms of defense or from exacerbation of virulence of
the microorganisms such that the host defense system
is overcome. Known factors which result in a weakened defense system include:
* reduced physical removal of inhaled microorganism
by the mucociliary mechanisms
* dysfunction of the macrophages
* alteration of the acellular lining material of the deep
lung ( surfactant) or the mucus in the upper airways
* presence of edema fluid or inflammatory exudates
in the airways
* pulmonary immunosuppression
* influence of environmental pollutants
* stress
* preexisting disease
* attenuated cytotoxic T cell function in malnourished
children, alcoholics and elderly individuals
* immunocompromised individuals such as HIV-positive patients or transplant patients receiving immunosuppressive therapy (1).
3
560
Respiratory Infections
fever. A variety of microorganisms are capable of producing toxins such as aflatoxins, botulism, ricin,
staphyloccal enterotoxin, mycotoxins and perfringen
toxins. Toxin inhalation may cause acute illness with
fever, sweating, muscle aches, rhinitis, and asthma.
Currently it is estimated that about 17 countries are
suspected of having offensive biological warfare programs, making the use of biological agents on military
and civilian populations a greater threat than ever. Biological agents are easy to acquire, to synthesize, and
to use. Only small quantities of microorganisms are
needed to cause respiratory disease in people in metropolitan areas, so it is relatively easy to conceal,
transport and disseminate them. These agents are difficult to detect or protect against since they are invisible, odorless, and tasteless, and their dispersal can be
performed silently.
3
3
3
3
Assessment of Risk
All microorganisms and chemical agents have the potential to be harmful under certain conditions of exposure. Examples of microorganisms causing respiratory
infection or sensitization when inhaled are presented
in Table 1.
The important issue is not just of toxicity but also of
risk. All humans accept some degree of risk in their
daily activities. However, it is important to determine
the probability that such an exposure will cause an
adverse effect under actual conditions of human exposure. The National Academy of Science/National Research Council provides a structured approach to the
process that has been widely used for assessing the
risk of health effects resulting from exposure to chemical agents. There has been interest in developing similar risk assessment models to evaluate the likelihood of adverse human effects from exposure to infectious microorganisms. While the methods presently
in use for chemicals are not directly appropriate for
assessing risk from exposure to airborne pathogens,
they do provide a conceptual framework for developing a similar process. Issues that are unique include
assessment of the pathogen-host interaction, consideration of secondary spread, the possibility of shortterm and long-term protective immunity, and assessment of the conditions that might allow microorganisms to propagate. Thus, the development of a process
for pathogenic risk assessment is complex and should
be expected to consist of several interrelated components, which are conceptually distinct steps.
Regulatory Environment
US government agencies with regulatory responsibilities, including the Environmenta Protection Agency
(EPA) and the Food and Drug Administration (FDA),
have recommendations for guidelines for immunotoxicity testing strategies (3,4). These are discussed in
561
greater detail in other chapters. Such testing is intended to provide information on changes in the functioning of the immune system, which might occur as a
result of exposure to a variety of agents. In the past, a
battery of different assays, structured in a multitiered
approach, has been used to assess immunotoxicity.
Some of these assays included models for detecting
respiratory susceptibility to bacterial and viral exposure.
Respiratory susceptibility to infectious agents can best
be measured if the immune system is asked to perform
its normal functions (that is to defend against infectious agents) and to correlate changes in various host
resistance animal models with changes in specific immune parameters. Since an increase in the incidence
and/or severity of infection has been consistently identified as one of the hallmark indicators of immune
malfunction, a great deal of research has been conducted to design and characterize such host resistance
models (5). These studies have consistently indicated
that changes in specific respiratory immune functional
parameters are associated with the changes in host resistance models (making the host more susceptible to
pulmonary infection). While these animal model systems are sensitive indicators for examining the enhancement of microbial infection of the lungs from
exposure to airborne agents, it is now generally accepted that such host resistance models are not feasible
choices as initial predictors of immunotoxicity because
of their high complexity and cost. It is thought, therefore, that these models are best positioned in the second tier of a testing strategy.
