P
p53
When bound to mdm-2, p53 can no longer function as
an activator of transcription.
Cancer and the Immune System
3
PAH
3
A tumor suppressor gene that codes for a protein of
approximately 53 K molecular weight that controls
cell death and cell cycling; this gene is mutated in
many human cancers allowing transformed cells to
escape cell death. p53 is often activated in response
to DNA damage produced by genotoxic agents leading
to inhibition of the cell cycle and induction of apoptosis.
Polycyclic Aromatic Hydrocarbons (PAHs) and the
Immune System
Polycyclic Aromatic Hydrocarbons (PAHs) and the
Immune System
3
Paramagnetic Cell Selection
Antigen-Specific Cell Enrichment
3
p53 Tumor Suppressor Protein
Parturition
The act of giving birth to offspring.
Developmental Immunotoxicology
3
Passive Cutaneous Anaphylaxis
3
p53 tumor suppressor protein (p53) is a labile protein
located in the nucleus. Agents which damage DNA
induce p53 to become very stable by a post-translational mechanism, allowing its concentration in the nucleus to increase dramatically. The functional structure
includes a strong transcriptional activation domain at
the amino terminus, a central evolutionary highly conserved sequence-specific DNA binding domain and a
tetramerization domain. p53 is a potent transcription
factor and once activated it represses transcription of
genes containing p53-binding sites (several of which
are involved in stimulating cell growth) while stimulating expression of other genes involved in cell cycle
control. The wild-type form of p53 exhibits strong
anti-oncogenic properties. It arrests the cell cycle in
response to DNA damage, thereby allowing DNA repair before the replication of the genome, and induces
apoptosis if the damage to the cell is too severe. A loss
of p53 function (oncogenic transformation) is therefore a key step in the neoplastic progression. It is
also capable of strongly inhibiting transcription from
many genes lacking p53-binding sites. Several oncogenic DNA viruses express viral gene products that
associate with and inhibit the transcriptional activation
function of p53. In cells, p53 can associate with a 90kD protein, identified as the product of the mdm-2
oncogene, which is amplified in some types of tumors.
Assays for Antibody Production
Passive Immunotherapy
Immunotherapy based on the administration of preformed therapeutic agents of immunological origin,
for example antibodies or cytokines. Passive immunotherapy entailing transfer of cells of the immune
system is called “adoptive”. In principle, passive immunotherapy does not depend on the immune system
of the host, but in practice therapeutic efficacy frequently depends on the active participation of host’s
immune responses. For example the activity of monoclonal antibodies can depend on host’s complement or
cell-mediated cytotoxicity. See also Active immunotherapy.
Passive Lymph Pump
3
Passive Lymph Pump
Called also as an “extrinsic lymph pump.” This term
describes a sum of different forces which influence
lymph flow but do not originate from intrinsic lymphatic contractions. Depending on the current conditions, these forces could sometimes generate pressure
gradients in the lymphatic network supportive for central lymph flow. These passive lymph-driving forces
include lymph formation, contractions of skeletal muscles, and fluctuations in central venous pressure, the
influence of respiratory movements, pulsations of adjacent arteries, and influences of gravitational forces.
Lymph Transport and Lymphatic System
PEF
Peak expiratory flow, a measurement of the rate of
exhalation of air.
Asthma
Pentavalent Vanadium
Pentavalent vanadium is the ionic form of vanadium
when the maximal number (five) of the outer shell
electrons (the two from 4s and all three from 3d)
have been shed, thereby giving the atom an overall
charge of +5.
Vanadium and the Immune System
3
Tumor, Immune Response to
3
494
3
Peptide Regulatory Factors
The ability to produce a disease or morbid condition.
Streptococcus Infection and Immunity
3
Pathogenicity
Cytokines
Peptides
Immunotoxicology of Biotechnology-Derived Pharmaceuticals
3
3
PBS
Phosphate-buffered saline.
Maturation of the Immune Response
Percent of Living Cells
3
3
Passive Cutaneous Anaphylaxis
3
PCDDs
Perforin
A protein that, upon secretion, polymerizes to form
membrane-spanning pores. Perforin monomers are
synthesized by cytotoxic T lymphocytes (CTL) and
natural killer (NK) cells and stored in cytoplasmic secretory granules.
Cell-Mediated Lysis
Cytotoxic T Lymphocytes
Cancer and the Immune System
3
PCA
Viability, Cell
3
3
PCR
Polymerase Chain Reaction (PCR)
3
Dioxins and the Immune System
Peripheral Tolerance
Tolerance mechanisms taking place in the blood,
spleen, lymph node and the mucosal immune system.
3
Phosphatases
Lymphoid aggregates found in the wall of the small
intestines which are mainly composed of B lymphocytes with many blasts organized in germinal centers.
The predominant non-IgM isotype of B blasts in
Peyer’s patches is IgA, making these organs important
players in mucosal immune responses.
Immunotoxic Agents into the Body, Entry of
Mucosa-Associated Lymphoid Tissue
3
3
PHA
Phagocytosis involves the engulfment of insoluble
particles such as bacteria or erythrocytes resulting in
their uptake into the cell cytoplasm where they would
normally be degraded. Phagocytosis is commonly
measured ex vivo using a microscopic or flow cytometry technique.
Canine Immune System
Pharmacodynamics
A branch of pharmacology dealing with the interactions between drugs and living systems.
Therapeutic Cytokines, Immunotoxicological Evaluation of
3
3
Peyer’s Patches
Phagocytosis Assay
3
Tolerance
495
Polyclonal Activators
3
Pharmacokinetics
Cells that have the capability of ingesting bacteria,
foreign material, and other cells.
Respiratory Infections
The study of bodily absorption, distribution, metabolism, and excretion of drugs. Developing the pharmacokinetic profile of drugs is an important component
of setting safe and effective dose levels.
Therapeutic Cytokines, Immunotoxicological Evaluation of
3
Phagocytic Cells
3
Phagocytosis
Phenotype
The combination of behavioral, physiological, and
structural changes that occur in a genetically engineered animal. Phenotypes in knockout mice may be
obvious, subtle, or not apparent, depending upon the
presence of one or more compensatory genes (which
can compensate for the engineered mutation) and the
assays selected to assess the animals.
Knockout, Genetic
3
Phosphatases
Phosphatases are enzymes responsible for the removal
of phosphate groups from proteins. In many cases they
are responsible for shutting down signaling pathways
or preventing activation of signaling pathways in a
cell.
Signal Transduction During Lymphocyte Activation
3
Phagocytosis describes the ingestion of particles of
more than 0.5 μm. This ability is largely restricted to
specialized cells called phagocytes such as polymorphonuclear granulocytes or monocytes/macrophages,
whereas pinocytosis (the uptake of small particles
less than 0.5 μm) is a capacity of many cells. Phagocytosis depends on membrane receptors, including
many receptors of the innate immune system or products of the adaptive immune system (e.g. antibodies).
Phagocytosed biological materials including microbes
are degraded and in most cases thereby inactivated.
Phagocytosis by antigen-presenting cells is the prerequisite for the generation of small peptides, which are
bound by MHC class II molecules and presented to
T lymphocytes. Thus phagocytosis forms a link between innate and adaptive immunity.
Immune Response
Fish Immune System
Mucosa-Associated Lymphoid Tissue
Streptococcus Infection and Immunity
Humoral Immunity
P
3
3
3
3
3
496
Phosphorylative Balance
Phosphorylative Balance
Phosphorylative balance is a descriptor of the status
maintained by cellular proteins as a result of the activities of cellular phosphatases and kinases being regulated in a manner such that neither overwhelmingly
predominates under normal conditions.
Vanadium and the Immune System
Photoreactions
Thomas Maurer
Toxicology
Swissmedic
Erlachstrasse 8
CH-3000 Bern 9
Switzerland
3
Synonyms
Photoactivation
Photoreactions
photosensitization, photodermatology, photoactivation, photoallergy
Definition
The interactions of sunlight and skin may lead to
beneficial effects such as vitamin D production, as
well as adverse effects such as sunburn or cancer.
Photoreactions may be due to direct effects of sunlight
or due to the combination of an exogenous chemical
and sunlight. The mechanisms underlying photoreactions are different and the immune system may or may
not be involved. The sunlight effects are dependent on
the wavelength of radiation and as a consequence on
the penetration into the skin. Because of those influences it is clear that various types of reactions exist.
3
3
Photoallergic Contact Dermatitis
Photoreactive Compounds
3
Photoallergy (Photoallergic Contact
Dermatitis)
Photoallergy is an acquired immunological reactivity
which does not occur after the first exposure of skin to
certain chemicals (also after systemic administration)
and subsequent exposure to light or UV radiation. It
needs 1–2 weeks before a photoallergen-specific skin
reactivity can be demonstrated after re-exposure and
light or UV irradiation. It is a delayed hypersensitivity
response manifested in the skin as eczema.
Local Lymph Node Assay (IMDS), Modifications
Photoreactions
Photoreactive Compounds
3
3
3
Photodermatology
Photoreactions
3
Photoirritation
Photoirritation is an acute toxic response elicited after
the first exposure of skin to certain chemicals (also
after systemic administration) and subsequent exposure to light or UV radiation.
Local Lymph Node Assay (IMDS), Modifications
Characteristics
The solar spectrum is divided into the ultraviolet (UV)
part, the visible part, and the infrared part. In the field
of photodermatology, the ultraviolet region is the most
important. In Table 1 ultraviolet is divided according
to the “Commission Internationale d’Éclairage” and
some characteristics for the three parts of ultraviolet
radiation are given (1).
In many publications, the separation of UV-A and UVB is made at 320 nm. In addition, the division between
UV-B and UV-C is often made at 290 nm, due to the
fact that shorter wavelengths are not present in terrestrial sunlight.
The laws of photochemistry are important in all photoreactions:
* Grotthus-Draper law: only radiation that is absorbed is capable of initiating a photochemical process.
* Bunsen-Roscoe law: the photochemical effect depends on dose (intensity × time) and not dose rate
(intensity).
* Stark-Einstein law: each photon absorbed by a molecule activates one molecule in the primary step of
a photochemical process.
* Planck’s law: the energy of a photon is related to its
wavelength.
A light-absorbing molecule, called chromophore, can
3
Photoreactions
497
Photoreactions. Table 1 Ultraviolet
Light
Type
% Solar Radiation on
Earth
Wavelength
Depth of Penetration
Window Glass
Penetration
UV-C
0%
280–100 nm
Epidermis
−
UV-B
1.7%
315–280 nm
Epidermis to papillary
dermis
−
UV-A
6.3%
400–315 nm
Papillary to reticular der- +
mis
Visible
92%
400–800 nm
Reticular dermis to sub- +
cutis
be present in the skin (such as DNA, proteins, lipoproteins, blood components, urocanic acid) or be exogenously applied (directly to the skin or be distributed
to the skin after oral or parental administration).
Molecules absorbing radiation get activated and can
act directly by the oxidation of molecules, by radical
or toxic photoproduct formation or by the formation of
allergens based on the photoactivated binding of
newly formed haptens with proteins.
Terms Often Used in Photodermatology
Photosensitization
This is often used as a general term for a chemicalinduced reaction in the presence of ultraviolet or visible radiation including the terms phototoxicity and
photoallergy.
Phototoxicity
Phototoxicity has been defined as an increased reactivity of the skin to UV radiation and/or visible light
produced by a chemical agent on a non-immunological basis. It is an acute reaction which can be caused
by a single exposure to a chemical and UV or visible
light. In some publications the term photoirritation has
been used specifically for phototoxic reactions after
topical application of compounds. Phototoxic reactions may be induced in all persons exposed to a certain chemical and the appropriate dose of radiation.
Photoallergy
This is defined as an increased reactivity of the skin to
UV radiation produced by a chemical agent on an immunological basis. The skin reaction does not occur
after the first combined exposure to a chemical and
light. An induction period of minimally 1 week or
2 weeks is required before skin reaction may be elicited. As in contact allergy, not all persons exposed to
a combination of a certain chemical and UV radiation
will react. The energy needed for the provocation of a
photoallergic reaction is generally much lower than
that needed for a phototoxic reaction.
Preclinical Relevance
Predictive testing of chemicals for their phototoxic or
photoallergic potential has been performed for many
years, even before official guidelines were available.
However, these tests were only performed for certain
classes of chemicals, such as optical brighteners, cosmetics, tetracyclines, non-steroidal anti-inflammatory
drugs, etc. Testing for photomutagenicity or photocarcinogenicity has also been performed.
More details on the compounds involved in photoreactions are given in a separate chapter of this encyclopedia.
Relevance to Humans
Sunlight Effects Without Involvement of the Immune
System
Acute and chronic effects in man are known due to
direct exposure to sunlight. Acute effects, such as sunburn, occur in everyone if enough of the appropriate
wavelength is absorbed. In the case of exposure to
UV-B, a dose of 10–50 mJ/cm2 can be sufficient to
elicit a sunburn reaction. The erythema starts a few
hours after exposure, leads to erythema and edema
formation, and finishing with desquamation and
long-lasting pigmentation.
Sunburn induced by the UV-A radiation range also
occur. However, the dose needed lies in the range of
50–100 J/cm2. The erythema starts earlier and the pigmentation induced is not of long duration.
The characteristic long-term clinical effects of sunlight
exposure are wrinkling, atrophy, hyperpigmentation,
actinic keratoses, and skin cancer formation (2,3).
Sunlight Effects with Involvement of the Immune
System
The various forms of photoimmunologic effects in
man are described in the monograph of Krutmann
and Elmets (4). Photoreactions with an immunological
mechanism are polymorphous light eruptions, chronic
actinic dermatitis, lupus erythematosus, and solar urticaria.
The polymorphous light reaction is the most frequent
P
Photoreactions
form of photoreaction. The reaction is mainly elicited
by UV-A radiation.
Photourticaria is a rare type of photoreaction and often
a chemical is additionally involved. The action spectrum varies and the reactions may be depend on UV-A,
and visible light, as well as UV-B.
Sunlight may not only stimulate photoreactions in the
skin in combination with allergens but also can suppress the immune system of the skin (5). Especially
when the skin is exposed to UV-B, the function of the
Langerhans cells may be inhibited and/or the number
of cells in the skin is reduced. UV immunosuppressive
effects may lead to the inhibition of contact allergic
reactions and are involved in the promotion of skin
cancer (4,5).
Sunlight Effects in Combination with Chemicals
Phototoxic reactions in man are much more frequent
than photoallergic reactions. However, photoallergic
reactions may lead to persistent light sensitivity and
are, therefore, more relevant for the patients involved.
Chemicals known to be involved in photoallergies are
antipsychotic drugs, antihistamines, antidiabetics,
diuretics, antibiotics, halogenated salicylanilides, UV
filters, etc.
Photoallergic reactions are difficult to categorize according to the classification of Coombs and Gell. Due
to the fact that they are seen 24 hours after photoprovocation, it is thought that most of the photoallergic
reactions are of type IV. In most cases the photoallergic reactions are dependent on UV-A and the irradiation dose for the elicitation of a photoallergic reaction
is generally much lower than that eliciting a phototoxic reaction or sunburn.
The combination of radiation and a chemical to induce
a toxic effect has been used in recent years for therapy
in dermatology and oncology (6). Compounds used
for photodynamic therapy are hematoporphyrin derivative[s], aminolevulinic acid, benzoporphyrin derivative[s], or phthalocyanines. These chemicals are activated by wavelength of the low energetic visible part.
This light part penetrates better, to deeper tissues, and
should elicit phototoxic reactions only in the tissue
where the chemical had been distributed but not in
the normal tissue. With flexible lasers it is even possible to treat cancers in the body (e.g. cancers in the
urinary bladder).
Regulatory Environment
For many years the only guideline including a list of
methods to test for photoallergenic potential was the
Guideline for Toxicity Studies of Drugs Manual of
Japan. Eight methods were included in the list of possible methods without giving priority to any one of the
eight methods.
Last year a new CPMP guideline of the European
Agency for the Evaluation of Medicinal Products
(EMEA) came into force: CPMP/SWP/398/01: Note
for Guidance on Photosafety Testing (http://www.
emea.eu.int). It is requested to test all new drugs,
which absorb light between 290 nm and 700 nm and
which are used topically or reach the skin or eyes
following systemic exposure. The testing should include phototoxicity, photoallergy, photomutagenicity
and photocarcinogenicity. The 3T3 NRU in vitro test
is recommended for phototoxicity. This test has been
validated and a draft OECD protocol is available; the
final acceptance by the OECD is expected soon. For
the other parts of phototesting general recommendations are made.