Control and Prevention
The detection, prevention and management of airborne
respiratory infectious disease depend on preventing
the exposure to the biological agents. The ultimate
aim must be to quickly identify the causative agent
and to establish reliable approaches for prevention
and control. A well-designed, well-implemented surveillance program can detect unusual clusters of respiratory disease, document an outbreak, estimate the
magnitude of the problem, and identify factors responsible for its emergence. It is important to eliminate the
reservoir of the microorganisms, to interrupt the transmission of the infection, and to increase the resistance
of the individual to the microorganism. Personal hygiene and cleanliness in the living environment may
lessen the spread of the disease, as well as using personal protective equipment in living and work places,
proper disposal of waste (especially those suspected of
microbial contamination), and the proper design and
construction of buildings to avoid buildup of fungal
growth. Special care is necessary in the case of immunocompromised and particularly sensitive or susceptible individuals. Drug-resistant bacterial, viral, and pro-
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Respiratory Infections
Respiratory Infections. Table 1 Airborne microorganisms causing infection or sensitization
Bacterial disease
Causative Organism
Primary Reservoir
Pneumonia
Nosocomial pneumonia
Pneumonia
Walking pneumonia
Q fever
Ornithosis, psittacosis
Brucellosis*
Legionnaires disease
Tuberculosis
Hypersensitivity pneumonitis
Diphtheria
Pertussis (whooping cough)
Inhaled anthrax
Bubonic plague*
Tularemia
Streptococcus pneumoniae
Klebsiella pneumoniae
Haemophilus influenzae
Mycoplasma pneumoniae
Coxiella burnetii
Chlamydia psittaci
Brucella melitensis
Legionella pneumophila
Mycobacterium tuberculosis
Thermoactinomyces
Corynebacterium diphtheriae
Bordetella pertussis
Bacillus anthracis
Yersinia pestis
Francisella tulaensis
Humans
Humans
Humans
Humans
Animals
Birds
Animals
Water
Humans
Water, soil, compost
Humans
Humans
Animals
Animals
Animals
Viral diseases
Influenza
Severe acute respiratory syndrome
(SARS)
Croup
Bronchiolitis pneumonia
Mumps*
Measles
Common cold
Chicken pox*
Small pox*
Humans
Influenza A, B and C viruses
Humans, animals
Coronaviruses
Humans
Parainfluenza viruses
Humans
Respiratory syncytial virus
Humans
Mumps virus
Humans
Rubella virus
Rhinoviruses, coronaviruses, parainfluen- Humans
za
Humans
Varicella zoster virus
Humans
Variola virus
Fungal diseases
Asthma, rhinitis
Asthma, rhinitis
Asthma, rhinitis
Pulmonary aspergillosis
Coccidioidomycosis
Histoplasmosis
Alternaria
Cladosporium
Penicillium
Aspergillus
Coccidioides immitis
Histoplasma capsulatum
Outdoor air, dead plants
Outdoor air, dead plants
Damp organic material
Soil, compost
Soil of arid regions
Animals, soil, feathers
Protozoa
Algae
Water reservoirs
Water reservoirs
Protozoan/ algal diseases
Hypersensitivity pneumonitis
Asthma, rhinitis
* Disease transmitted via respiratory tract, but signs of infection and disease are seen elsewhere in body.
tozoan pathogens pose a serious and growing problem
for all people, regardless of their age, gender, or socioeconomic background.
Microorganisms that are resistant to antibiotics cause
the vast majority of infections that people acquire in
hospitals. Drug resistance is accumulating and accelerating, thereby reducing the ability of drugs to combat infectious disease. While the emergence of microbial resistance cannot be stopped, the National Academy of Science/Institute of Medicine has addressed
the urgency of this problem at the recent Forum on
Emerging Infections. A number of specific suggestions were identified that will aid better understanding
of microbial resistance, mitigating its impact on
human health, thus transforming this growing threat
into a manageable problem.
References
1. Gardner DE (2001) Bioaerosols and disease. In: Bingham
E, Cohrssen B, Powell CH (eds) Patty’s Industrial
Hygiene and Toxicology, Volume 1, 5th ed. Wiley, New
York, pp 679–711
2. Cohen MD, Zelikoff JT, Schlesinger RB (eds) (2000)
Pulmonary Immunotoxicology, Kluwer, Norwell
3. US Food and Drug Administration (2002) Immunotoxicology Evaluation of Investigational New Drugs. FDA
October 2002, pp 1–35
Rheumatoid Arthritis and Related Autoimmune Diseases, Animal Models
4. Environmental Protection Agency (1998) Health Effects
Test Guidelines. OPPTS 870.7800 Immunotoxicity. EPA
712-C-96-351 October 1998, pp 1–11
5. Conn CA, Green FH, Nikula KJ (2000) Animal models
of pulmonary infection in the compromised host. Inhal
Toxicol 12:783–827
Responder Cell
Specific cell types present in complex cell mixtures
can be identified based on their functional response
to specific stimulation.
Limiting Dilution Analysis
563
Rheumatoid Arthritis and Related
Autoimmune Diseases, Animal Models
Jeanne M Soos
Immunologic Toxicology, Preclinical Safety
Assessment
GlaxoSmithKline R&D
709 Swedeland Road
P.O.Box 97605
King of Prussia, PA 19406-0939
USA
Synonyms
Arthritis models, autoimmune models
3
Short Description
Responder Cell Frequency
In limiting dilution analysis, the frequency of a specific cell type in a cell mixture is estimated based on
its functional response to specific stimulation.
Limiting Dilution Analysis
3
Reverse Enzyme-Linked Immunospot
Assay
RELISPOT is a synonym for ELISPOT.
Enzyme-Linked Immunospot Assay
3
Reye’s Syndrome
Fatal, fulminating hepatitis with cerebral edema.
Anti-inflammatory (Nonsteroidal) Drugs
3
Rheumatic Fever
Late complication of dermatological or pharyngeal infection. Some serologic subtypes of β-hemolytic
streptococci lead to the production of antibodies
against the bacterial cell wall protein (M protein).
Some of these antibodies cross-react with myocardial
sarcolemmal proteins, leading to carditis.
Dermatological Infections
Animal models of rheumatoid arthritis are experimental models of induced joint and digit inflammation that
can be utilized to investigate the mechanisms contributing to arthritic inflammation, to investigate potential therapies for rheumatoid arthritis, and to assess the
potential for substances to either induce or downregulate autoimmune inflammatory responses. Autoimmune arthritis can be induced by injection or immunization by several different types of antigens in multiple susceptible strains of mice, rats, and higher animals (1).
Characteristics
Each of the arthritis models described below is characterized by induction of disease through immunization with either a self antigen or injection with a mixed
antigen preparation. Assessment of arthritis in animal
models is achieved through visual inspection of the
front and hind paws. Inflammation can occur in the
ankle and throughout the digits depending on the severity of disease. The scoring system for assessing the
severity of inflammation is presented in Table 1. The
scores for each joint are added and thus the maximum
score for an individual animal is 16.
Rheumatoid Arthritis and Related Autoimmune
Diseases, Animal Models. Table 1 Scoring system
for visual evaluation of experimental arthritis
Score
Pathology on Visual Inspection
0
Normal size and structure of paw
1
Swelling observed in a single digit
2
Swelling observed in more than one digit
3
Swelling observed in the joint
4
Complete distention and swelling of digits
and joint
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3
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Rheumatoid Arthritis and Related Autoimmune Diseases, Animal Models
Additional methods for assessing development of disease in models of arthritis include the measurement of
paw thickness using a caliper, mercury or water plethysmography, histopathology of the joint, and radiographic evaluation of the joint.
Collagen-Induced Arthritis (CIA)
Collagen-induced arthritis can be induced in a variety
of rodent strains as well as nonhuman primates.
Type II collagen, the specific self antigen used for
disease induction, is a major component of cartilage.
Similar to human rheumatoid arthritis, susceptibility to
collagen-induced arthritis is linked to the expression of
certain major histocompatability complex (MHC)
class II molecules (2). Disease is monophasic and
can result in full resolution of disease or ankylosis of
the joint. Pathogenesis of disease in this model involves both T cell and B cell responses.
Adjuvant Arthritis (AA)
Adjuvant arthritis can be induced in a variety of rat
strains with the Lewis rat most commonly used. Rats
are immunized with heat-killed Mycobacterium tuberculosis, strain H37Ra, in incomplete Freund's adjuvant
and evaluated over time for development of disease.
The disease tends to be monophasic and often results
in irreversible joint ankylosis. Disease can also be induced by adoptive transfer of mycobacteria-specific
T cells or lymph node cells of immunized rats.
Streptococcal Cell Wall Arthritis (SCW)
Streptococcal cell wall arthritis is inducible in a wide
variety of rat strains and, depending on the strain, the
susceptibility to disease development and severity of
disease can vary. Streptococcal cell wall group A peptidoglycan-polysaccharide polymer preparation isolated from Streptococcus pyogenes cell walls is injected intraperitoneally for development of disease. The
course of the disease is characterized by acute onset
during the 48 h after injection followed by a chronic
phase.
Measurement of Cellular Responses and Cytokines in
Arthritis Models
Humoral responses can be evaluated by measuring antibody titers to the antigens used for induction of disease in the models described above. T cell responses
can be measured by cellular proliferation assays as
well as by the generation of antigen-specific T cell
lines and clones. Cytokine analysis can be a valuable
method for evaluating the mechanisms for modulation
of disease. A list of the characteristics of each of these
models is presented in Table 2.
Pros and Cons
There are some advantages of the individual models of
arthritis. The use of a specific self antigen, such as
type II collagen in the CIA model, allows for the dissection of multiple cellular inflammatory mechanisms
by the methods described above. Induction of disease
in the SCW arthritis model illustrates molecular mimicry and allows for study of the initial pathogenesis
and potential triggers of the autoimmune response (3).
Also the SCW is a chronic model of disease that more
closely reflects the course of rheumatoid arthritis in
humans.
Disadvantages also exist for these models. The course
of disease is limited only to an acute phase, with no
observations of a longer, chronic phase in the collagen-induced and adjuvant arthritis models. Further, for
both the collagen-induced and SCW models, great attention must be paid to the quality and purity of the
antigen preparation used to induce disease.
Predictivity
These models are the methods of choice for initial
pharmacological studies, having good predictive
value. However they may not be fully predictive of
what may be observed in humans. For example, blockade of an inflammatory cytokine was shown to be
therapeutic in animal models of autoimmunity (4,5)
while blockade of that same cytokine induced autoimmunity in clinical studies (6,7). Immunological mechanisms leading to autoimmunity in humans are
more complicated and less well understood than the
prevention of autoimmunity (i.e. by blockade of
T helper type 1 cytokines) in these animal models.
For immunotoxicology, these models of inflammation
provide a valuable means for evaluating potential for
proinflammatory activities of new drugs and substances.