A guidance for industry paper on photosafety testing
from the FDA (CDER) came into force in May 2003
(http://www.fda.gov/cder/guidance/index.htm). In the
introduction, the following statement was made:
"Use of the principles expressed in this guidance
should reduce unnecessary testing while ensuring an
appropriate assessment of photosafety". The guidance
paper does not recommend specific methods. It mentions general aspects in photosafety testing which have
to be considered and explains possible testing strategies that depend on the duration of use of a drug.
Discussions on the influence of formulations on photoreaction are also included.
3
498
References
1. Diffey B-L (2002) What is light? Photodermatol Photoimmunol Photomed 18:68–74
2. Epstein JH (1999) Phototoxicity and photoallergy. Semin
Cutan Med Surg 18:274–284
3. Berneburg M, Plettenberg H, Krutmann J (2000)
Photoaging of human skin. Photodermatol Photoimmunol
Photomed 16:239–244
4. Krutmann J, Elmets C-A (1995) Photoimmunology.
Blackwell Science, New York
5. Meunier L (1999) Ultraviolet light and dendritic cells.
Eur J Dermatol 9:269–275
6. Ceburkov O, Gollnick H (2000) Photodynamic therapy in
dermatology. Eur J Dermatol 10:568–576
Photoreactive Compounds
Human Safety Department
Procter & Gamble Company
P.O.Box 538707
Cincinnati, OH 45253-8707
USA
Synonyms
photoallergy, photoallergic contact dermatitis, contact
photoallergy
Definition
Photosensitivity is the broad term that is used to
describe abnormal or adverse reactions to the sun or
artificial light sources. These response may be phototoxic or photoallergic in nature. Photoallergy is a cellmediated immunologic reaction to a chemical that has
been made antigenic by the interaction with ultraviolet
(UV) or visible radiation (1). This reaction is similar to
allergic contact dermatitis, but differs in that chemicals
require activation by light to elicit the response. Clinically, photoallergenic skin responses resemble phototoxic reactions but may be distinguished by the increased severity with repeat exposure and time course
for eliciting a response. Further, photoallergenic responses are less frequent than phototoxic reactions.
Histologically, the response is characterized by epidermal edema and vesicle formation and a dense perivascular infiltrate. The action spectrum for most photoallergic reactions is 310–400 nm, primarily UVA radiation. Photoallergy is prevented by protection from or
avoiding light exposure. Many of the known human
photoallergens are also phototoxic.
3
Molecular Characteristics
The common point of initiation for any biological response to light is absorption of photon energy by a
chromophore. The probability of light absorption is
dependent on the molecular structure of the chromophore and the wavelengths of light. The wavelengths
of light of most concern are UV (100–400 nm) and, to
a lesser extent visible radiation (400–760 nm). Because wavelengths below 290 nm are absorbed by
the ozone layer and do not reach the surface of the
earth, UVC radiation from sunlight is of little concern. It is important to keep in mind that the energy of
UVR is inversely proportional to its wavelength. This
relationship is meaningful when considering the probability and consequences of photoactivation.
Absorption of UV or visible photons results in electronically excited molecules; dissipation of this energy
may result in an adverse phototoxic effect on biolog-
Putative Interaction with the Immune System
Photoallergy is a cell-mediated immunologic reaction
to chemicals that histologically and mechanistically
resembles allergic contact dermatitis (1). The critical
factor differentiating these reactions is that the chemicals that produce photoallergy reactions require activation by UV radiation in order to induce and elicit the
response. Two mechanisms have been postulated to
explain formation of the photoproducts responsible
for the induction of photoallergic reactions (2). In
the first mechanism, a photoallergen in its excited
state reacts with proteins to form an allergen. In the
second, the excited state of the photoallergen is converted into a simple contact allergen that binds to protein. Following formation via either pathway, it is believed that the photoallergen is processed by epidermal
Langerhans cells, which transport the processed antigenic determinant (photohapten) to regional lymph
nodes. Therein, antigen-specific T helper cells recognize the antigen bound to Langerhans cells and are
triggered to proliferate and promote the dissemination
of effector and memory T cells that are able to elicit a
cutaneous response upon subsequent encounter with
the inducing antigen. In support, it has been shown
that photoallergic contact dermatitis can be adoptively
transferred with immune cells from animals with
photosensitization to naive animals (3). Moreover,
it has been demonstrated that photohapten-modified
Langerhans cells are capable of stimulating lymphocytes from photoallergic animals in an antigen-specific
manner (4).
Guinea pig and mouse models have been developed
for predicting the photoallergic potential of photoreactive chemicals (5,6). Currently, there are no in vitro
methods available for screening chemicals for photoallergy. However, an in vitro 3T3 Neutral Red Uptake
assay has been developed for phototoxicity testing
that has been proposed as being useful in tier testing
for photoallergy (7).
3
Frank Gerberick
ical systems. For a molecule to have a direct photobiological effect, it must absorb photons and dispense
with this gain of energy in some manner. Otherwise,
any impact of a xenobiotic on UVR-induced responses
in the skin would be attributed to some secondary
mechanism or response modifier, that is, changing
the spectrum of light to which the skin is exposed.
Thus, understanding the potential mechanism (s) of
the interaction between UV and a chemical is critical
when considering the phototoxicologic impact of a
response (2). However, it is not possible at this time
to predict the photoallergic potential of a compound
from its molecular structure alone.
3
Photoreactive Compounds
499
Relevance to Humans
Photoallergy clinical responses characteristically range
P
3
3
500
Photosafety
from a simple erythema to a severe vesiculobullous
eruption. The diagnosis of photoallergy is suspected
by the clinical picture, including the character and distribution of the eruption (e.g. eruption is most notably
in sun-exposed areas such as face and hands). Involvement extending beyond the exposed site frequently
occurs. Similar to the identification of a contact allergen, the contact photoallergen is identified by photopatch testing by the dermatologist.
Over the years, photoallergic responses in humans
have been clearly established with a number of compounds (Table 1). For example, antimicrobial agents,
fragrances, plant derivatives, sunscreens, and some
drugs have been reported to produce photoallergy in
humans. Specifically, halogenated phenolic compounds, coumarins, musk ambrette, promethazine,
chlorpromazine and p-aminobenzoic acid have been
reported to be photoallergenic in humans (8).
3.
4.
5.
6.
7.
References
Photoreactive Compounds. Table 1 Substances
reported to produce photoallergic contact dermatitis in
humans
Classes and Compounds
Antimicrobial
agents
3,3,4’,5-Tetrachlorosalicylanilide
3,4’,5’-Tribromosalicylanilide
3,4,4’-Tribromocarbanilide
Hexachlorophene
8.
Photosafety
Side effects due to a combination of parts of sunlight
and a light absorbing chemical have different mechanisms (e.g. non-immunological or immunological based
reactions). To cover all aspects of possible reactions, it
is therefore necessary to test a chemical in various
models. Guidelines for photosafety testing covers in
general discussions on phototoxicity, photoallergy,
photomutagenicity and photocarcinogenicity.
Photoreactions
3
1. Stephens TJ, Berstresser PR (1985) Fundamental concepts in photoimmunology and photoallergy. J Toxicol
Cutan Ocular Toxicol 4:193–218
2. Kornhauser A, Wamer WG, Lambert LA (1996) Cellular
and molecular events following ultraviolet irradiation of
skin. In: Marzulli FN, Maibach HI (eds) Dermatotoxicology. Taylor & Francis. Washington DC, pp 189–220
Harber LC, Harris H, Baer RL (1966) Photoallergic
contact dermatitis due to halogenated salicylanilides and
related compounds. Arch Dermatol 94:225–230
Gerberick GF, Ryan CA, von Bargen EC, Stuard SB,
Ridder GM (1991) Examination of tetrachlorosalicylicylanilide (TCSA) photoallergy using in vitro photohaptenmodified Langerhans cell-enriched epidermal cells. J
Invest Dermatol 97:210–218
Gerberick GF (1994) Predictive models for assessment of
contact allergy. In: Dean JH, Luster MI, Munson AE
(eds) Immunotoxicology and immunopharmacology.
Raven Press, New York, pp 681–692
Ulrich P, Homey B, Vohr HW (1998) A modified murine
local lymph node assay for differentiation of contact
photoallergy from phototoxicity by analysis of cytokine
expression in skin-draining lymph node cells. Toxicology
125:149–168
Spielmann H, Balls M, Dupuis J et al. (1998) The
international EU/COLIPA in vitro phototoxicity validation study: Results of phase II (blind trial). Part 1: The
3T3 NRU Phototoxicity Test. Toxicol In Vitro 12:305–
327
Kaidbey K (1991) The evaluation of photoallergic contact
sensitizers in humans. In: Marzulli FN, Maibach HI (eds)
Dermatotoxicology. Taylor & Francis, Washington DC,
pp 595–605
Bithionol
Fentichlor
Fragrances
Musk ambrette
Photosensitivity
6-Methylcoumarin
Balsam of Peru
Wood mixture
Lichen mixture
Sunscreens
p-Aminobenzoic acid (PABA)
Octylbenzones
Butyl
methoxydibenzoylmethane
Drugs
Sulfanilamide
Chlorpromazine
Promethazine
Enoxacin
Used to describe a state of heightened reactivity to
photons and may be due to photoallergy, phototoxicity
or an unknown mechanism. This term is often used
when the mechanism underlying the abnormal reaction
is unknown.
Photoreactive Compounds
3
Plant derivatives
Plaque Assay
Pineal Gland
Corpus pineale; epiphysis, epiphysis cerebri, pineal
body, ductless gland; a small endocrine gland in the
brain, situated beneath the back part of the corpus
callosum, which produces the hormones melatonin
and serotonin.
Serotonin
Pinocytic Uptake
A form of endocytosis whereby a small liquid droplet,
a minute particle, and/or solute is ingested by a cell via
a process that utilizes nonspecific membrane invagination and subsequent formation of endocytic vesicles
containing the exogenous material.
Chromium and the Immune System
3
3
3
Although a general term for compounds able to induce
photoreactions, i.e. photoirritation, photoallergy,
photogenotoxicity, or photocarcinogenicity, it is, however, often used to substitute the term photoirritation,
which broadly refers to photon-induced damage to
cells or tissues. Thus, its use is usually restricted to
damage that does not occur via an immune mechanism. Phototoxicity is used clinically to refer to the
presence of morphological evidence of acute changes
in the skin (e.g. erythema, edema, and scaling) following exposure to UV radiation with or without an exogenous photosensitizer.
Photoreactive Compounds
Local Lymph Node Assay (IMDS), Modifications
Porcine Immune System
3
3
Phototoxicity
Pig
3
Local Lymph Node Assay (IMDS), Modifications
Photoreactions
Photoreactive Compounds
A plant lectin that can activate human and rodent T
lymphocytes. It is a mitogen for T cells.
Polyclonal Activators
Nutrition and the Immune System
3
Thie term has two meanings:
* Light sensitivity produced after photons are absorbed by an exogenous chromophore or an abnormally large amount of an endogenous chromophore.
* Describes the physical process of increasing the
(skin) sensitivity to the effects of light, mainly
UVA- and UVB-absorbing substances (photosensitizer). When used to describe the reaction of skin to
an exogenous chemical and UV or visible radiation,
the term includes both photoirritation and photoallergic reactions.
Phytohemagglutin (PHA)
3
Photosensitization
501
3
3
Pituitary Gland
These are the many properties which can be used to
describe a chemical, such as its formula, its melting
point, pKa, lopP, molecular orbital data, electrophilicity and so on. Data on these properties is used to
build quantitative structure–activity relationships.
Chemical Structure and the Generation of an Allergic Reaction
A small endocrine gland located at the base of the
brain that regulates growth and metabolism. The
gland is divided into the posterior and anterior pituitary, each producing its own unique hormones.
Stress and the Immune System
3
Physicochemical Properties
3
Plaque Assay
Plaque-Forming Cell Assays
Plaque Versus ELISA Assays. Evaluation of Humoral Immune Responses to T-Dependent Antigens
P
3
3
502
Plaque-Forming Cell Assays
Plaque-Forming Cell Assays
Gregory Ladics
DuPont Haskell Laboratory
1090 Elkton Road
Newark, DE 19714
USA
Synonyms
Humoral immune assay, hemolytic plaque assay,
plaque assay, antibody-forming cell, AFC, enzymeELISPOT, sheep red blood
linked immunospot,
cells, SRBC, enzyme linked immuno sorbant assay,
ELISA, spot-forming cells, SFC
3
Short Description
The aim of such assays is to evaluate the ability of an
individual to mount a humoral immuneresponse (i.e.
antibody response) to a particular antigen, typically
sheep red blood cells (SRBC). Following exposure
to SRBCs or other T-cell dependent antigens, the
generation of an antigen specific antibody response
requires the cooperation and interaction of several immune cell types: antigen presenting cells (e.g. macrophages), T cells, and B cells. Thus, there are a number
of steps in the process where alterations in the function
of specific cells can impair the ability of B cells to
produce antigen-specific antibody. Because of this
complex interaction of cells, the quantification of the
plaque-forming cell (PFC) response (i.e. the specific
antibody-forming cell (AFC) SRBC response) was
found to provide one of the best predictors of immunotoxicity in mice (1,2). The PFC response to SRBC
uses immunocompetent cells from lymphoid organs,
primarily the spleen. In addition, other antigens,
known as T cell-independent antigens (e.g. TNP-lipopolysaccharide, requires B cells and macrophages or
DNP-Ficoll, requires B cells only) can be used in the
PFC assay to identify the primary immune cell type (s)
targeted by a particular compound. These antigens bypass the need for T cells in eliciting production of
antibody by B cells. Despite the antigen used, the
PFC assay is based on SRBC, which include the antigen or conjugated hapten for the specific antibody
being produced by B cells. A modification of the
PFC assay that allows for the measurement of AFCs
that produce antibody of different isotypes (immunoglobulins IgM, IgG, IgE, or IgA) is the ELISPOT
assay. The ELISPOT assay is similar in methodology
to the ELISA.
3
3
Characteristics
The hemolytic plaque assay was originally developed
by Jerne and Nordin in 1963 as a means to measure
the number of IgM antibody-forming cells specific to
SRBC (3). In this method which has been subsequently modified, spleens from SRBC immunized animals are removed (for rodents, typically 4–5 days
following immunization) and cells are then mixed
with SRBC and complement in a semisolid media
(e.g. agar). This mixture is then plated onto Petri
dishes, covered with a glass coverslip, and incubated
for 3 h at 37°C. During the incubation, B cells produce
IgM antibody specific for SRBC, which then bind to
SRBC membrane antigens and cause complementmediated lysis of the SRBC and the subsequent formation of plaques (i.e. clear areas of hemolysis around
each antibody - forming cell) which can then be
counted visually. Data are usually expressed as IgM
AFC (or PFC)/spleen or IgM AFC (or PFC)/million
spleen cells. The secondary immune response (i.e. IgG
AFC) can be evaluated using a minor modification of
this same assay.
The PFC assay may also be conducted entirely in vitro
with immunocompetent cells obtained from either chemically treated or naive animals using modifications of
the Mishell-Dutton culture system (4). In the latter
case, the immunocompetent cells are exposed to the
test article (or metabolites if test article incubated with
a metabolic activation system prior to in vitro exposure of cells) and SRBC for the first time in tissue
culture. This approach also allows for separation-reconstitution studies with the cells involved in generating a primary humoral immune response (i.e. macrophages, B cells, and T cells) to identify the primary
cell type (s) targeted by a test article.
The ELISPOT assay is a modification of the PFC
assay that allows for the measurement of AFCs that
produce antibody of different isotypes (IgM, IgG, IgE,
or IgA). The ELISPOT assay is similar in methodology to the ELISA. Antigen is allowed to adhere to a
solid support (e.g. plastic or nitrocellulose). A blocking solution is then added to bind any remaining protein-binding sites and subsequently decrease nonspecific binding of reagents. Immunocompetent cells are
then added and during an incubation period at 37°
C/5% CO2 for 4–20 h, AFCs secrete antibody that
binds to surrounding antigen. The AFCs detected by
the ELISPOT assay are called spot-forming cells
(SFC). To determine the isotype of SFC, an enzyme
(e.g., alkaline phosphatase) linked anti-immunoglobulin antibody conjugate specific for different heavy
chains is used. A substrate is then added and an insoluble product produced in areas where antibody is
bound to antigen. Each spot produced by the insoluble
product represents an AFC. The spots are counted
under magnification (15–25×) and compared to negative controls (no cells or no antibody). Data are expressed as SFC/million cells or SFC/organ (e.g. small
intestine or spleen).
Plaque-Forming Cell Assays
503
Plaque-Forming Cell Assays. Figure 1 Immunoglobulin M plaque-forming cell assay.