Relevance to Humans
Animal models of arthritis serve as a means for evaluating new therapies for human rheumatoid arthritis.
Regulatory Environment
The field of autoimmunity and the use of autoimmune
models such as rheumatoid arthritis do not require
regulation, but the value of these models is recognized
for understanding the mechanisms of immunotoxicologic potential and for risk assessment by the FDA in
the guidance for Immunotoxicology Evaluation of Investigational New Drugs.
Relevant Guidance
FDA (CDER) Immunotoxicology Evaluation of Investigational New Drugs, 2002
Ricin
565
Rheumatoid Arthritis and Related Autoimmune Diseases, Animal Models. Table 2 Characteristics of the most
widely employed animal models of experimental rheumatoid arthritis
Animal
Models
Species/Strains
Collagen-induced arthritis
Chicken or bovine type II
Mouse: primarily DBA/1,
strains of the I-Aq and I-Ar collagen
class II allelles
Rat: variety of strains from
multiple class II Rt alleles
Monophasic Mouse: days 21–35
Rat: high responder days
8–10
Rat: low responder days
30–60
Adjuvant arthritis
Lewis rat
Monophasic Days 10–17
Heat-killed Mycobacterium
tuberculosis H37Ra
Disease
Course
Streptococcal cell wall
Biphasic
group A peptidoglycan-polysaccharide polymers
3
3
Rhinitis
Inflammation of the nasal mucus membrane, marked
by sneezing, lacrimation and watery mucus.
Respiratory Infections
3
Ribonucleic Acid (RNA)
A biomolecule that has an informational, structural,
and enzymic role. The structure is of ribose units
joined in the 3' and 5' positions through a phosphodiester linkage with a purine or pyrimidine base attached
to the 1' position. (RNA = ribonucleic acid).
Southern and Northern Blotting
3
Ricin
A highly toxic lectin and hemagglutin occurring in
seeds of castor beans. Used as a chemical warfare
agent.
Respiratory Infections
3
A common inflammatory disease caused by an autoimmune reaction directed to joints. It is mostly
mediated by humoral immune reactions and immune
complex deposits. So-called rheumatoid factors, i.e.
autoantibodies against the constant region (Fc) of antibodies, have been described for this disease for the
first time. This chronic potentially disabling arthritic
condition with a female predominance, is character-
3
Rheumatoid Arthritis (RA)
ized by peripheral symmetric polyarthritis with or
without other associated systemic involvements, occurring predominantly in individuals who are HLADR4/DR1 positive.
Hypersensitivity Reactions
Systemic Autoimmunity
Fatty Acids and the Immune System
Complement Deficiencies
Molecular Mimicry
Cyclosporin A
3
1. Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM,
Stober W (eds) (1991) Current Protocols in Immunology.
John Wiley and Sons, New York
2. Wooley PH, Luthra HS, Stuart JM, Cavid CS (1981)
Type II collagen-induced arthritis in mice. I: Major
histocompatibility complex linkage and antibody correlates. J Exp Med 154:688–700
3. Taylor JE, Ross DA, Goodacre JA (1994) Group A
streptococcal antigens and superantigens in the pathogenesis of autoimmune arthritis. Eur J Clin Invest
24:511–521
4. Mori L, Iselin S, de Libero G, Lesslauer W (1996)
Attenuation of collagen-induced arthritis in 55-kDa TNF
receptor type 1 (TNFR1)-IgG1-treated and TNFR1deficient mice. J Immunol 157:3178–3182
5. Korner H, Goodsall AL, Lemckert FA et al. (1995)
Unimpaired autoreactive T-cell traffic within the central
nervous system during tumor necrosis factor receptormediated inhibition of experimental autoimmune encephalomyelitis. Proc Natl Acad Sci USA 92:11066–11070
6. Sicotte NL, Voskuhl RR (2001) Onset of multiple
sclerosis associated with anti-TNF therapy. Neurology
57:1885–1888
7. Mohan N, Edwards ET, Cupps TR et al. (2001)
Demyelination occurring during anti-tumor necrosis
factor alpha therapy for inflammatory arthritides. Arthritis
Rheumatol 44:2862–2869
Acute phase: 48 hours
Chronic phase: days 10–
21 with continued disease for months
3
References
Induction of Disease
3
Streptococcal Lewis rat primarily
cell wall arMultiple rat strains with
thritis
varying susceptibility
Antigen for Immunization
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Risk Assessment
Risk Assessment
Statistics in Immunotoxicology
3
Rodent Immune System, Development
of the
Kenneth S. Landreth
Department of Microbiology, Immunology, and Cell
Biology
West Virginia University Health Sciences Center
Morgantown, WV 26506
USA
Synonyms
Murine immune system, mouse immune system, rat
immune system
Definition
Our understanding of cells and tissues that make up
the immune system of vertebrates came from seminal
studies of avian species. However, rodents have become the animal model of choice for studies of immunocompetence, and most of our knowledge of the development of the immune system has come from studies using mice or rats. The rodent immune system
develops through a set of critical windows of vulnerability during embryonic and adult life that can be
used for evaluating effects of environmental exposure
to potentially toxic compounds (1). Most of the information in this review comes from studies of mice and,
in all cases tested, closely parallels that found in other
rodents, including rats.