Pros and Cons
The PFC has some advantages.
* The quantification of the PFC response (i.e. the
specific IgM antibody-forming cell response) was
found to provide one of the best predictors of immunotoxicity in mice (1,2).
* The PFC assay is well-characterized and has been
validated in a number of laboratories for its sensitivity and reproducibility and is recommended by
the US National Toxicology Program to assess the
humoral immune response.
* ELISPOT can measure isotypes and subclasses.
* The PFC assay can also be conducted entirely in
vitro. This approach allows for separation-reconstitution studies to identify the primary cell type (s)
targeted by a test article as well as a means to
potentially distinguish between test article-induced
direct or indirect (e.g. neuroendocrine alterations)
effects on immunocompetent cells.
There are also some disadvantages.
The PFC does not quantitate the amount of antibody produced, but rather the number of specific
antibody-producing plasma cells in a particular tissue (e.g. spleen) and therefore does not account for
antibody produced in other sites (e.g. bone marrow,
lymph nodes).
* In addition, the PFC assay requires the sacrifice of
the animal and thus does not allow for multiple
samples to be taken from the same animal. As a
result, a time course of humoral immune function
cannot be conducted; a recovery period following
*
*
*
test article administration cannot be evaluated; upon
rechallenge with antigen a secondary IgG-mediated
immune response cannot be measured.
The PFC assay is expensive and very labor intensive.
The PFC assay does not lend itself to be automated,
although the ELISPOT assay, which can be conducted in nitrocellulose bottom 96-well plates,
may be automated.
Predictivity
As indicated above quantification of the PFC response
was found to provide one of the best predictors of
immunotoxicity in mice (1). In a subsequent study,
51 chemicals were assessed in mice using a panel of
immunotoxicologic assays. Of the assays evaluated,
the highest correlations with immunotoxic potential
for the chemicals were observed for the splenic IgM
PFC response and cell-surface marker analysis (2).
Therefore, the PFC response to SRBC may be the
most sensitive immune parameter available to assess
chemical-induced alterations to the immune system.
The basis for this sensitivity, as previously discussed,
stems from the fact that the generation of an antigenspecific antibody response requires the cooperation
and interaction of several immune cell types: antigen
presenting cells (e.g. macrophages), T cells, and
B cells. Thus, there are a number of cells that a chemical can target to alter the ability of B cells to produce
antigen-specific antibody.
With respect to ELISPOT, nothing is known up to now
P
504
Plaque-Forming Cell Assays
about predictivity for the immunotoxic potential of a
chemical.
Relevance to Humans
Due to the invasive nature (i.e. injection of antigen,
acquisition of immunocompetent cells) of the PFC
assay, predictive testing in humans is not conducted.
However, limited predictive testing can be performed
on human peripheral blood. Serum concentrations of
each of the major immunoglobulin classes (IgM, IgG,
IgA, and IgE) can be measured and natural immunity
(i.e. antibody levels to ubiquitous antigens such as
blood group A and B antigens, heterolysins, and antistreptolysin) can be assessed by ELISA. However,
quantifying total immunoglobulin levels lacks the predictive value of assays that measure specific antibody
responses following challenge with an antigen. Additionally, antibody responses following immunization
to proteins (e.g. diphtheria, tetanus, poliomyelitis)
and polysaccharides (e.g. pneumococcal, meningococcal) can be measured.
For the most part, the tests available for evaluating
humoral immunity in humans only assess the secondary recall response, rather than a primary response to a
new antigen. Primary immune responses, however, are
a more sensitive measure of immune alteration compared to secondary responses (5). The clinical relevance of moderate or transient alterations in humoral
immune function is also not known. Human data is
limited to severe and longlasting immunosuppression
resulting from therapeutic drug treatments. Furthermore, what human data are available are difficult to
interpret due to the idiosyncrasies of the immune
system. The age, sex, or genetic background of an
individual, and a number of other factors such as
stress, malnutrition, chronic infections, or neoplasia
can effect a “normal” immune response. Risk assessment is further complicated due to a lack of human
exposure data to xenobiotics in general. Additionally,
a biologically significant change in immune function
does not necessarily produce a clinical health effect
until the patient encounters a stress or insult. Further
problems arise when evaluating dose-response relationships due to the immune systems’ reserve or redundant capacity.
Regulatory Environment
Relatively recently the regulatory agencies have begun
to require the evaluation of the primary antibody response. In the USA, for example, in 1998 the Environmental Protection Agency EPA published guidelines requiring chemicals used as pesticides to undergo
an evaluation of the primary humoral immune response to a T-dependent antigen (i.e. SRBC) using
either the PFC or ELISA following the administration
of a test article to mice and/or rats for 28 days. Testing
the ability of pharmaceuticals to alter the antigen-specific antibody response is determined by a number of
conditions. In the US, for example, the Food and Drug
Agency (FDA) suggests considering follow-up studies
to investigate mechanism (s) of immunotoxicity that
may include evaluating the antibody response to a Tdependent antigen among other endpoints if:
* there is evidence of immunotoxicity in repeat-dose
toxicology studies
* the test article or metabolites accumulate or are
retained in reticuloendothelial tissues (i.e. there
are pharmacokinetic effects)
* the test article is used for the treatment of HIV or a
related disease
* there are effects suggestive of immunosuppression
that occur in clinical trials.
In Europe, conventional pharmaceuticals (not biotechnology derived or vaccines) under CPMP guidance
must undergo an initial 28-day screening study in
which the primary humoral immune response to a Tdependent antigen (e.g. SRBC) is conducted if an
analysis of lymphocyte subsets and natural killer cell
activity are unavailable. Additional studies are conducted on a case-by-case basis, which consist of functional assays to further define immunological changes
and may include a measure of the primary antibody if
not evaluated in the initial screening study. The relevant guidelines are:
* FDA (CDER) Immunotoxicology Evaluation of Investigational New Drugs, 2002
* EPA OPPTS 870.7800 Immunotoxicity, 1998
* CPMP/SWP/2145/00 Note for Guidance on NonClinical Immunotoxicology Testing of Medicinal
Products, 2001.
References
1. Luster MI, Munson AE, Thomas P et al. (1988)
Development of a testing battery to assess chemicalinduced immunotoxicity: National Toxicology Program’s
guidelines for immunotoxicity evaluation in mice.
Fundam Appl Toxicol 10:2–19
2. Luster MI, Portier C, Pait DG et al. (1992) Risk
assessment in immunotoxicology. I. Sensitivity and
predictability of immune tests. Fundam Appl Toxicol
18:200–210
3. Jerne NK, Nordin AA (1963) Plaque formation in agar by
single antibody producing cells. Science 140:405–412
4. Kawabata TT, White KL Jr (1987) Suppression of the in
vitro humoral immune response of mouse splenocytes by
benzo(a)pyrene and inhibition of benzo(a)pyrene-induced
immunosuppression by α-naphthoflavone. Cancer Res
47:2317–2322
5. National Research Council. Biologic Markers in Immunotoxicology (1992) National Academy Press, Washington DC, pp 1–206
Plaque Versus ELISA Assays. Evaluation of Humoral Immune Responses to T-Dependen
Kimber L White
Dept. of Pharmacology & Toxicology
Virginia Commonwealth University
P.O.Box 980613
Richmond, VA 23298
USA
Synonyms
Plaque Assay, Antibody-Forming Cell Assay, SRBC
ELISA, KLH ELISA, T-Dependent Antibody-Forming
Cell Response, Evaluation of Humoral Immunity
Short Description
In other sections of this encyclopedia are descriptions
on the Plaque assay and various ELISA assays used to
evaluate the humoral immune response to T-dependent
antigens. This essay will compare the Plaque assay to
the ELISA assay with regards to the information obtained from each, focusing on the strengths and weaknesses of the various assays, proper data interpretation,
and the state of validation and predictability obtained
from the different assay techniques. It may be helpful
for the reader to review the sections on the methodology of the assay in the encyclopedia to better appreciate the comparison of the two assays as they relate to
evaluating humoral immune responses to T-dependent
antigens.
Characteristics
The Plaque assay to the T-dependent antigen sheep
erythrocytes (SRBC) has been the “gold standard”
for evaluating potential immunotoxic effects of compounds on the immune system. While it is used primarily to evaluate effects on the humoral immunity,
the complex nature of developing an antibody response to a T-dependent antigen requires multiple
cell types, including antigen presenting cells, such as
macrophages and dendritic cells, T cells including Thelper cells, specifically Th2 T-helper cells, and B
cells that are capable of proliferating and differentiating into plasma cells capable of secreting antibody to
the SRBC antigen. The Plaque assay has been validated in both mice and rats in national and international ring studies, and it has been shown to be the most
predictive of the functional assays for identifying immunomodulatory compounds (see below) (1,2)
Several regulatory agencies have indicated that in
evaluating responses to T-dependent antigens, assays
other than the Plaque assay can be used. These agencies often site the work of Temple et al. (3) as the basis
for these alternative assays. In the Temple paper, the
effects of two potent immunosuppressive compounds,
benzo(a)pyrene [ B(a)P] and cylophosphamide, were
evaluated in both female B6C3F1 mice and Fischer
344 rats. Both the Plaque assay and the SRBC
ELISA were conducted on the optimum day for each
assay in the two rodent species. The results of these
studies showed similar immunosuppressive results for
both the Plaque assay and the SRBC ELISA in mice
and rats. Due to the similar responses observed, the
SRBC ELISA and, subsequently, ELISAs to other Tdependent antigens have been accepted by various
regulatory agencies as appropriate for evaluating the
potential immunotoxicity of drugs and chemicals.
While both the Plaque assay and ELISAs to T-dependent antigens are capable of evaluating the effects on
the humoral immune response, one must be careful in
the interpretation of the data, which are obtained from
each of the assays. The key points of the data interpretation are shown below in Table 1.
The Plaque assay, conducted on splenocytes, measures
only the production of antibody cells in the spleen.
Accordingly, it only reflects effects that occur on
spleen cell populations. The endpoint evaluated is
not antibody levels, but the number of antibody-forming cells (AFC) also called plaque-forming cells (PFC)
present in the spleen. Each of the AFCs is capable of
producing one plaque, which is actually enumerated in
the assay. In contrast, the ELISA to a T-dependent
antigen measures the antibody levels in the serum
(serum titer) and is either expressed in mass units, if
a standard is available, or as a titer, which is routinely
defined as the reciprocal of the dilution which meets
the criteria of the laboratory’s ELISA assay. Such criteria can include an “Endpoint Titer”, which is the
serum dilution of the test sample that does not differ
from background control ± either two or three standard
deviations of the background. Midpoint Titers are another approach for defining the concentration of antibody present in the serum. Often the Midpoint Titer is
defined as the reciprocal of the dilution of serum obtained at an optical density of O.5, provided the value
falls on the linear portion of the ELISA curve.
Regardless of how the serum titer is calculated, common to all approaches is the interpretation of data.
Titers obtained from serum represent the antibody levels resulting from the production of antibody in the
spleen, lymph nodes and the bone marrow. While most
immunotoxicologist are familiar with the fact that
spleen and lymph nodes are capable of producing antibody, the fact that the bone marrow is a major source
of antibody production and the primary source for secondary antibody production is less well known. Accordingly, the two assays are actually measuring two
different endpoints. In the case of the Plaque assay, the
effect of the compound on the spleen only is being
3
Plaque Versus ELISA Assays.
Evaluation of Humoral Immune
Responses to T-Dependent Antigens
505
P
506
Plaque Versus ELISA Assays. Evaluation of Humoral Immune Responses to T-Dependen
Plaque Versus ELISA Assays. Evaluation of Humoral Immune Responses to T-Dependent Antigens.
Table 1 Immunological Interpretation of Plaque and ELISA Data
Assay
Antibody Production Site
Endpoint Evaluated
Plaque Assay
Spleen
Effects on Spleen Only
Serum Titers
(ELISA)
Spleen, Lymph Nodes, and Bone
Marrow
Holistic Evaluation of Antibody Production in the
Animal
studies we have never seen an effect on the IgG response, which did not also have an effect on the IgM
response. In contrast to the primary IgM response, the
secondary IgG response (following two injections of
antigen) is extremely insensitive to modulation by
compounds, if the primary response was allowed to
develop intact. For example, B(a)P is a potent immunosuppressive compound that will significantly decrease the primary IgM response when administered
before or during the time of antigen sensitization.
However, if B(a)P is administered before or during
the time of the second antigen administration, but
after the primary response developed, B(a)P will not
suppress the development of the secondary response.
During the primary response high avidity antibody
(IgM) is primarily produced. In the secondary response low avidity antibody (IgG) is the primary
isotype formed to T-dependent antigens. Although
IgM is a high avidity immunoglobulin, following a
single injection the affinity of the antibody is usually
very low. The high avidity and low affinity favor conducting a Plaque assay over an ELISA. The high avidity enhances interaction with SRBC in the Plaque
assay and, even though the affinity is low, it is sufficient to bind the SRBC antigen and activate complement producing cell lysis. In contrast, a low affinity
IgM antibody can be easily washed away in the multiple washing steps of an ELISA.
As indicated previously much of the secondary (IgG)
antibody secretion after two sensitizations occurs in
the bone marrow. Accordingly, evaluation of secondary response would favor using the ELISA approach
over the Plaque assay. The high affinity antibody,
which results following two injections as the antibody
matures, also favors the use of ELISA over the Plaque
assay for evaluating the secondary response.
3
3
determined, while in the ELISA the effect of the compound on the spleen, lymph nodes and bone marrow is
what is being evaluated. Thus, it is not surprising that
compounds that affect either the spleen or bone marrow have the potential for producing different results
in the two assays.
In studies conducted by the US National Toxicology
Program (NTP), differential effects have been observed in the Plaque assay and the SRBC ELISA in
the same animals treated with the test compound. For
example, with AZT, a drug known to affect bone
marrow, no effect was observed in the Plaque assay,
while a statistically significant decrease in the serum
titers, as determined by the SRBC ELISA, was observed. Similarly, with the drug thalidomide, which
produces numerous changes in the spleen, a statistically significant enhanced response was observed in
the Plaque assay, while no effect was observed in the
serum antibody levels as determined by SRBC ELISA.
Recently, Keyhole Limpet Hemocyanin (KLH), another T-dependent antigen, has been receiving considerable use in evaluating the effects on humoral immunity using ELISA technology to measure antibody levels to KLH in serum. KLH has several advantages
over SRBC as a T-dependent antigen in conducting
immunotoxicological evaluations. These are addressed
in the next section. While the use of KLH is increasing, recent studies have suggested that it may be less
sensitive than the Plaque assay (4). By increasing the
amount of KLH antigen used to sensitize the test species, at least for mice and rats, sensitivity can be increased somewhat but it still is less than observed with
the Plaque assay.
Since the Plaque assay and ELISA actually measure
two different endpoints, the selection of what assay
should be used depends in part on the question
being asked. On occasion, regulatory agencies have
requested evaluation of both the primary (IgM response following single sensitization) and secondary
(IgG response following two sensitizations) responses.
The characteristics of these responses are shown in
Table 2.
In general the primary response, following a single
injection of a T-dependent antigen, results predominately in the production of IgM. The primary response
is very sensitive to modulation by compounds. In our
Pros and Cons
Each assay used to evaluate the T-dependent antibody
response, the Plaque assay and ELISA, have their
strengths and weaknesses. The Plaque assay is well
established, numerous ring studies have validated the
assay in mice and rats, and it has been shown to be
predictive for immunotoxicological effects of drugs
and compounds. The assay is relatively simple and
does not require expensive equipment. Furthermore,
3
Plaque Versus ELISA Assays. Evaluation of Humoral Immune Responses to T-Dependen
507
Plaque Versus ELISA Assays. Evaluation of Humoral Immune Responses to T-Dependent Antigens.
Table 2 Characteristics of Primary and Secondary Responses
Primary Response
Secondary Response
Sensitive to Modulation by Compounds
Insensitive to Modulation by Compounds
High Avidity Antibody (IgM)
Low Avidity Antibody (IgG)
Low Affinity Antibody
High Affinity Antibody
Favors Conducting
Favors Conducting
Plaque Assay
ELISA Assay
below, only limited validation of the ELISA to T-dependent antigens has been conducted.
Predictivity
Concordance
Studies conducted by the NTP in mice demonstrated
that the Plaque assay was the most predictive of the
functional immunotoxicological assays evaluated. The
concordance with the Plaque assay alone was 78%.
When a second assay was added, i.e., NK, the concordance increased to 94% and, with the plaque assay
and two additional assays, a 100% concordance could
be achieved with the proper selection of assays. As of
this time the predictability of either the SRBC ELISA
or the KLH ELISA has not been established in rodents
or other species.