Characteristics
Embryonic development. Embryonic development of
the immune system in rodents initiates with formation
of multipotential hematopoietic stem cells (HSC) in
intraembryonic spanchnoplure surrounding the heart
and in association with endothelial cells of the extraembryonic yolk sac (2). Hematopoietic stem cells undergo a temporal migration from intra-embryonic mesenchyme to fetal liver, fetal spleen, and ultimately to
final residence in bone marrow and thymus: organs
which continue to produce immunocompetent cells
throughout life (Figure 1).
HSC first appear in embryonic mice during the 7th day
of gestation. These cells develop in both intraembryonic splanchnopleuric mesenschyme surrounding the
heart, a tissue often identified as the aorto-gonadomesonephric region (AGM), and in extraembryonic blood
islands of the yolk sac (3). However, HSC in these two
embryonic tissues differ dramatically in their developmental fate. Intraembryonic stem cells, but not those
that arise in the yolk sac, contribute to sustained blood
cell development and functional immune-responsive
cells in postnatal rodents. The standard experimental
assay for HSC has been the in vivo spleen colony
forming cell (CFU-S) assay. This assay relies on the
unique ability of rodent stem cells to migrate to the
spleen after adoptive transfer and to initiate formation
of macroscopic colonies of hematopoietic cells that are
clonally derived. More recent in vitro assays for multipotential hematopoietic cells have been developed and
enumerate cells capable of forming colonies in a semisolid matrix that contain multiple blood cell lineages.
The initial period of stem cell formation for the hematopoietic system culminates as newly developed stem
cells migrate to the embryonic liver and spleen.
At approximately day 10 of gestation in mice, HSC
relocate from the AGM to the developing fetal liver. In
fetal liver, HSC develop into more differentiated and
lineage-restricted stem cells. These lineage-restricted
stem cells further differentiate in this tissue site to
form more mature progenitor cells which retain the
ability to proliferate but are more restricted in their
developmental potential. Lineage-restricted stem cells
are operationally defined by their proliferation and/or
differentiation in response to specific hematopoietic
cytokines.
Progenitor cells are routinely enumerated using in vitrocolony forming unit (CFU) cell assays. The availability of recombinant cytokines and use of these assays have been invaluable in enumerating specific progenitor cells in embryonic tissues that are otherwise
indistinguishable by morphologic analysis. These assays are central to any study of direct effects of toxic
compounds on hematopoietic tissues and blood cell
formation in the developing embryo or in postnatal
animals.
Fetal Liver. Fetal liver continues to be the principal
hemato-lymphopoietic organ until near the end of gestation, however, few morphologically or functionally
identifiable mature leukocytes are found in the embryo
until near the time of birth (4). B lymphocyte production in the rodent fetal liver has been well characterized and serves as a prototype of fetal development of
the immune system (5). Cells with immunoglobulin
(Ig) gene rearrangements are first found in the liver
on gestational day 11 and increase rapidly to easily
detectable numbers by gestational day 13. Cells with
cell surface Ig (B lymphocytes) are not detected in
fetal liver until day 18 of gestation, and remain at
low frequency until birth. Numbers of hematopoietic
cells decline in the fetal liver as the bone marrow
assumes primary hematopoietic function at gestational
day 18. HSC and lineage-restricted lymphoid progenitor cells are found in the developing spleen on gesta-
3
Rodent Immune System, Development of the
567
Rodent Immune System, Development of the. Figure 1 Organs which continue to produce immunocompetent
cells throughout life.
Adapted from: Dietert RR et al. (2000) Environ Health Perspect 108 [Suppl 3]:483–490
tional day 13, approximately the same time they are
found in the fetal liver and remain detectable in that
tissue until a few weeks after birth. Unlike the bone
marrow, lymphopoiesis rapidly wanes in the spleen
after birth and can not be demonstrated in adult mice.
Thymus. Organogenesis of the thymus initiates from
the 3rd and 4th pharyngeal pouches in mice on gestational day 11. The thymus is immediately colonized by
immigrant HSC, which are detectable on day 11 of
gestation. The thymus continues to be a source of
lymphoid cells (T lymphocytes) in postnatal rodents
until somewhat after sexual maturity when the thymus
regress and ceases function.
Lymph nodes. Lymph nodes form in the developing
embryo by endothelial budding of the venous circulatory system, a process that initiates on gestational day
10.5 in mice. The formation of these organs is dependent on interaction of immature lymphoid cells with
developing endothelial cells. Peyer’s patches develop
from clusters of cells on the proximal end of the intestine on gestational day 15.5 and nasopharyngeal
lymphoid tissues form after birth in mice.