3
the assay can be conducted rapidly if multiple technicians are utilized. Data are obtained the same day the
study is conducted. The data collected only consist of
a small number of pages; i.e., for a 50-animal study
two pages are generated. This facilitates data review,
particularly when studies are conducted under Good
Laboratory Practices (GLP). The major weakness with
the assay is the reliance upon SRBC as the T-dependent antigen. Sheep must be screened to ensure they
produce a good response and are not bled too frequently. On the other hand there are a few small companies providing SRBC commercially, e.g. Dr. Merk
& Kollegen, Germany, or the Colorado Serum Company, Denver, USA. Another weakness of the assay is
that the blood has a limited shelf life for sensitization.
The ELISA assay, particularly the SRBC ELISA, has
been shown to produce similar results as the Plaque
assay when evaluated using potent immunosuppressive compounds. The SRBC ELISA, like all ELISAs,
has the advantage in that the assay does not have to be
conducted the same day animals are sacrificed. Serum
or plasma can be collected and frozen away for evaluation at a future date. As with the Plaque assay the
major weakness is related to the reliance on SRBC as
the sensitizing T-dependent antigen as well as for preparing the SRBC membrane preparation needed for
the ELISA. For the KLH ELISA, the KLH is available
commercially which assures lot-to-lot consistency of
the antigen. This is a major problem when SRBCs are
used to prepare membrane preparation for use in the
SRBC ELISA. More importantly, standards for both
mice and rats of anti-KLH IgM and IgG antibodies are
available which allow the data from KLH ELISA to be
expressed in mass units, i.e., μg/ml of anti-KLH antibody.
Among the weaknesses of all ELISAs is the time required to properly analyze and evaluate the data.
Furthermore, when conducting studies under GLP, a
significant amount of ELISA data must be generated
in order to ensure an adequate data trail. Unlike a 50animal Plaque assay, which will generate two sheets of
paper, the ELISA evaluation for the same 50 animals
would generate two notebooks of data in order to meet
the GLP requirements (see Figure 1). As addressed
P
Plaque Versus ELISA Assays. Evaluation of
Humoral Immune Responses to T-Dependent
Antigens. Figure 1 ELISA Curve illustrating why
multiple sample dilutions (Mulitpoint Analysis) are need
when conducting ELISA. In the Prozone, area of high
antibody concentration, the optical density (OD) actually increases as the sample is diluted due to antigenantibody complex formation. In the linear region, the
OD halves with each dilution when samples are serially
diluted 1:2. Slope in the linear region is equal to 1. As
the concentration of antibody decreases in diluted
samples the OD approaches background. Using a
single sample dilution (Single Point Analysis) can
produce erroneous results.
508
Plasma Cell
Assay Validation
Relevant Guidelines
The Plaque assay has undergone numerous validation
studies, at both the national and international level.
Numerous studies have shown that in both mice and
rats, the Plaque assay was capable of detecting effects
of immunosuppressive compound in the various laboratories conducting the test. In these validation studies, as expected, reduced variability was observed in
those assays, which had the greatest experience in
conducting the Plaque assay. However, even those laboratories with little or no experience were capable of
detecting effects of immunosuppressive compounds
once they had the assay up and running. In contrast,
only one validation study has been conducted using
the SRBC ELISA (5). In this study, the Plaque assay
was compared to the SRBC ELISA in both CD-1 mice
and Sprague Dawley rats. The conclusion of this study
was that the Plaque assay detected suppression at
lower concentrations compared to the SRBC ELISA
in both rats and mice. While numerous laboratories are
using the KLH ELISA to assess humoral immune responses, as of this date, no validation study has been
completed. Several validation studies for KLH are
being discussed for both rodents and non-rodent species.
EPA OPPTS 880.3800 Biochemicals Test Guidelines,
Immune Response, 1996
OECD 407, Guideline for 28-Day Repeated Dose Oral
Toxicity Test in the Rat, 1998
The European Agency for the Evaluation of Medicinal
Products. Evaluation of Medicines for Human Use,
1999
Relevance to Humans
Results from studies using the Plaque assay have been
predictive of effects observed in clinical studies. However, the development of antibody levels in humans is
evaluated by measuring antibody levels in the serum
or plasma of the patient. This is routinely done using
ELISA. Thus in terms of methodology, evaluating humoral immune responses in humans is more consistent
with the evaluation of humoral immune responses in
animals by ELISA than the Plaque assay, although a
primary response by in vitro stimulation of PBC with
SRBC would theoretically also be possible.
Regulatory Environment
Draft Guidelines
FDA (CDER), Immunotoxicology Evaluation of Investigational New Drugs, Draft Guidance, 1002
ICH Guidelines on Immunotoxicology (in preparation)
References
1. Luster MI, Portier C, Pait DG, White KL Jr, Gennings C,
Munson AE, Rosenthal GJ (1992) Risk assessment in
immunotoxicology. I. Sensitivity and predictability of
immune tests. Fund Appl Toxicol 18:200–210
2. Dayan AD, Kuper F, Madsen C, Smialowicz RJ, Smith E,
Van Loveren H, Vos JG, White KL Jr (1998) Report of
validation study of assessment of direct immunotoxicity
in the rat. The ICICIS Group Investigators. Toxicology
125:183–201
3. Temple L, Kawabata TT, Munson AE, White KL Jr
(1993) Comparison of ELISA and plaque-forming cell
assay for measuring the humoral immune response to
SRBC in animals treated with benzo(a)pyrene or cyclophosphamide. Fund Appl Toxicol 21:412–419
4. Shea JA, Peachee VL, White KL Jr (2003) Characterization of Keyhole Limpet Hemocyanin (KLH) as an
alternative t-dependent antigen for ELISA immunotoxicological evaluations in mice. Toxicologists 72:504
5. Loveless SE, Ladics GS, Smith C, Holsapple MP,
Woolhiser MR, Anderson PK, White KL Jr, Musgrove
DL, Smialowicz RJ, Williams W (2002) Interlaboratory
study of the primary antibody response to sheep red blood
cells in outbred rodents. Toxicologist 66:1164
Plasma Cell
Terminally differentiated B lymphocyte that synthesizes and secretes antigen-specific immunoglobulin.
Germinal Center
B Lymphocytes
Lymphocytes
3
3
3
In the past the Plaque assays were considered the
“gold standard’ for evaluating the effect of a test article on the humoral immune response. However, the
US Environmental Protection Agency (EPA) initially
and other regulatory agencies followed, in accepting
the antibody response to a T-dependent antigen, evaluated using endpoints other than the Plaque assay.
Among other endpoints considered acceptable was
the SRBC ELISA, as well as ELISAs to other T-dependent antigens, including Keyhole Limpet Hemocyanin (KLH), Tetanus Toxoid, and nitrophenyl-chicken gamma globulin. The use of ELISPOT technology as a method of evaluating the humoral immune
response has also been suggested by some regulatory
agencies.
Plasmodium
3
3
A parasite that causes malaria, which infects erythrocytes.
Host Resistance Assays
3
Polybrominated Biphenyls (PBBs)
Little plates or plaques; specifically, blood platelets or
thrombocytes; irregularly shaped disks, containing
granules but no definite nucleus; about one-third to
one-half the size of an erythrocyte, and containing
no hemoglobin; called also Hayem’s hematoblast,
Zimmermann’s corpuscles or particles; number from
200,000–800,000/cu mm of blood; fragments of the
cytoplasm of older megakaryocytes.
Serotonin
Blood Coagulation
Pluripotential Stem Cell
A midechelon stem cell, ancestor of committed myeloid and lymphoid cell lines.
Colony-Forming Unit Assay: Methods and Implications
3
Platelets
509
Pneumococcal Disease Models
3
Pneumonia
Inflammation, or an acute infectious disease, of the
lungs.
Klebsiella, Infection and Immunity
Respiratory Infections
3
3
The fraction of all cells seeded into a clonogenic semisolid culture assay which will give rise to colonies.
Hematopoietic Stem Cells
3
Plating Efficiency
Streptococcus Infection and Immunity
3
3
The immune response going on in a lymph node is
measured quantitatively using parameters, such as cell
count, cell proliferation or expression of cell surface
markers. The PLN index is calculated by dividing the
value for the lymph node from the hind footpad treated
with the test substance by the value for the lymph node
from the other hind footpad treated with the vehicle (or
left untreated). Similarly, the PLN index can be calculated from PLN parameters from a group of test chemical-treated animals and from a control group.
Popliteal Lymph Node Assay, Secondary Reaction
Pokeweed Mitogen
A lectin (glycoprotein) extracted from the pokeweed
(Phytolacca Americana). In immunology it is used to
stimulate the division of lymphocytes, and can therefore be used to test their proliferative capacity. Unlike
concanavalin A and phytohemagglutinin, it stimulates
B cells and plasma cells, as well as T cells, and is
therefore frequently used to investigate functions of
those particular types of cell, including antibody production. See also Concanavalin A; Phytohemagglutinin.
Lymphocyte Proliferation
Polyclonal Activators
3
PLN Index
3
3
PLNA
Polarization
3
33
3
Pluripotent
A state in which a primitive stem cell can differentiate
into any of multiple daughter cell lineages. Partially
committed stem cells can develop into only one or a
few cell lineages.
Lymphocytes
Differentiation capacity.
Maturation of the Immune Response
3
Reporter Antigen Popliteal Lymph Node Assay
Popliteal Lymph Node Assay
Popliteal Lymph Node Assay, Secondary Reaction
Polybrominated Biphenyls (PBBs)
PBBs and their chlorinated analogues (PCBs) are
highly lipid soluble and environmentally persistent
compounds which were used in a variety of applications. They were among some of the first materials
investigated for potential immunotoxicity in both lab-
P
3
510
Polychlorinated Biphenyls (PCBs) and the Immune System
oratory animals and man, following accidental exposure of populations.
Lymphocyte Proliferation
3
Polychlorinated Biphenyls (PCBs) and
the Immune System
John L Olsen
Stony Brook University Medical School
327 Sheep Pasture Road
Setauket, NY 11733
USA
Synonyms
chlorobiphenyl, Aroclor (USA), Clophen (Germany),
Kanechlor (Japan), polychlorinated diphenyls
Definition
Polychlorinated biphenyls (PCBs) belong to the halogenated aromatic hydrocarbon family of chemicals
that also includes dioxins and polychlorinated dibenzofurans (PCDFs). PCBs are a mixture of up to 209
individual chlorinated compounds, known as congeners, which are no longer produced in the USA but
are among the most widespread of environmental pollutants. Electrical transformers, capacitors, lighting
fixtures, hydraulic oils, paints, inks, and home appliances may contain PCBs if they were manufactured
before 1977. Burning of some wastes can also release
PCBs into the environment. Commercial PCB mixtures are known in the USA by the trade name Aroclor
(Monsanto, St Louis MO, USA). There are no known
natural sources of PCBs. Human exposure to PCBs
occurs primarily through food contamination. Occupational exposure was reported to produce acute health
effects as early as 1936. Indicators of exposure to
PCBs in humans may include chloracne, altered hepatic enzyme levels, liver malignancy, decreased pulmonary function, susceptibility to lung infections,
changes in the menstrual cycle, lower birth weights,
eye irritation, immunosuppression, elevated neutrophil
counts, and deficits in vision and intelligence measures.
Molecular Characteristics
A PCB has the formula C12H10−nCln with the general
structure as shown in Figure 1.
PCBs are either oily liquids or solids that are colorless
to light yellow. They are inert chemicals, resistant to
biological and chemical degradation. Some PCBs can
exist as a vapor and can travel long distances in the air
to be deposited in areas where air currents are cooler
than where the PCBs were released. In water, PCBs fix
to organic particles and bottom sediments, though a
fraction will remain undissolved. Aroclors are identified by a four-digit number. The first two digits indicate the number of carbon atoms; the last two digits
refer to the percent weight of chlorine in the mixture.
For example, Aroclor 1248 denotes 12 carbon atoms
with 48% chlorine by weight. Aroclors 1232, 1248,
1260 and 1270 have an average number of chlorines
per PCB molecule of 2, 4, 6, and 10, respectively. The
pattern of chlorination of a congener determines its
physical, chemical, and biological properties. For example, chlorines in the meta (carbon atoms 3, 3’, 5 and
5’) or para (carbon atoms 4 and 4’) positions allow the
PCB molecule to remain planar and to bind the aromatic hydrocarbon receptor (AhR), achieving effects
similar to—but less potent than—2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin). However, halogen
substitution at the ortho positions (carbons 2 and 6) on
one phenyl ring interfere with ortho halogens in the
other phenyl ring (at carbon atoms 2’ and 6’) creating
a non-planar molecule that cannot bind the AhR.
Coplanar dioxin-like PCBs, including 2,3,3',4,5,5'hexachlorobiphenyl and 2,3,3',4,5'-pentachlorobiphenyl, are assumed to exert immunotoxic effects through
an initial action of binding the cytosolic AhR, which is
a member of the thyroid hormone/steroid superfamily
of transcription factors. Over 400 environmental toxicants and endogenous compounds, including by-products of aspartate aminotransferase metabolism, bind
the AhR (1). After ligand binding, AhR releases its
chaperone heat shock proteins, moves into the nucleus
and forms a dimer with the AhR nuclear translocator
protein, Arnt. The basic region of AhR performs DNA
binding and contains a nuclear localization sequence,
whereas its helix-loop-helix and Per-Arnt-Sim (PAS)
homology domains confer protein-binding capability.
In the nucleus, the AhR/Arnt complex binds xenobiotic-responsive elements (XREs) in the genes of the
so-called AhR battery involved in development, reproduction, toxicity response, oncogenesis, and oxidative
stress, including phase I genes (p4501A1, 1A2, 1B1)
and phase II genes (GSTα, NADPH-quinone-oxidoreductase, UDP-glucuronosyltransferase). Deregulation
of retinoid and thyroid hormone homeostasis is an
important consequence of induction of AhR battery
genes. The drug-metabolizing enzymes of the battery
carry out the destruction of their own inducing ligands
or ligand metabolites. PCBs are metabolized mainly
by mixed-function oxidases into a wide range of metabolites, but enzymatic activation is not necessary for
PCB toxicity.
c-Src protein is functionally attached to the AhR and is
activated by ligand binding in numerous animals, including rodents and humans. This activation is rapid,
often potent at very low doses, and longlasting. Down-
Polychlorinated Biphenyls (PCBs) and the Immune System
stream targets of this activity include increased protein
kinase C (PKC) activity in the thymus and upregulation of thyroid hormone receptor mRNA. p450 regulation is likely src-independent. The AhR also forms
complexes with the retinoblastoma tumor suppressor
in human MCF-7 cells, suggesting a role for the AhR
in cell cycle regulation and G1 arrest.
Non-dioxin-like PCBs account for the bulk of the
PCBs found in biological and environmental samples.
Immunotoxic effects mediated independently of the
AhR include:
* PCB metabolism to arene oxide intermediates that
can alkylate cellular macromolecules to become
toxic adducts
* congener-specific antagonistic or synergistic interactions between non-coplanar PCBs and dioxin
* some PCBs and PCB metabolites are thought to
bind transport proteins in plasma, including transthyretin, thus altering the transport and plasma concentrations of endogenous molecules
* PCB-induced liver enzyme induction and metabolism of parent substrates (including medications or
environmental pollutants) to daughter compounds
that may have enhanced or diminished immunotoxic or therapeutic properties.
Regarding the second point, Aroclors 1248, 1254,
1260, and several congeners have been shown to antagonize the immunosuppressive effects of dioxin.
Whether this antagonism is mediated by means of
displacing dioxin from its binding site on the AhR
or by actually counterbalancing the effects of dioxin
through an alternate pathway is not clear at this point.
The use of the term 'non-dioxin-like PCBs' is not necessarily practical. They are not a single class of chemicals and have multiple toxicities with discrete structure-activity relationships. Further congener-specific
research may better classify these compounds and
identify their specific targets in human tissues.
Putative Interaction with the Immune System
The potential for PCBs to be immunomodulatory has
been the subject of extensive experimental investigations. Accumulating evidence indicates that the im-
Polychlorinated Biphenyls (PCBs) and the Immune
System. Figure 1 General structure of polychlorinated
biphenyls (PCBs).
511
mune system is probably one of the most sensitive
targets for PCB-induced toxicity.
Antibody Titers and Lymphocyte Populations
In mice, humoral suppression is dependent on a PCB
congener’s affinity for the AhR. In C57Bl/6 mice the
coplanar, non-ortho-substituted 3,3’,4,4’-tetrachlorobiphenyl (TCB) exhibits a high affinity for the AhR
and causes severe humoral antibody suppression. But
in the genetically different DBA/2D2 mice, for which
TCB shows a lower binding affinity for the AhR, this
humoral suppression does not occur. The essentially
non-coplanar, di-ortho-substituted 2,2’,5,5’-tetrachlorobiphenyl binds the AhR weakly in both animals and
does not suppress humoral immunity in either strain.
Several animal studies using Aroclor mixtures have
shown that it is the more highly chlorinated PCBs
(Aroclors 1260, 1254, 1248) that are responsible for
diminished plaque-forming cell responses to sheep red
blood cell challenge, decreased IgG titers to KLH antigen, and decreased spleen and lymph node cellularity. TCB and other AhR-binding PCBs cause thymus
atrophy in animal models, specifically targeting immature thymocytes. Interestingly, PCBs given in less than
immunotoxic levels to mice may reduce the effects of
2,3,7,8-TCDD-induced inhibition of the plaque-forming cell response to sheep erythrocytes.
Chronic exposure of rhesus monkeys to Aroclor 1254
was associated with decreased titers to SRBC; the
calculated lowest observable adverse effect level
(LOAEL) for this effect was 5 μg/kg/day. Rhesus
monkeys exposed to Aroclor 1248 had reduced cortical and medullary areas in the thymus, inapparent germinal centers in lymph nodes and spleen, and hypocellular bone marrow. It is important to note that PCB
suppression of humoral antibody responses can be
both T cell-dependent (an SRBC response) and
T cell-independent (an LPS response), indicating that
PCBs can alter B cell differentiation into plasma cells
independently of thymic alterations.
Data is limited and often conflicting regarding PCBinduced suppression of cell-mediated immunity in laboratory animals. Dogs exposed to Aroclor 1248 had a
decreased delayed-type hypersensitivity reaction to intradermal injection of tetanus toxoid compared to controls. This, along with several in vitro experiments,
indicates that a subpopulation of T-cells may be affected.
Effects of PCB exposure in humans were documented
following ingestion of contaminated rice bran oil in
Yusho, Japan in 1968, and of rice in Yu-Cheng, Taiwan in 1979). Contamination of the PCBs with PCDFs
confounds the interpretation of these studies. Longterm studies of the more than 2000 people who were
exposed during these events revealed increased mortality due to the food poisoning. In both of the studies,
P
512
Polychlorinated Biphenyls (PCBs) and the Immune System
normal IgG levels were seen, but IgM and IgA were
decreased, returning to normal after 4 years. Reductions were seen in total, active, and helper T cells, as
well as delayed-type hypersensitivity to tuberculin antigen.
The immunologic effects of in utero exposure have
been studied in Yu-Cheng children born between
1978 and 1987 (2). Serum immunoglobulins and cell
surface markers were not different compared to controls. It has been reported that children exposed to
dioxin-like PCBs in breast milk have decreased numbers of CD8 cells. In a study of Dutch children monitored for prenatal and childhood exposures, PCBs
were associated with lower antibody levels to
mumps and measles at preschool age (3).
Infection and the Innate Immune Response
The maturation of the immune system is particularly
vulnerable to PCBs. In the Yu-Cheng study (2) children exposed in utero exhibited immunosuppression,
higher frequencies of bronchitis in the first 6 months
of life, and higher frequencies of respiratory tract and
ear infections in a 6-year follow-up. In the Dutch study
(3) prenatal PCB exposure was associated with less
shortness of breath with wheeze, and at 42 months
of age PCB levels were associated with a higher prevalence of recurrent middle-ear infections and chicken
pox, yet a lower prevalence of allergic reactions. It is
therefore possible that exposure to dioxin-like PCBs in
utero and during early childhood might lead to the
recurrence of common infections that help to prevent
the development of allergy. So far, though, proof of a
causal relationship requires more evidence and improved reporting of confounding factors.
In experimental animals, mononuclear phagocytes are
altered functionally by PCBs, showing decreased phagocytic activity and splenic clearance of bacteria and
increased sensitivity to bacterial endotoxins. Interestingly, rhesus monkeys administered Aroclor 1254
have increased serum complement activity (CH50).
Mice have a decreased ability to clear Listeriamonocytogenes when given PCBs by acute peroral gavage
or subacute parenteral injection, and this seems to be
sex specific, as females are less sensitive to the effects
of PCBs.
Studies in non-human primates, dogs, and human
adults and stillborn babies exposed to PCBs show
many common defects to structures that are important
to the maintenance of innate immunity: modulation of
skin and nail beds, eye exudate, inflammation of Meibomian glands, and neutrophil levels and function.
Gastric hyperplasia, which could affect host mucosal
immunity, has been found in monkeys but not in dogs.
The natural killer (NK) cell is a cellular bridge between innate and adaptive immunity. In rat studies,
NK cell activity was depressed by Aroclor 1254.
Paradoxically, Aroclor may be protective for tumor
development in cancer-cell transplantation studies. Aroclor 1254 reportedly reduced mortality in mice following injection of Ehrlich’s tumor cells. Rats transplanted with Walker 256 carcinosarcoma showed retarded tumor cell growth if also treated with Aroclor
1254.
In mice, many PCB congeners and methylsulfonyl
metabolites bind the bronchial mucosa and are selectively retained in lung Clara cells by a PCB-binding
protein that is homologous to human uteroglobin. The
uteroglobin gene encodes a cytokine-like anti-inflammatory protein that has a potent-inhibitory activity
against phospholipase A2. Lung tissues from PCBpoisoned Yusho patients harbored 16 different PCB
methylsulfonyl PCBs. Clara cells are a key site of
activities that are dependent on p450 enzymes that
convert various substrates to reactive products. When
compared to control mice, uteroglobin knockout mice
fail to accumulate methylsulfonyl PCBs in lung. The
role of uteroglobin in PCB-induced respiratory immunotoxicity has not been fully elucidated in humans.
Relevance to Humans
PCBs have been found in at least 500 of the 1600
National Priorities List sites identified by the US Environmental Protection Agency. As in other species,
PCBs accumulate in humans in fatty tissues because of
their lipophilic properties and resistance to degradation. Average levels in adipose tissue are 1 p.p.m.
and 10 p.p.b. in blood, and have been declining in
the population since the 1980s. In general, fish consumption is the major source of exposure to PCBs.
The human health effects of exposure to PCBs in
fish are a function of congener toxicity, PCB concentration in fish, and fish consumption rates. Some risk
factors for having a high PCB body burden include
older age, dermal and ingestion exposure, as well as
complex metabolic factors including lactation, diet,
and exercise activity. As a significant percentage of
PCBs are liberated from drying soil, humans may be
exposed via inhalation. There may be sex-specificity
for some biological outcomes.
It has been reported that breastfed infants of mothers
who ate high amounts of Great Lakes fish had a greater amount of microbial infections compared to control
infants of mothers who did not eat fish. Studies of
bacterial aerobic degradation of PCBs (removal of
chlorines) in the environment indicate that this process
decreased the risk of PCB-induced immunotoxicity, as
measured by splenocyte proliferation assays.
PCBs elicit a wide spectrum of biochemical responses,
some of which are similar to those caused by dioxin.
Because dioxin-like compounds normally exist in biological and environmental samples as complex mixtures, the concept of toxic equivalency factors (TEF)
Polychlorinated Dibenzodioxins
between a high level of background exposure to PCBs
and development of non-Hodgkin lymphoma (5).
References
1. DeVito MJ, Birnbaum LS (1995) Dioxins: model
chemicals for assessing receptor-mediated toxicity. Toxicology 102:115–123
2. Yu ML, Hsin JW, Hsu CC, Chan WC, Guo YL (1998)
The immunologic evaluation of the Yucheng children.
Chemosphere 37:1855–1865
3. Weisglas-Kuperus N, Patandin S, Berbers GA et al.
(2000) Immunologic effects of background exposure to
polychlorinated biphenyls and dioxins in Dutch preschool
children. Environ Health Persp 108:1203–1207
4. Ahlborg UG, Becking GC, Birnbaum LS et al. (1994)
Toxic equivalency factors for dioxin-like PCBs: Report
on WHO-ECEH and IPCS consultation, December 1993.
Chemosphere 28:1049–1067
5. Rothman N, Cantor KP, Blair A et al. (1997) A nested
case-control study of non-Hodgkin lymphoma and serum
organochlorine residues. Lancet 350: 240–244
6. Ahlborg UG, Becking GC, Birnbaum LS et al. (1994)
Toxic equivalency factors for dioxin-like PCBs: Report
on WHO-ECEH and IPCS consultation, December 1993.
Chemosphere 28:1049–1067
Polychlorinated Dibenzodioxins
3
has been developed to simplify risk assessment and
regulatory management. Relative toxicities of dioxinlike compounds in relation to the reference compound,
TCDD, were determined on the basis of results obtained in in vivo and in vitro studies. A dozen congeners in particular exhibit dioxin-like effects
(Table
1).
3,3',4,4',5-pentachlorobiphenyl,
3,3',4,4',5,5'-hexachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl have TEFs in humans of 0.1, 0.01, and
0.0001, respectively. Maximum dioxin-like activity is
obtained when there are no ortho, two or more meta,
and both para positions are occupied. A single chlorine in the ortho position weakens dioxin-like activity
but increases AhR-independent toxicity of the compound. In population studies, TEFs should be calculated for PCBs, dioxins, and polychlorinated dibenzofurans. However, the antagonism between certain
high-dose PCB congeners/mixtures and dioxin-like
chemicals may confound models that assume TEFs
can be additive.
Because PCBs cause immune dysregulation experimentally, it is plausible that they may alter cancercell surveillance. Consistent with animal studies,
workers occupationally exposed to PCBs show increased deaths from several cancers including those
of the liver, melanoma, thyroid, pancreas, and gastrointestinal and central nervous system neoplasms. However, it is very possible that these diseases affect PCB
levels due to wasting and contraction of the body lipid
compartment, so that retrospective studies need to be
interpreted with caution. An intriguing prospective
study reported a clear positive dose-response relation
513
Dioxins and the Immune System
Polychlorinated Biphenyls (PCBs) and the Immune System. Table 1 Polychlorinated biphenyl (PCB)
congeners that resemble dioxin*
Congener number
IUPAC name
77
3,3',4,4'-tetrachlorobiphenyl
81
3,4,4',5-tetrachlorobiphenyl
105
2,3,3',4,4'-pentachlorobiphenyl
114
2,3,4,4',5-pentachlorobiphenyl
118
2,3',4,4',5-pentachlorobiphenyl
123
2,3',4,4',5'-pentachlorobiphenyl
126
3,3',4,4',5-pentachlorobiphenyl
156
2,3,3',4,4',5-hexachlorobiphenyl
157
2,3,3',4,4',5'-hexachlorobiphenyl
167
2,3',4,4',5,5'-hexachlorobiphenyl
169
3,3',4,4',5,5'-hexachlorobiphenyl
189
2,3,3',4,4',5,5'-heptachlorobiphenyl
*Congeners that show structural similarity to dioxin, bind to the AhR, induce dioxin-specific responses, and are persistent and
accumulate in the food chain (Ahlborg UG, Becking GC, Birnbaum LS, et al (1994) Toxic equivalency factors for dioxin-like
PCBs: Report on WHO–ECEH and IPCS consultation, December 1993. Chemosphere 28: 1049–67).
P
514
Polychlorinated Diphenyls
Polychlorinated Diphenyls
Polychlorinated Biphenyls (PCBs) and the Immune
System
3
Polyclonal
Describes the proliferation or products of different
cells.
Mitogen-Stimulated Lymphocyte Response
3
Polyclonal Activators
Stephen B Pruett
Department of Cellular Biology and Anatomy
Louisiana State University
Health Sciences Center
Shreveport, Louisiana 71130
USA
Synonyms
Mitogens, polyclonal mitogens, PHA, phytohemagglutin, ConA, concanavalin A, LPS, lipopolysaccharide, SEB, streptococcal enterotoxin B, MLR, mixed
lymphocyte response, pokeweed mitogen, PWM,
major histocompatibility complex, MHC
Definition
Polyclonal activators are agents that activate a significant proportion of B or T lymphocytes (or both), inducing their proliferation and generally at least some
of the functions that are induced by antigen-mediated
cellular activation (e.g. cytokine production).
Characteristics
There are several classes of polyclonal activators,
which differ in structure and mechanism of action.
Several plant-derived lectins (which bind oligosaccharides on various cell surface proteins) activate T or B
lymphocytes. For example, phytohemagglutin (PHA)
and concanavalin A (ConA) activate T lymphocytes,
and PWM activates T and B lymphocytes. These
agents generally act by cross-linking surface molecules and initiating signaling events leading to cellular
activation. Bacterial lipopolysaccharide (LPS) at high
concentrations (usually greater than 10 μg/ml) polyclonally activates B lymphocytes, acting through Tolllike receptor 4 (1). Superantigens are agents that bind
T cell receptors on a broad subset of T cells (e.g. all
which express the Vb8 region on their T cell receptor)
and also bind the MCH class II protein. This functions
to link MHC II proteins on antigen-presenting cells
and T cell receptors on T cells in a manner that allows
T cell activation. Most superantigens are protein products of bacteria, and examples include staphylococcal
enterotoxins, streptococcal pyrogenic toxins, and
toxic-shock syndrome toxin (2). Staphylococcal
enterotoxin B (SEB) is one of the most potent and
most widely used superantigens. It could be argued
that superantigens represent a more physiologically
relevant method for activating lymphocytes, because
the receptors that normally are involved in activation
by antigen are involved in superantigen-induced activation. Antibodies specific for the antigen receptors or
signal transduction components of T cells (TCR or
CD3) or B cells (surface Immunoglobulin) can be
used to activate these cells. In the case of B cells,
anti-immunoglobulin antibodies plus interleukin(IL)4 are sufficient to achieve substantial B cell activation.
In the case of T cells, accessory cells (macrophages or
dendritic cells) must be present, or the anti-CD3 antibody must be coated onto the surface of the culture
plate to achieve full activation. These approaches are
often regarded to be similar to antigen-induced activation of these cells, but it should be noted that there are
likely several costimulatory signals induced during
natural activation of cells by antigen, which are not
induced by these stimuli. Finally, inactivated lymphocytes from allogeneic animals can be used as a polyclonal T cell activator. This is referred to as the mixed
lymphocyte reaction or mixed lymphocyte response
(MLR). It is based on the fact that a substantial percentage of T lymphocytes recognize and respond to
major histocompatibility proteins found on allogeneic
lymphocytes. If these “stimulator” cells are treated to
prevent their reciprocal response (i.e. by radiation or
DNA synthesis inhibitor), the response of the “responder” lymphocytes can be assessed by measuring their
proliferation.
Preclinical Relevance
The preclinical relevance of polyclonal activators in
immunotoxicity testing is indicated both by the rationale for their use and by experimental data. The rationale for evaluating responses to polyclonal activators
is that the activation and proliferation of T cells and
B cells are required for immune responses, and most
of the cellular events and molecular components required for these processes are also required for cellular
responses to polyclonal activators. Experimental results from studies sponsored by the National Toxicology Program demonstrate that the effects of a wide
variety of drugs and chemicals on lymphocyte responses to polyclonal activators (PHA, ConA, and
LPS) are strongly concordant with effects on other
Polycyclic Aromatic Hydrocarbons (PAHs) and the Immune System
important immunological end points, including T celldependent antibody responses (3).
515
Polycyclic Aromatic Hydrocarbons
(PAHs) and the Immune System
Relevance to Humans
Scott W Burchiel
Clinical immunologists routinely use polyclonal activators to test T and B lymphocyte function in humans.
Abnormal responses to these agents can be used in the
diagnosis of hereditary or acquired immunodeficiency
conditions.
College of Pharmacy Toxicology Program
University of New Mexico
Albuquerque, NM 87131-0001
USA
Synonyms
Regulatory Environment
Cellular responses to polyclonal activators are not specifically required or recommended as immune function
tests in immunotoxicity testing in Guidance documents from the US Environmental Protection Agency
(4) or US Food and Drug Administration (5). However, they are included in the “extended studies” required
by the European Agency for the Evaluation of Medicinal Products (EMEA) to characterize the effects of
drugs that seem to have immunotoxic potential (6).
In addition, they are often included in research studies,
because they are simple, reproducible, and generally
correlate well with other measures of immunotoxicity (3).