Bone marrow. Long bones of the embryo mineralize,
and the central marrow cavity is excavated to create a
marrow cavity on gestational day 17.5 in mice. This
new site is immediately populated by hematopoietic
cells derived from AGM (not yolk sac) (2). This population of cells establishes the hematopoietic bone
marrow which serves as a reserve of HSC, blood
cell development, and production of immune-responsive cells for the remainder of postnatal life.
Relevance to humans
Observations made on development of human lymphoid tissues and cells suggest that the temporal sequence of events described here for rodents is largely
duplicated in human development (Figure 1). However, there are notable differences between the gestational appearance of immune function in rodents and
humans. In general, immune responsive cells appear
relatively earlier in gestation in human embryos, and
tissues of the immune system are more mature at birth
than in rodents. Of particular importance, cytokines
that stimulate lymphoid cell proliferation and differentiation are conserved between humans and rodent species and, in many cases, retain sufficient homology to
be active on cells from either species.
Regulatory Environment
Rodents are the preferred model for studies of immunology and immunotoxicology and most regulatory
testing is carried out on these species. Despite the
fact that we know significantly more about the development of the immune system in mice, rats continue to
be a preferred model for immunotoxicology testing for
historical rather than scientific reasons.
References
1. Dietert RR, Etzel RA, Chen D, Halonen M, Holladay S,
Jarabek AM, Landreth K, Pedan D, Pinkerton K,
Smialowicz RJ, Zoetis T (2000) Workshop to identify
critical windows of exposure for children’s health:
immune and respiratory systems work group summary.
Environ Health Perspect 108 [Suppl 3]:483–490
2. Cumano A, Godin I (2001) Pluripotent hematopoietic
stem cell development during embryogenesis. Curr Opin
Immunol 13:166–171
3. Metcalf D, Moore MAS (1971) Haemopoietic Cells. In:
Neuberger A, Tatum EL (eds) Frontiers of Biology, Vol.
24. North-Holland, Amsterdam, pp 172–271
4. Landreth KS (2002) Critical windows in development of
the rodent immune system. Hum Exp Toxicol 21:493–
498
5. Landreth KS (1993) B lymphocyte development as a
developmental process. In: Cooper EL, Nisbet-Brown E
(eds) Developmental Immunology. Oxford University
Press, New York, pp 238–273
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Rodents, Inbred Strains
Rodents, Inbred Strains
Ina Hagelschuer
PH-R ZfV, Geb. 516
Bayer HealthCare AG
Aprather Weg 18
D-42096 Wuppertal
Germany
Synonyms
Inbred strains, inbreds, genetically defined rodents
Definition
General items
Most of the current definitions of inbred strains are
established from rats and mice. These rodents have
short-generation intervals and sufficient numbers of
offspring to allow the application of close inbreeding.
In contrast, only a few strains being inbred by definition (see below) are available from hamsters or guinea
pigs.
Inbred strain
A strain shall be regarded as inbred when it has been
mated brother × sister (hereafter called b×s) for 20 or
more consecutive generations. Parent × offspring matings may substitute b×s matings provided that, in the
case of consecutive parent × offspring matings, the
mating in each case is to the younger of the two parents. Inbred strains are designated by three to five
capital letters (for example the mouse strain CBA or
the rat strain BN). Before creating new designations
relevant databases have to be checked in order to
avoid duplications. Present information on nomenclature rules can be drawn from the Jax Mice Catalogue (1).
*
a combination of both if more than one substrain is
developed in the same laboratory, e.g. CF/1Ztm,
CF/2Ztm.CF/4Ztm for Central Laboratory Animal
Facility, Medical University Hannover.
Coisogenic strain
Inbred strains are coisogenic to each other if they differ in only one allelic character. This difference can be
only caused by a mutation and subsequent fixation of
the mutated allele.
Designation of a coisogenic strain
Symbol of the background strain followed by the differentiating allele in italic letters, separated by a hyphen, e.g. C.B-Igh-1b/IcrTac-Prkdcscid (formerly C.B17-Prkdcscid).
This strain is an example of a mutation within a congenic strain.
scid
* symbol: Prkdc
* symbol name: severe combined immune deficiency
(the mutation scid)
* gene name: protein kinase, DNA-activated catalytic
polypeptide (Prkdc)
* symbol description: mutation in the gene encoding
the catalytic subunit of DNA-activated protein kinase, Prkd. Arose in the C.B-17 congenic strain.
Substrain
Inbred strains are divided into substrains when there
are known or assumed genetic differences due to residual heterozygosity during colony set-up, mutation,
or genetic contamination. These reasons are likely
when:
* the strains are separated before inbreeding generation F40
* the present strain has been bred separately for 50 or
more generations
* genetic differences have been proven.
Congenic strain
A congenic strain is developed by transfer of a chromosomal segment (consisting of the differentiating
locus and flanking genes) from a donor strain to another strain (inbred background or recipient strain).
The genetic background of the initial crosses have to
be purified by at least 10 backcrosses with the recipient strain. Afterwards the original inbred strain and
the congenic strain (recipient strain) should only differ
in this introduced segment. A congenic strain should
be mated b×s after having finished the backcross process (see Fig. 1).