PAHs, benzo-a-pyrene, BaP, 7,12-dimethylbenz-a-anthracene, DMBA, 3-methylcholanthrene, 3-MC, dibenz-a, h-anthracene, benz-a-anthracene, BA, BP-quinones, BPQ, BP-7,8-diol, BP-7,8-diol, 9,10-epoxide,
BPDE, anthracene, pyrene, benzo-e-pyrene, BeP,
nitro-PAH, oxy-PAH
Definition
Polycyclic aromatic hydrocarbons are a class of diverse compounds that consist of three or more fused
aromatic rings, primarily based on anthracene and pyrene. Environmentally, PAHs are formed during the
combustion of fossil fuels and high temperature reactions of hydrocarbons, benzene and other aromatic
compounds.
Molecular Characteristics
References
1. Kawai T, Adachi O, Ogawa T, Takeda K, Akira S (1999)
Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11:115–122
2. Kuby J (1994) Immunology. WH Freeman, New York,
pp 218–219
3. Luster MI, Portier C, Pait DG et al. (1992) Risk
assessment in immunotoxicology. I. Sensitivity and
predictability of immune tests. Fundam Appl Toxicol
18:200–210
4. US EPA(1998) Health Effects Test Guidelines OPPTS
870.7800 Immunotoxicity. http://www.epa.gov/opptsfrs/
OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Series/870-7800.pdf
5. US FDA (2002) Guidance for Industry. Immunotoxicology evaluation of investigational new drugs. http://www.
fda.gov/cder/regulatory/applications/guidance.htm
6. European Agency for Evaluation of Medicinal Products
(2000) Note for Guidance on repeated dose toxicity.
http://www.emea.eu.int/pdfs/human/swp/104299en.pdf
Polyclonal Mitogens
Polyclonal Activators
The biologic and toxicologic activities of PAHs are
largely dependent on their ability to interact with
AhR receptors present in many mammalian cells and
tissues. In general, PAHs are semivolatile compounds
that are quite lipophilic and exert both specific and
non-specific membrane effects. Highly specific structure-activity relationships have been observed for the
effects of PAHs on cells and tissues. Specific effects
generally relate to the expression of AhR and the varying ability of individual PAHs to activate AhR-dependent gene promoter regions, referred to as dioxin response elements (DRE). The dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a pure AhR agonist
that is approximately 100 times more potent than PAH
in binding to AhR and activating DRE. TCDD and
related agents are discussed elsewhere in this book.
PAHs are metabolized by cytochrome P450-dependent
and - independent pathways. The P450-dependent
pathways, most notably CYP1A1, CYP1A2 (liver
only) and CYP1B1, are induced following binding
of AhR and activation of DRE. In the case of BaP
and other PAHs, these enzymes form oxidative metabolites, such as epoxides, which in the presence of
epoxide hydrolase-1 (EPHX1) form dihydrodiols that
can undergo another round of metabolism to form
diol-epoxides such as benzo-a-pyrene(BaP)-diol-epoxide (BPDE). BPDE and several other BaP metabolites,
P
3
516
Polycyclic Aromatic Hydrocarbons (PAHs) and the Immune System
such as the BP-quinones (BPQ; discussed below) are
strong electrophiles that trigger the activation of secondary metabolizing enzymes including glutathioneS-transferases (GST) and N-quinone oxidoreductase
(NQO1) via interaction with electrophile response elements (EpRE) that are also referred to as antioxidant
response elements (ARE) (1). Electrophilic BaP metabolites, most notably BPDE, covalently bind to DNA
leading to genetic mutations responsible for tumor initiation. In addition, the formation of BPDE-DNA adducts leads to induction of the p53 pathway, which
may trigger cell cycle changes and apoptotic pathways
in target cells.
Several oxidative metabolites of PAHs, such as BPquinones and DMBA-quinones are known to redoxcycle, leading to production of reactive oxygen species
(ROS) (2). PAH-quinones are formed via P450-dependent and independent reactions, such as by peroxidases or by ultraviolet light (3). Redox-cycling results
in the formation of ROS, including superoxide anion
and hydrogen peroxide, which are known to activate
lymphocytes. Redox-cycling requires reducing equiva3
Polycyclic Aromatic Hydrocarbons (PAHs) and the
Immune System. Figure 1 Potential molecular mechanisms of polycyclic aromatic hydrocarbon (PAH)
immunotoxicity via Ah receptor(AhR)-dependent and
AhR-independent pathways. Several AhR-dependent
pathways produce immunotoxic metabolites of benzoa-pyrenes (BaP) (such as BPDE) that appear to act via
both p53-dependent (genotoxic) and p53-independent
pathways. BPDE has also been shown to signal Ca2+dependent pathways, and BaP metabolites such as
BP-quinones (BPQ) also signal via Ca2+ due to redoxcycling and mitochondrial oxidative stress. The immunotoxicity of BPQ has not been fully evaluated. Two
secondary enzyme systems, glutathione-S-transferase
(GST) and N-quinone oxidoreductase (NQO1), reduce
the concentrations of PAH metabolites. Thus, the
effects of PAHs such as BaP and DMBA involve
complex interplay between bioactivating and inactivating pathways. The metabolites act via non-genotoxic
(signaling and apoptotic pathways) and genotoxic
pathways. (DRE: dioxin response elements.)
lents generally supplied by NADPH, and thus BPQ
and related agents interact with the mitochondrial electron transport chain, leading to oxidative stress and
ATP depletion. The immunotoxicologic effects of
BPQ, other oxy-PAHs, and nitro-PAHs, have not
been fully evaluated.
Thus, the biologic and toxicologic action of PAHs is a
complicated interplay between the ability of specific
PAHs to bind to endogenous AhR, the formation of
oxidative and electrophilic metabolites, and the removal of reactive molecules via secondary metabolic
processes (Fig. 1). The effects of these agents result
from activation of both genotoxic and non-genotoxic
pathways. Therefore, the toxicity of PAHs to target
tissues is dependent upon the exposure of cells and
tissues to circulating parent compounds and metabolites, their expression of AhR, and their propensity to
form bioactive versus detoxified metabolites.
Putative Interaction with the Immune System
Carcinogenic PAHs have been found to suppress the
immune system of animals (4,5). Initial studies
showed that BaP (but not BeP), DMBA, and 3MC
suppress humoral immunity, and later studies showed
that many immune cells are targets of PAH action (as
described below). The humoral immune response to Tdependent antigens is considered to be a sensitive indicator of immune suppression, although suppression
of T cell and B cell proliferation has been observed at
similar exposure levels. In general, there is a correlation between the carcinogenicity of a PAH and its
immunotoxicity, which is likely due to requirements
for AhR binding activity and metabolic activation
pathways. The immunosuppression produced by BaP
and DMBA has both similarities and differences. BaP
is a moderate to strong AhR ligand, whereas DMBA is
a weak AhR ligand. In addition (as discussed below)
much of the immunotoxicity of BaP is due to P450
CYP1A1-dependent metabolism. CYP1A1 is expressed only at low levels in lymphocytes until AhR
are activated. In the case of DMBA, the major metabolizing enzyme is P450 CYP1B1, which is expressed
in many tissues constitutively.
Immune System Targets of PAH Toxicity
Bone Marrow
Both DMBA and BaP exert important effects on the
bone marrow altering the formation of B cells. The
mechanism of pre-B cell bone marrow suppression
appears to be P450 CYP1B1-dependent and may be
caused via pre-B cell apoptosis (6).
Thymus
Thymic atrophy is observed in mice treated with high
concentrations (50–100 mg/kg) of DMBA. Interestingly, there does not appear to be a correlation be-
Polycyclic Aromatic Hydrocarbons (PAHs) and the Immune System
tween AhR phenotype and the ability of DMBA to
decrease thymus weights (7).
Spleens
Many studies have been performed with spleens obtained from PAH-treated mice. In these studies both
DMBA and BaP have been found to be immunosuppressive for humoral and cell-mediated immunity.
DMBA was also found to be immunosuppressive
when exposures occurred in vitro. In general, the
most sensitive target cells for PAHs appear to be B
and T helper cells for humoral immunity and cytotoxic
T cells (CTL) for cell-mediated immunity, although
macrophages and antigen-presenting cells (APC)
have also been implicated. Splenic natural killer
(NK) cells have also been shown to be suppressed
by DMBA.
Peripheral Lymphoid Tissues
A few peripheral lymphoid tissues have been examined for the effects of PAHs following intratracheal or
intragastric administration of BaP. It was found that
BaP suppressed the antibody response of lung-draining lymph nodes in rats exposed to BaP intratracheally, and suppressed the response of murine mesenteric
lymph nodes (MLN) and Peyer’s patches following
gavage.
Mechanisms of Immunosuppression by PAHs
AhR Ligands Suppress the Immune System
As mentioned above, many PAHs are moderate-tostrong AhR ligands. It has been previously noted
that there is generally a positive correlation between
the carcinogenicity and immunotoxicity of a PAH.
This correlation likely exists because both carcinogenicity and immunotoxicity are largely dependent on
AhR binding, increased P450 expression, and formation of bioactive metabolites. Direct AhR-dependent
immunosuppression has been reported in both T and
B cells for the pure AhR agonist, TCDD. Presumably
these same pathways would be activated by BaP and
other AhR ligands; however, a key difference is that
the persistence of PAHs in the body is reduced due to
metabolism.
PAH Metabolism Required for Immunotoxicity
P450 metabolism is very important for immunotoxicity of PAHs. Metabolism occurs in central (liver,
lung) and peripheral tissues (lymph nodes, spleen,
and bone marrow). The local metabolism of PAHs is
complicated by the expression of AhR and constitutive
levels of P450s such as CYP1B1. CYP1A1 is highly
inducible in some white blood cells. The highest levels
of P450 activity have been detected in monocytes and
macrophages, and low levels have been detected in B
and T cells. Most human and murine B and T cell lines
517
used for in vitro modeling studies have little P450
activity.
Further support for the role of P450 metabolism in the
immunotoxicity of PAHs has been obtained in studies
that demonstrate that the Ah receptor antagonist and
P450 CYP1A1/CYP1B1 inhibitor α-naphthoflavone
(ANF) prevents the immunotoxicity of BaP and
DMBA in murine spleens cells and human peripheral
blood T cells (8). The CYP1A1 and CYP1B1 metabolite responsible for murine spleen cell and human
T cell immunotoxicity due to BaP is likely BPDE.
The role of metabolism for DMBA bone marrow toxicity in mice has been established through the use of
CYP1B1 null (knock-out) mice. DMBA-induced preB cell toxicity was nearly totally ablated in mice that
did not express CYP1B1 (6).
Genotoxic Mechanisms of PAH Metabolite-Induced
Immunotoxicity
The mechanisms whereby PAHs produce immunotoxicity should be divided into two general categories:
genotoxic and non-genotoxic (Figure 1). Many PAHs
are bioactivated to reactive metabolites that bind to
DNA and exert genotoxicity. These compounds can
be identified in an Ames assay. The mechanism
whereby genotoxic chemicals produce immunotoxicity is likely p53-dependent. It is beyond the scope
of this essay to discuss mechanisms of p53 signaling,
but it is known that p53 plays a critical role in cellcycle regulation and the induction of apoptosis. Bulky
PAH-DNA adducts induce p53, which is turn inhibit
cell cycling and induce apoptosis in many cells.
DMBA has been found to produce immunotoxicity
in bone marrow via the induction of apoptosis, so it
is not surprising that some recent studies showed p53
knock-out mice are resistant to the bone-marrow suppressive effect of DMBA (9). It will be interesting to
examine the p53-dependence for immunotoxicity produced by other PAHs, because it has been shown that
DMBA induces apoptosis in several peripheral lymphoid organs (10). It is also known that many genotoxic chemicals that induce p53 are immunosuppressive. Therefore, p53 is probably an important pathway
for immunotoxicity of numerous agents. It should be
noted that in some studies good agreement has been
found between the functional immunotoxicity observed and change in immunophenotypic markers,
while in other studies there has been a lack of agreement. These differences probably relate to the amount
of apoptosis and cell death that was produced by
PAHs, as it is unlikely that functional changes would
be detected simply by immunophenotypic analysis of
spleen cells. Thus, immunophenotyping is not likely a
stand-alone sensitive marker for immunotoxicity produced by many xenobiotics at non-cytotoxic concentrations.
P
518
Polycyclic Aromatic Hydrocarbons (PAHs) and the Immune System
Non-Genotoxic Mechanisms of PAH Immunotoxicity
Several studies have shown that PAHs can activate or
interfere with lymphocyte signaling pathways in both
murine and human B and T cells. Many xenobiotics
produce biphasic effects on immune responses with
low concentrations stimulating responses and high
doses producing inhibition. Agents that mimic or
alter signaling pathways may manifest these characteristics.
PAHs have been found to activate protein tyrosine kinase activity in lymphocytes leading to Ca2+-dependent signaling in B and T cells in mice and humans.
Two reviews have appeared on this topic (4,5). DMBA
has been found to directly activate human T cells;
however BaP metabolites are implicated in the activation of protein tyrosine kinases in human B and
T cells. The consequences of altered B and T cell signaling may be a persistent suppression of immune response resulting from a tolerance-like mechanism on
lymphocytes. One of the consequences of T helper cell
tolerance would be the lack of production of interleukin-2, which is a key cytokine for both humoral and
cell-mediated immunity. It has been found that DMBA
does prevent the formation of IL-2 and that IL-2 can
partially overcome immunosuppression.
Relevance to Humans
Humans are exposed to complex mixtures of PAHs via
the air breathed (including significant quantities that
are present in wood smoke, cigarette smoke, and various emissions) and via the diet. There is also occasional dermal exposure, usually associated with occupational exposures to tars, soots, and vapors. PAH
exposures can be monitored using major urinary metabolites. Although there have been some reports,
there is little epidemiologic evidence that PAHs produce immunosuppression under conditions of environmental or industrial exposure in humans. The concentrations of PAHs that are required to suppress humoraland cell-mediated immunity in mice is extremely high,
typically in the range of 10–50 mg/kg of BaP. These
high concentrations have also been found to suppress
humoral immunity in fish. In general, the concentrations of PAHs that are required to produce immunotoxicity are higher than those required to produce cancer. Nevertheless, it is clear that human white blood
cells, and lymphocytes in particular, are sensitive to
immunosuppression produced by BaP exposures in
vitro. Cellular signaling pathways are also affected in
human peripheral blood leukocytes (HPBL). In fact, in
vitro data suggests that HPBL may be more sensitive
than murine spleen cells in the in vitro suppression of
T cell mitogenesis produced by BaP (8). Given that
there are several genetic polymorphisms that may affect a person’s susceptibility to PAHs, further research
is necessary to determine who might be at risk for
PAH immunosuppression.
References
1. Primiano T, Sutter TR, Kensler TW (1997) Antioxidantinducible genes. Adv Pharmacol 38:293–328
2. Zhu H, Li Y, Trush MA (1995) Characterization of
benzo[a]pyrene quinone-induced toxicity to primary
cultured bone marrow stromal cells from DBA/2 mice:
potential role of mitochondrial dysfunction. Toxicol
Appl Pharmacol 130:108–120
3. Reed M, Monske M, Lauer F, Meserole S, Born J,
Burchiel S (2003) Benzo[a]pyrene diones are produced
by photochemical and enzymatic oxidation and induce
concentration-dependent decreases in the proliferative
state of human pulmonary epithelial cells. J Toxicol
Environ Health 66:1189–1205
4. Davila DR, Davis DP, Campbell K, Cambier JC,
Zigmond LA, Burchiel SW (1995) Role of alterations
in Ca2+-associated signaling pathways in the immunotoxicity of polycyclic aromatic hydrocarbons. J Toxicol
Environ Health 45:101–126
5. Burchiel SW, Luster MI (2001) Signaling by environmental polycyclic aromatic hydrocarbons in human
lymphocytes. Clin Immunol 98:2–10
6. Heidel SM, MacWilliams PS, Baird WM et al. (2000)
Cytochrome P4501B1 mediates induction of bone
marrow cytotoxicity and preleukemia cells in mice
treated with 7,12-dimethylbenz[a]anthracene. Cancer
Res 60:3454–3460
7. Thurmond LM, Lauer LD, House RV, Cook JC, Dean JH
(1987) Immunosuppression following exposure to 7,12dimethylbenz[a]anthracene (DMBA) in Ah-responsive
and Ah-nonresponsive mice. Toxicol Appl Pharmacol
91:450–460
8. Davila DR, Romero DL, Burchiel SW (1996) Human T
cells are highly sensitive to suppression of mitogenesis
by polycyclic aromatic hydrocarbons and this effect is
differentially reversed by alpha-naphthoflavone. Toxicol
Appl Pharmacol 139:333–341
9. Page TJ, O'Brien S, Holston K, MacWilliams PS,
Jefcoate CR, Czuprynski CJ (2003) 7,12-Dimethylbenz
[a]anthracene-induced bone marrow toxicity is p53dependent. Toxicol Sci 74:85–92
10. Burchiel SW, Davis DA, Ray SD, Archuleta MM,
Thilsted JP, Corcoran GB (1992) DMBA-induced cytotoxicity in lymphoid and nonlymphoid organs of
B6C3F1 mice: relation of cell death to target cell
intracellular calcium and DNA damage. Toxicol Appl
Pharmacol 113:126–132
Polymerase Chain Reaction (PCR)
John L Olsen
Stony Brook University Medical School
327 Sheep Pasture Road
Setauket, NY 11733
USA
Synonyms
PCR, competitive PCR, real-time PCR, real-time and
quantitative PCR, RTqPCR, real-time reverse transcription PCR, semiquantitative PCR
Short Description
The purpose of the polymerase chain reaction (PCR) is
to detect and characterize genes. PCR is an in vitro
method for amplifying a DNA sequence using a heatstable polymerase and two primers, one complementary to the (+) strand at one end of the sequence to be
amplified and the other complementary to the (−)
strand at the other end. The newly synthesized DNA
strands then serve as templates for the same primers
and successive rounds of primer annealing, strand
elongation and dissociation produce a highly specific
amplification of the sequence. PCR can be used in
environmental monitoring assays to detect the existence or absence of a DNA sequence in a sample—
for example, a gene specific for an infectious viral
particle or bacterium. In semiquantitative PCR, an
analysis of the intensity of the stained PCR end products separated by gel electrophoresis can give a rankorder assessment of the original copy amount of the
gene among several samples. Real-time and quantitative PCR (RTqPCR) is a technique useful to determine
cellular mRNA expression changes for genes regulated by toxic compounds, drugs, infectious agents,
or biological processes. RTqPCR can also be used to
directly detect and quantify the number of DNA gene
copies in a sample, useful when monitoring bacterial
load.