The designation of a congenic strain contains the symbols or abbreviations of the recipient and donor strain,
separated by a dot. The transferred gene segment is
added in italic letters separated by a hyphen.
Thus, for C.B-Igh-1b/IcrTac (formerly.C.B-17)
* donor strain: C57BL/Ka strain (B)
* background strain: BALB/c, (C)
b
* symbol name: immunglobulin H-1
b
* gene name: heavy chain allele (Igh-1 )
b
* [C.B-17 = BALB/c.C57BL/Ka-Igh-1 ] (number 17
originated from the backcross number 17).
Designation of a substrain
Parental strain or symbol for differentiation (examples
of symbols):
* numbers, e.g. DBA/1 or DBA/2
* laboratory codes, e.g. A/J for Jackson Laboratory
For B10.129P-H12b/(6M)SnJ
* donor strain: 129P3/J
* background strain: C57BL/10n
* symbol name: histocompatibility 12b
* gene name: histocompatibility 12.
Rodents, Inbred Strains
F1-hybrid
The cross of two inbred strains leads to an F1 hybrid.
All F1 animals of the same parental strains are genetically identical. They are heterozygous at all loci having different alleles in the parental strains. F1 hybrids
share the advantages of inbred strains. In addition they
are robust against environmental influences resulting
in low quantitative character variability. An important
limitation is the discontinuation of breeding. The designation of a F1-hybrid consists at first of the symbol
of the female parent followed by that of the male
parent. Short or full symbols may be used. For example, B6C3F1 is the result of a female C57BL/6 mouse
crossed to a C3H male mouse.
Characteristics
Advantages of inbred strains
Inbred animals of the same strain can be regarded as
identical twins and can be reproduced unlimited.
Long-term genetic stability
Inbred strains can be assumed to be genetically constant for a long period of time, supposed they are
correctly bred and genetically monitored. Under
these conditions background data on strain characteristics may be comparable for many years.
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Isogenicity
All individuals of a strain are isogenic. Thus skin
grafts and tumors may be transplanted within an
inbred strain without immunological rejection. Genetic
data can be accumulated within the same strains. Of
special interest for immunotoxicologists are data on
major histocompatibility complex (MHC) (Fig. 2).
Homozygosity
Inbreds are homozygous at virtually all loci, and thus
will breed true within the strain.
International distribution
Most of the used inbred strains have an international
distribution, thus the genetic results of research can be
easily compared.
Identification/monitoring
Inbreds can be identified by their strain-specific genetic profiles consisting of DNA polymorphisms, immunologic markers (e.g. MHC haplotypes in the mouse;
RT1 haplotypes in the rat) and biochemical markers.
The regular monitoring of genetic profiles can reduce
the risk of unnoticed genetical contamination. The
strain monitoring has to be performed of course by
competent laboratories.
Uniformity
Genetic variation is reduced to nearly zero within
strains by constant inbreeding. The use of inbred
strains enable a much better standardization of the
test conditions. Application of inbreds can improve
the quality, the repeatability and comparability of results. The extent of analysis may be reduced or kept at
a minimum by selecting an appropriate genetic model.
Such a principle is in line with animal welfare legislation calling for reduction and refinement of animal
experiments.
Individuality
Each inbred strain represents an unique genotype. This
genotype can lead to a phenotype of biomedical interest (for example, inbreds with high or low tumor incidence, or high or low disease resistance).
Limitations of inbred strains
Individuality
Each inbred strain represents only one genotype. To
extrapolate experimental results different inbred
strains have to be considered, ideally including also
their F1 hybrids.
Rodents, Inbred Strains. Figure 1 Congonic strains.
From: Cruse JM, Lewis RE (2004) Atlas of Immunology.
CRC Press, Boca Raton.
Isogenicity and uniformity
The variability of quantitative characters within a single strain is exclusively due to non-genetic factors, like
environmental or methodological factors.
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Rodents, Inbred Strains
Rodents, Inbred Strains. Figure 2 H-2 histocompatibility system is the major histocompatibility complex in the
mouse. From: Cruse JM, Lewis RE (2004) Atlas of Immunology. CRC Press, Boca Raton.
Examples of commonly used inbred strains in the field
of immunotoxicology
Brown Norway rat (BN) (RT-1 haplotype n)
The BN rat is characterized by high basal level of
serum IgE which can be induced massively by immunization with proteins. The inducibility of the IgE response is currently under evaluation as an indicator for
allergic asthma and for chemically induced autoimmunity. As a reason for this high responsiveness the reduced antioxidant levels in the BAL cells is considered, resulting in reduced ability to adapt to oxidative stress induced by allergen induction.
In 2002 Vohr et al reported on a model that uses the
reactions of regional lymph node as indicators for the
induction of respiratory allergy.
The BN rat was also found to be a suitable model in a
modified local lymph node assay (LLNA), self-limiting increase of IgE, reduced endogenous antioxidant
level in BALF, and food allergy test.