Characteristics
PCR kinetics was introduced in 1985 by Kary Mullis
and takes advantage of DNA polymerase to amplify a
specific fraction of the genome (1). In conventional
PCR, nucleic acid is first extracted from cells or a
biological matrix. Several commercial kits and purification columns are available to extract DNA or mRNA
(Qbiogene; Promega; Qiagen). After nucleic acid is
purified, its purity and quantity are measured using a
spectrophotometer. If one is conducting a gene expression experiment and mRNA regulation is the target of
study, reverse transcriptase is used to generate complementary DNA (cDNA) from mRNA. For eukaryot-
ic cells, random hexamers, oligo d(T)s, or sequencespecific reverse primers may be used to generate
cDNA during reverse transcription. cDNA can be
used in the PCR reaction in a similar fashion to DNA.
A requirement for amplifying a gene sequence is to
know the nucleotides flanking the segment of DNA so
that specific oligonucleotide primers can be designed
and synthesized. A well designed primer should consist of about 18 to 30 bases and hybridize to its short
sequence with insignificant hybridization to other sequences in the reaction tube. Two primers are included
in the reaction tube, one for each of the complementary DNA strands. The complete target sequence spanning the region between the two primers is generally
less than 600 base pairs.
PCR amplification of a DNA or cDNA sequence is
carried out in a reaction tube with an optimized salt
buffer. It is a three-step process, referred to as a cycle,
that is repeated 30 to 40 times in an automated thermal
cycler. The first step is denaturation of doublestranded DNA to single strands by heating the sample,
usually to greater than 90 °C. The second step is annealing of the two primers to their targets on singlestranded DNA, achieved between 40 °C and 65 °C.
Selecting the most favorable annealing temperature is
dependent on the length and base sequence of the
primers. The third step of the cycle is extension.
Once the primers are annealed, the temperature is elevated to approximately 72 °C and the enzyme
Taq DNA polymerase catalyses primer extension (beginning at the 3' end of the primer) with complementary nucleotides (dNTPs) in the 5’ to 3’ direction.
Taq DNA polymerase is a recombinant heat-stable
DNA polymerase derived from Thermus aquaticus.
The thermal cycler carries out the reaction through
30 or more cycles of denaturing, annealing, and extension. In conventional PCR, an endpoint analysis is
done by looking at the fluorescently stained PCR products separated by gel electrophoresis.
Fluorescence-based real-time and quantitative PCR
was developed in the mid 1990s to better confirm,
characterize, and quantify nucleic acids in different
sample populations. The genetic material to be analyzed can be either DNA or cDNA produced following
reverse transcription of a sample’s mRNA. An oligonucleotide probe ( TaqMan; Roche, Mannheim, Germany) containing a reporter fluorescent dye on the
5’ end and a quencher dye on the 3’ end is synthesized. This probe is designed to hybridize to the gene
of interest in a central region of the amplicon. In an
intact probe the quencher produces Förster resonance
energy transfer (FRET), which lessens the reporter
dye’s fluorescence. However, during the PCR reaction,
when the polymerase replicates a template on which
the Taqman probe is bound, the 5´ nuclease activity of
the DNA polymerase cleaves the probe, separating the
3
Polymerase Chain Reaction (PCR)
519
P
520
Polymerase Chain Reaction (PCR)
reporter and quencher dyes to bring about increased
fluorescence of the reporter. RTqPCR detects accumulating levels of DNA indirectly by monitoring the increase in fluorescence of the probe (see Figure 1).
Software calculates the number of cycles necessary
to detect a signal, generally in the exponential phase
of the PCR reaction, and can determine the starting
level of the gene. The sample starting amount can be
interpolated either by comparison with a serially diluted standard curve where the target gene copy number
(in plasmid or gene fragment form) per well is known
(absolute quantitation) or by comparison with a housekeeping gene, such as β-actin, amplified from the
same sample (fold-change or ddCt quantitation). Careful consideration should be given when choosing internal reference housekeeping genes for data normalization, as a popular gene used for this purpose,
GAPDH, plays a role in several cellular functions
and its mRNA levels do not consistently remain constant.
Regularly used alternatives to the Taqman method directly measure DNA using a fluorescent DNA
( SYBR green) that hybridizes nonspecifically to
double-stranded DNA or by using probes—known as
molecular beacons and scorpions—that specifically
hybridize with the gene of interest. The increase in
fluorescence is measured real-time and analyzed similarly to the Taqman-based method described above.
When working with tissue samples, RNA template can
be derived from homogenized biopsy material or,
when it is preferable to study the RNA and/or protein
3
Polymerase Chain Reaction (PCR). Figure 1 In the
PCR reaction the polymerase replicates a template on
which the Taqman probe is bound. The 5´ nuclease
activity of the DNA polymerase cleaves the probe,
separating the reporter and quencher dyes to bring
about increased fluorescence of the reporter. RTqPCR
detects accumulating levels of DNA indirectly by
monitoring the increase in fluorescence of the probe.
of a specific cell type, cells are purified by flow cytometry or laser capture microdissection (LCM) of
paraffin-embedded tissue sections mounted on a
glass slide. RNA expression has been quantified
even after immunohistochemical staining of tissue sections using LCM, opening up the possibility of studying any archived tissue specimen. This combination of
LCM and RTqPCR is particularly useful for studying
the genetic regulation of tissue infiltration by tumor
cells.
Pros and Cons
RTqPCR has several advantages over other methods
of RNA quantitation. The assays are highly specific
and sensitive to genetic regulation, and are easier and
cheaper to perform than traditional hybridization techniques, such as northern blotting. The PCR reaction is
carried out in a 96-well plate, making it a highthroughput method to test numerous conditions in
the same experiment. The starting amount of RNA
can be very small (nanograms) and single cells or
tens of cells can be studied. SYBR green has the advantage of being applicable to any PCR product, doing
away with the need to design specific probes for each
gene target, but at the same time increasing the possibility of falsely positive amplification. The RT and
PCR reactions may be done in the same tube (multiplex) but because of competition for reagents in the
well, this protocol is less sensitive than the two-tube
method and the results of these experiments should not
be directly compared to two-tube protocols. Careful
consideration should be given when choosing an
RNA purification method, as different manufacturers’
kits may result in RNA preparations that yield dissimilar amounts of cDNA template. Probes and primers
made by different companies, or different lots from the
same manufacturer may have different chemistries,
requiring end-users to verify these reagents with
cDNA standards.
Some other factors should be considered when performing RTqPCR. Dispensing the low-volume solutions can become tedious, and the cost of robotic preparation is out of reach for most laboratories. Also,
though most protocols call for DNase treatment of
RNA preparations, many researchers hesitate to use
DNase because of the possible residual RNases that
might degrade their sample. In order to protect against
contamination, significant laboratory space and dedicated equipment is necessary to physically separate the
different stages of the PCR process. The potential presence of inhibitors of PCR in the matrix of biological
samples is another limitation.
PCR detection methods for pathogens offer the capacity of replacing immunoassays (e.g. ELISA) and cell
culture. Viral samples can be characterized immediately without the need for culturing. PCR can detect
Polymerase Chain Reaction (PCR)
3
3
Predictivity
One of the major advantages of RTqPCR is its ability
to detect very rare events (one copy of a specific
gene/107 cells). This high sensitivity makes the assay
vulnerable to false-positives caused by DNA or RNA
contamination. Also, computer quantitation of gene
copy number can be sensitive to operator-dependent
effects. Results can be produced with insufficient
knowledge of the statistical analyses being performed
by the software that comes bundled with the RTqPCR
machines, and some authors are not forthcoming in
describing their entire statistical analysis and normalization protocols. To make results meaningfully predictive of human health outcomes, investigators
should ensure validation of reagents and RNA samples
and move towards standardized and universal methods
and reagents.
Relevance to Humans
The RTqPCR assay has become the gold standard and
most commonly used mRNA quantification method
for monitoring transcription in vitro and for observing
the effects of receptor signaling.The majority of the
RTqPCR immunotoxicology experiments up to this
point have been performed either using animal models
or using human or animal cell lines. The goals of these
and human in vivo experiments are to define biomarkers (any plausible and measurable biological endpoint relevant to the toxicity of a compound), or
surrogates (statistically validated endpoints with
medical importance) of clinical illness, and to develop
therapies for these processes. For example, Spink and
coworkers used RTqPCR to investigate the effects of
estrogen on aryl hydrocarbon responsiveness in dioxin-treated human breast cancer cells (2). RTqPCR can
be used to validate in a quantitative manner the findings of microarrays, which measure genome-wide responses to toxic compounds but also generate overwhelming data sets that are cumbersome for rapid toxicology screens. Morgan and coworkers used cDNA
microarrays to explore the effects of a diverse group of
toxicants including ouabain and hydrogen peroxide in
a human hepatocellular carcinoma cell line (3). Their
array data found a subset of seven genes that can be
used in an RTqPCR platform to test compounds for
cellular oxidative stress.
Real-time quantitative PCR is applied extensively in
the study of cancer and the response of cancer cells to
drugs. It has been used to monitor the toxicology and
dissemination of adenoviral vectors synthesized to
treat cancers. It can also effectively detect small numbers of blood-borne tumor cells and lymphangiogenesis, but is in general still in the developmental stage
of accurate cancer diagnosis. PCR-based assays can
identify specific chromosome aberrations, such as
have been shown to be associated with benzene exposure. Biernaux et al. used nested RT-PCR to detect the
BCR-ABL fusion in RNA from the t(9;22) (q34; q11)
translocation (4). Appropriately designed primer pairs
and probes can preferentially amplify mutant genomic
DNA and single nucleotide mutations even in the presence of a 1000-fold excess of wild-type DNA.
Several important factors should be taken into account
when designing and interpreting PCR-based toxicology experiments. First, because of its high sensitivity,
RTqPCR can detect small changes in genetic regulation. However, RTqPCR does not give information
about RNA stability, protein production, or biological
activity of proteins, so that the presence of transcriptional changes should be verified by protein analysis.
Second, given the variability of expression of genes in
3
pathogens that cannot be cultured in the lab. When
considering the analysis of viruses, RTqPCR cannot
determine directly viral infectivity and may not detect
the new nucleotide sequences of emerging viruses.
Useful technologies to enrich for viruses in sample
material include using host cell receptors or monoclonal antibodies to capture virus particles for further genetic analysis. Microbiologists have also developed
PCR-based DNA fingerprinting methods to distinguish between various sources of fecal bacteria.
Allele-specific discrimination is accomplished by
using two different Taqman probes, each with a unique
fluorescent dye attached. These assays help to genotype individuals and have been used to analyze gene
polymorphisms that contribute to the susceptibility to
toxic insult, including cytochromes and glutathione Stransferases. Genetic variants of cytokine receptors
have also been characterized in human populations
using RTqPCR allelic discrimination technology.
Most assays discriminate between two alleles of single
nucleotide polymorphisms (SNPs). An initial mutation
screening study to identify different variants of a gene
is performed, using direct sequencing, PCR-single
strand conformation polymorphism (PCR-SSCP), or
denaturing high performance liquid chromatography
(D-HPLC). Once polymorphisms have been identified,
allele-specific probes are designed for both alleles A
and B (assuming a bi-allelic system for the sake of
illustration, though the method is capable of discriminating an unlimited number of alleles). Binding of the
probe for allele A to allele B DNA is suboptimal because of the mismatch within the Taqman probe and
the target sequence. For a bi-allelic system, an unknown sample of DNA will be identified as having
present allele A, B, or both A and B. These genotypes
(AA, AB, and BB) are designated based on real-time
PCR growth curves and can be validated by direct
DNA sequencing of PCR products separated by agarose gel electrophoresis.
521
P
Polymeric Immunoglobulin Receptor (pIgR)
normal subjects ('natural variation') and the presence
of highly polymorphic genes such as CYP2D6, which
has at least 70 allelic variants with a range of biological activities and a high degree of interethnic variability ('population-specific variation'), it is advised to
have large sample sizes and background information
about the normal allelic variation of the gene under
consideration. Third, the extent of tissue sampling
within an organ should be sufficient to accommodate
for any local differences in genetic distribution. One
lung lobe may have a different blood perfusion and
airflow than another, so care should be taken to consistently harvest similar biological material.
Regulatory Environment
While PCR detection methods clearly play an important role in hazard identification, risk assessment and
contaminant regulation, a major problem associated
with its use in the regulatory context is how to properly interpret positive and negative results. Regulatory
agencies decide whether detection-only of pathogens
can be used for risk assessment or if that data must be
coupled with evidence of infectivity. While positive
findings of a genetic mutation in a tissue sample indicate the presence of that altered gene, the medical
implication is not always clear. In Biernaux’s study,
the BCR-ABL translocation was found in 40% of normal healthy subjects, complicating the health risk assessment (5). Negative results need to be interpreted in
light of the manner of sample collection, processing
and storage methods that might degrade genetic material, and the detection limit of the assay.
Lack of standardization of PCR methods is a major
concern for developing health-impact assessments.
Quality assurance and quality control measures, sample preparation protocols, and internal controls need to
be tested in many laboratories and standardized in
order to move beyond the developmental stage.
Many PCR primers are constructed by different laboratories and this may confound pathogen occurrence
monitoring. PCR methods are rapidly changing, so a
performance-based approach with benchmark values
is needed to know if the data being generated is helping to address specific health problems.
As gene array-based approaches remain semiquantitative, RTqPCR assays are still the most common method to quantitate gene expression patterns in different
populations. Microarrays produce a comprehensive
picture of the genomic-wide response to toxic compounds. Microarray techniques are most appropriate
for individual compound studies rather than for
screening of multiple chemicals. Real-time PCR is
designed to monitor single endpoints or small sets of
endpoints among several samples. RTqPCR is therefore the method of choice to verify and quantify microarray results or resolve questions when discrepan-
cies appear in direction of gene change on microarray
plates produced by different companies. Microarray
and RTqPCR methodologies are used together to develop molecular signatures for classes of toxicants
with distinct actions on specific sets of genes. These
molecular signatures are valuable when predicting the
toxicity of other compounds that have incomplete toxicological information.
Until the advent of RTqPCR and cDNA microarrays,
molecular toxicology studies relied almost exclusively
on rodent models and in vitro human systems to identify cellular changes that may be important in the
human population. Those toxicological techniques required complicated methodologies to deal with dose
extrapolation, interspecies extrapolations and interindividual differences. With the integration of RTqPCR
into immunotoxicology risk assessment, epidemiological biomarkers can now be accurately quantified in
order to resolve some of the uncertainties involved
with previous approaches.