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B6C3F1 and C57BL/6 (h-2 haplotype b/k and b)
Historically both strains have been used extensively in
the National Toxicology Program to set up an immunotoxicologic database which consists of a vast variety
of tests of over 50 compounds (for a review article see
Luster et al 1994 ). Both strains gave acceptable results in comparison study of mouse strains in a LLNA.
BALB/c (h-2 haplotype d)
This strain was used in the first LLNA. Its feasibility
has been confirmed recently by Woolhiser et al in
2000, and Hüsler et al in 2003. BALB/c mice are
IgE high responders and they are occasionally used
for investigations of the regulation and induction of
this antibody subclass. Because of this they have also
been the object of intensive investigations for estab-
lishing short-term models for the differentiation between respiratory and (skin) contact allergy.
CBA/Ca and CBA/J (h-2 haplotype k)
The strain CBA/Ca was recommended by Kimber and
Weisenberger in 1989 for the murine LLNA as a result
of comparison of four murine strains. Young adult
male or female mice of CBA/Ca (or CBA/J strain in
the USA) are also recommended for the LLNA in the
OECD guideline 429, 2002. The Fischer rat (F344-RT1 haplotype lv1), Lewis rat (LEW-RT-1 haplotype l),
and A/J-mouse (h-2 haplotype a) are also widely used
in different immunotoxicity studies.
Preclinical Relevance
Inbred strains have made an essential contribution to
biomedical research. Much progress in research, especially in the field of immunology, has followed from
the development of inbred strains. Use of inbred
strains enables much better standardization of the conditions. Inbreds help to improve the quality and reproduction of the results obtainable. A comparability of
the results is given. Selecting an appropriate genetic
model reduces the analysis needed, and may even keep
it to a minimum. These points are in line with the
animal welfare legislation calling for reduction and
refinement of animal experiments.
On the other hand, the increased variability of immune
responses of non-inbred strains (so-called outbred
stocks) does reflect the human situation in a more
realistic way. Results obtained by the use of outbred
animals may thus have a better impact on risk assessment. Therefore, there is still debate about which of
the strains mentioned are to be used for immunotoxicologic investigations. The various inbred rat and
mouse strains show differences in their immunological
reactions. Many differences occur especially in the
RTqPCR
field of sensitivity (from high-responders to non-responders). Other 'normal' immunoreactions can also
differ, like the plaque-forming cell assay (PFCA)
against sheep erythrocytes (SRBC). Undesired hyperreactions or autoimmune reactions may also depend on
the genetic background, as well as host-resistance
analysis. So it is essential that a rat or mouse strain
must be checked for its immunological reactivity before using it for immunotoxicologic examinations. In
addition, positive controls have to be established.
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3. Festing M (1993) International index of laboratory
animals, 6th ed. The British Library, London
4. Foster HL, Small JD, Fox JG (1981) The mouse in
biomedical research, Volume I: History, genetics, and
wild mice. Academic Press, New York
5. Hedrich HH (1990) Genetic monitoring of inbred strains
of rats. Gustav Fischer Verlag Stuttgart, New York
6. Heinecke H (1989) Angewandte Versuchtierkunde. VEB
Gustav Fischer Verlag, Jena
7. Klein J (1986) Natural history of the major histocompatibility comple. John Wiley and Sons, New York
8. Krinke GJ (2000) The laboratory rat. Academic Press,
New York
Relevance to Humans
With the exception of the above-mentioned OECD
guideline 429 there are no recommendations for the
use of special inbred or outbred strains in other immunotoxicity guidelines.
These techniques are used to identify or isolate particles or cells bound by indicator cells or erythrocytes.
For example, mixing sheep erythrocytes with human
blood cells results in rosetting of human T cells surrounding the sheep erythrocytes. This erythrocyte (E)
rosette was the first technique in separating T from B
cells. The receptor responsible for this binding is the
CD2 molecule on T cells. The rosettes are named after
the central particle, i.e. E-rosette (see above), erythrocyte antibody (EA) rosette, or erythrocyte antibody
complement (EAC) rosette.
Rosetting Techniques
RT1.B, RT1.D (Rat)
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Regulatory Environment
Rosetting Techniques
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Inbred strains serve as models of human diseases in
various disciplines like immunobiology, transplantation medicine, autoimmunity, and oncology. Inbred
mice and rats of one strain can be seen as identical
twins.
The rat can also be used as an experimental model for
nutrition research, with a reliable correlation of approximately 0.98 between rat and human in the digestibility of nutrients.
Research applications of the rat seems to be dominated
by assigning function to the complete genomic sequence, particularly with respect to those regions involved in common human diseases. All in all, the rat
offers the best 'functionally' characterized mammalian
model system.
Antigen Presentation via MHC Class II Molecules
RTqPCR
References
3
1. Jax Mice Catalogue (2001) http://www.informatics.jax.
org/menus/strain_menu.shtml
2. Baker HJ, Lindsey JR, Weisbroth SH (1979) The
laboratory rat, Vol. 1: Biology and diseases. Academic
Press, New York
Polymerase Chain Reaction (PCR)
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