References
1. Mullis KB (1990) The unusual origin of the polymerase
chain reaction. Sci Am 262:56–61
2. Spink DC, Katz BH, Hussain MM, Pentecost BT, Cao Z,
Spink BC (2003) Estrogen regulates Ah responsiveness
in MCF-7 breast cancer cells. Carcinogenesis 24:1941–
1950
3. Morgan KT, Ni H, Brown HR, Yoon L et al. (2002)
Application of cDNA microarray technology to in vitro
toxicology and the selection of genes for a real-time RTPCR-based screen for oxidative stress in Hep-G2 cells.
Toxicol Pathol 30:435–451
4. Biernaux C, Loos M, Sels A, Huez G, Stryckmans P
(1995) Detection of major bcr-abl gene expression at a
very low level in blood cells of some healthy individuals.
Blood 86:3118–3122
Polymeric Immunoglobulin Receptor
(pIgR)
Polymeric immunoglobulin receptor (pIgR) is a specialized receptor that binds IgA and translocates it
across epithelium into secretions.
Mucosa-Associated Lymphoid Tissue
3
522
Polymorphism
Polymorphism is a term often used to represent any
variation of a character or gene within a population. A
more precise definition is the existence of a character
in two or more variant forms in a population, where
the least common form is present in more than 1% of
Popliteal Lymph Node Assay
individuals. Consequently, a polymorphic character is
neither very rare nor very common.
AB0 Blood Group System
3
Polymorphonuclear Leukocyte
This is a collective term for granulocytes (basophils,
eosinophils, neutrophils) derived from the bone marrow and characterized by a multilobed nucleus. These
phagocytic cells migrate from the circulation to the site
of inflammation in response to chemotactic signals.
Streptococcus Infection and Immunity
Lymphocytes
523
Briefly, compounds are injected subcutaneously into
the hind footpad of either mice or rats and 6–8 days
later the draining popliteal lymph node (PLN) is excised and compared with popliteal lymph nodes from
vehicle-treated animals. Comparison is basically made
between the weight or cellularity of the nodes; the
ratio of these parameters from compound-treated
over vehicle-treated PLNs (designated the PLN
index) indicates whether or not the compound is immunostimulatory. Immunostimulation is regarded as
one of the possible prerequisites for pharmaceuticals
and other compounds to activate and sensitize T cells
and to induce systemic allergy or autoimmune-like
derangements.
3
3
Characteristics
Polymorphonuclear Neutrophil
Neutrophil
3
Polynucleotide Vaccines
DNA Vaccines
3
Polyunsaturated Fatty Acids
These are fatty acids which contain more than one
double bond.
Fatty Acids and the Immune System
3
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
PLNA.
Short Description
The popliteal lymph node assay (PLNA) is in use to
test the immunostimulatory capacity of low molecular
weight compounds, in particular pharmaceuticals.
In the late 1970s and early 1890s, Gleichmann and
colleagues realized that graft-versus-host (GvH) reactions might be the basis for pathogenetic mechanisms
behind the development of drug-induced allergy and
autoimmunity (1). To substantiate this hypothesis they
studied effects of the hypersensitizing drug diphenylhydantoin in the PLNA, which was already in use to
study GvH reactions (2). Kammüller and colleagues
(3) further explored the PLNA as a method to screen
for the immunostimulatory potential of chemicals by
comparing series of structural homologues of drugs (e.
g. hydantoin, zimeldine) and by studying of the immunological changes in the draining PLN induced by
a variety of immunostimulatory chemicals (among
these also contact sensitizers). Originally conducted
in mice, Descotes and colleagues (4) used the rat as
species to perform the PLNA and they found that the
rat responds equally well to a series of chemicals
(Note: some rats may have more than one popliteal
lymph node per paw).
Usually, experimental groups contain 4–6 animals per
group. The contralateral PLN can be used as internal
control, but because in some cases these nodes also
show signs of immunostimulation separate control
groups are recommended.
The volume of the solution injected (with a 24 G needle) in the paw of mice is usually 50 μl in the case of
aqueous solutions (both saline and PBS have been
used) and 10 μl (alone or mixed with 40 μl saline)
in the case of DMSO (for hydrophobic compounds).
In the rat up to 100 μl can be used as injection volume.
Specific buffers are used in case a compound is less
soluble or not soluble at physiological pH.
The dose of the chemicals injected is mostly about
1 mg per mouse or up to 5 mg per rat, but for new
chemicals this should be carefully evaluated as the
dose may strongly vary and depends on the toxicity
of the chemical (HgCl2 for instance is used in a dose
of only 50 μg per animal). Injection is either from toe-
P
524
Popliteal Lymph Node Assay
to-heel or from heel-to-toe which does not seem to
make any difference.
The size of the lymph node (by weight, cell number of
even diameter) is the most frequently used parameter
for lymph node activation because it is easy to detect.
However, this is not a very informative parameter and
certainly does not distinguish sensitizers from nonsensitizing irritant chemicals. For this, immunologically more relevant parameters need to be included. Immunohistology, for instance, can be used to detect the
presence of germinal centres, which are known to be
T cell-dependent. Cytokine production by isolated
PLN cells after mitogen-specific or hapten carrier-specific restimulation, either combined or not combined
with flow cytometrical analyses of the cells, can also
be used to provide information on T cell-dependency
and also on the type of immune response that is elicited by the compound.
Pros and Cons
The primary PLNA is a straightforward, objective and
cheap assay that allows fast screening of the immunostimulatory potential of compounds. Concentrationeffect relationships of series of structural homologues,
for instance of a newly synthesized pharmaceutical
compound, can be easily tested in the PLNA, and by
using immunologically more sophisticated read-out
parameters the PLNA also offers a means to investigate initiating mechanisms of chemically induced adverse immune reactivity.
An important disadvantage of the unmodified simple
PLNA, with only the PLN size as parameter, is that
strong irritant chemicals are false positive in the
PLNA. This is probably due to the fact that these
chemicals induce too much local damage and consequently elicit a strong inflammatory response. However, mere irritants can be excluded as non-sensitizers by
using more sophisticated variations of the PLNA (secondary PLNA, adoptive transfer PLNA, RA-PLNA).
In particular, pro-haptens that require metabolic conversion (most well known in this respect is the antiarrhythmic drug procainamide) are false negative in
the PLNA. However, some of these false negative
compounds can be detected by including a metabolizing system (addition of S9 mix or oxidizing polymorphonuclear cells) in the injection solution. Compounds
that do not induce adverse immune reactions by immunostimulation, but for instance by interfering with
immunoregulatory mechanisms, may also turn out to
be false negative in the PLNA. Important to note is
also that different strains of mice respond to a different
degree to some chemicals, indicating genetic dependency of the PLNA response.
A major limitation of the PLNA with regard to risk
assessment is that the exposure route (subcutaneous
exposure) is not the usual route of exposure for
drugs. In addition, subcutaneous paw injection limits
the volume that can be injected, so some poorly soluble chemicals are not fully dissolved and need to be
injected as suspensions. This may cause aspecific activation of macrophages and elicitation of inflammatory responses, so it is generally regarded as undesirable, although on the other hand aspecific activation
by poorly soluble compounds may also occur in the
gastrointestinal tract in case of those chemicals.
In all, it has to be realized that essentially the PLNA is
a screening assay to assess a chemical's potential to
stimulate the immune system. When extended with
immunologically relevant parameters the PLNA can
also indicate whether a chemical has the potential to
sensitize the immune system, that is it may cause allergy or autoimmune-like phenomena in susceptible
individuals under certain predisposing conditions. As
a research tool the PLNA has without doubt contributed considerably to the fundamental knowledge about
drug-induced immunostimulation.
It is important to note that over the past 20–25 years
more than 130 chemicals (including drugs and homologues, as well as other chemicals) have been tested in
the PLNA (5), yet the assay has never been formally
validated. But available data of chemicals that have
been tested in various laboratories have been compared, good reproducibility and similar predictivity,
with only a few false-negative or false-positive compounds.
Predictivity
When using the PLNA as a screening test one should
of course be aware of false-positive (e.g. strong irritants) and false-negative (e.g. pro-haptens) compounds. A false-negative compound that is indeed a
pro-hapten can be dealt with by using a metabolic
system as mentioned above. Strong irritants that induce severe inflammatory responses and necrosis at
the side of injection should not be tested, or should
be tested at a dose that has less aspecific effect. The
use of immunologically relevant parameters or modifications of the PLNA that allow detection of anamnestic immune responses may provide more information in the case of false-positive compounds and identify such chemicals.
When this is taken into account, the correlation between PLN enlargement and the ability of a chemical
to cause systemic autoimmune disorders is good to
very good, considering that of the 130 or so chemicals
tested only some 10 false-negative and false-positive
chemicals could not be explained (3,5).
Notably, also known allergens (e.g. contact or respiratory allergens) are positive in the PLNA. This stresses
that the PLNA may indicate the immunosensitizing
potential of compounds, but it does not provide information about the kind of adverse immune effect that
Popliteal Lymph Node Assay, Secondary Reaction
can be expected in humans. For that far more information is needed on the vast array of predisposing, immunoregulatory factors, as well as environmental factors, that influence the development of a sensitized
immune system to actual clinical symptoms.
525
Popliteal Lymph Node Assay,
Secondary Reaction
Peter Griem
Regulatory Environment
The PLNA has been successfully used as a screening
test for drug-induced adverse immune effects for over
two decades, but the test has never been formally validated. Because of its many advantages as a simple
screening assay and because of its added value to other
local lymph node tests such as the LLNA it is considered important to validate the PLNA. Attempts to do
this are ongoing in the framework of the International
Life Sciences Institute-Health and Enviromental
Sciences Institute (ILSI-HESI) Immunotoxicology
Technical Commitee (ITC).
References
1. Gleichmann E, Pal ST, Rolink AG, Radasziewicz T,
Gleichmann H (1984) Graft versus host reactions: clues
to the etiopathology of a spectrum of immunological
diseases. Immunol Today 5:324–332
2. Gleichmann H (1981) Studies on the mechanism of drug
sensitization: T-cell-dependent popliteal lymph node
reaction to diphenylhydantoin. Clin Immunol Immunopathol. 18:203–211
3. Kammüller ME, Thomas C, De Bakker JM, Bloksma N,
Seinen W (1989) The popliteal lymph node assay in mice
to screen for the immune dysregulating potential of
chemicals-a preliminary study. Int J Immunopharmacol
11:293–300
4. Verdier F, Virat M, Descotes J (1990) Applicability of the
popliteal lymph node assay in the Brown Norway rat.
Immunopharmacol Immunotoxicol 12:669–677
5. Pieters R, Albers R (1999) Screening tests for autoimmune-related immunotoxicity. Env Health Persp
107:673–677
Synonyms
PLNA, secondary PLNA, adoptive transfer PLNA
Short Description
The secondary PLNA (reviewed in (1–3)) aims at determining a secondary T lymphocyte response to a
low-molecular-weight chemical by injecting the chemical subcutaneously into one hind footpad of already
sensitized in mice or rats. The administered dose has
to be small enough not to elicit a primary immune
response in nonsensitized animals, i.e. it should not
suffice for stimulation of naïve T lymphocytes. The
immune response against the injected chemical or antigen is quantified by analyzing one or more parameters of the draining popliteal lymph node (PLN),
such as PLN weight, PLN cell number, cell surface
marker expression on PLN cells or PLN cell proliferation measured, for example, as 3H-methyl thymidine
incorporation.
Characteristics
In the direct PLNA, a single injection of the test chemical is administered subcutaneously into one hind footpad. The contralateral footpad serves as an internal
control and is usually injected with the vehicle (test
solution without test chemical) or left untreated. After
injection the test chemical is transported via the afferent lymphatics to the nearest draining lymph node,
the PLN, where a primary immune response may take
place. After 5–8 days, the left and right PLN are isolated and analyzed. PLN weight and/or other PLN
parameters like cell count, cell proliferation and expression of cell surface markers are determined. Results are usually expressed as a PLN index which is
the ratio of values obtained from the experimental and
control sides. A primary PLN response normally peaks
between days 4 and 10 and than returns to preinjection
level within 3–5 weeks, unless a persistent material,
such as silica particles, is injected.
While the primary PLNA is capable of distinguishing
immunostimulating chemicals from immunologically
inactive chemicals, this assay fails to distinguish haptens or neoantigen-forming chemicals from mere inflammatory irritants, unless more sophisticated ana3
The PLNA should be regarded as a test to screen for
the possible hazard of a compound to cause immunosensitization. As such, the assay can be regarded as a
first step to evaluate whether the compound has also
the potential to stimulate and sensitize the immune
system via the relevant (mostly oral) route of exposure.Unfortunately, models to assess the immunosensitizing potential of chemicals, in particular drugs, via
the okal route are lacking. Conceivably, for the time
being, assessment of the immunogenic potential of a
chemical may be assessed by combining limited animal data (e.g. PLNA data) with information about
dose-response relationships and predisposing conditions.
Toxikology/Regulatory Affairs
Clariant GmbH
Am Unisys-Park 1
D–65843 Sulzbach
Germany
3
Relevance to Humans
P
Popliteal Lymph Node Assay, Secondary Reaction
lyses are performed, such as determination of cytokine
production in PLN cells and/or expression of activation markers on the cell surface. If, however, a chemical elicits a specific T cell response part of the activated T lymphocytes (also called primed T cells) will
differentiate into memory T cells. The latter can be
detected by their capacity to mount a secondary immune response, which is characterized by faster kinetics and lower elicitation doses than those required
for a primary response (i.e. T cell priming during sensitization). In order to test for a secondary response, a
secondary PLNA can be performed in one of two
ways (1–3).
3
Secondary PLNA in sensitized animals
Sensitization of naive animals (usually mice) can be
accomplished by treating the animals as for the direct
PLNA. Upon complete resolution of the primary PLN
response (which is shown by satellite groups analyzed
at different time intervals after injection) the animals
are challenged in the same paw with a lower dose of
the same chemical, which is substimulatory in the direct PLNA. After 4–6 days assessment of the specific
secondary response is determined as PLN index by
measuring the same parameters as in the direct PLNA.
Results are expressed as PLN indices (mean and standard deviation of groups of at least five animals) and
statistical tests, such as Student’s t test or anova, are
used to determine significant differences between
treatment and control groups.
Alternatively, the animals can be sensitized not via the
hind footpad, but via a different route that is more
relevant for human exposure, e.g. intranasally, orally,
intramuscular or intravenously (see Table 1 for examples). The treatment during this sensitization phase can
be extended to the time period desired. The animals
are then challenged by injection of the test chemical
into the hind footpad and the PLN response is evaluated as above.
3
Adoptive Transfer PLNA
This test measures secondary T lymphocyte responses
from a chemically-treated donor animal following
their transfer to a syngeneic (genetically identical) recipient animal. The T cell donor animals can be treated
with the test chemical under conditions of exposure as
to dose, route, frequency, and duration that mimic the
human situation (see Table 1 for examples). After the
treatment period, spleen cells, total splenic
T lymphocytes, or a selected T cell subpopulation of
the donor animals are transferred by subcutaneous injection into one hind footpad of recipient animals,
using T cells from unexposed or vehicle-treated animals as a negative control. One day after the transfer,
recipient animals are challenged at the same site by
subcutaneous injection of the test chemical at a sub-
stimulatory dose. The secondary PLN response is assessed 3–6 days after the challenge using the same
read-out parameters as described above.
A positive response in the secondary PLNA indicates
that exposure of the (donor) animals had resulted in Tcell sensitization and subsequent generation of memory T cells. The adoptive transfer PLNA provides direct evidence for the T cell dependence of the response
to the test chemical. Table 1 provides a few examples
of secondary and adoptive transfer PLNAs published
in the literature.
Pros and Cons
The secondary PLNA allows determination in vivo of
secondary T cell responses which sometimes cannot be
measured in vitro, e.g. in the lymphocyte proliferation
assay. The secondary PLNA could be performed on
satellite groups of standard subacute and subchronic
toxicity studies to identify a possible sensitization hazard of the test chemical.
Chemicals that cause hypersensitivity or autoimmunity through activation of specific T cells can be distinguished from chemicals that cause ‘unspecific’ or
‘polyclonal’ immunostimulation, such as hexachlorobenzene.
An interesting aspect of the secondary PLNA is that
for challenge not only can the chemical used for sensitization be employed, but also a metabolite of the
chemical that is presumed to be the ultimate hapten
of the chemical. This metabolite may be a pure,
synthesized chemical, it may be contained in a metabolite-generating enzyme solution or cell homogenate,
or it may be contained in a homogenate of cells isolated from animals after nontreatment. In this way, the
secondary PLNA can be used to demonstrate sensitization against a reactive intermediate metabolite of a
chemical (see Table 1 for examples).
Performing the PLNA requires some technical skill
and expertise. Especially the adoptive transfer PLNA
is rather laborious and requires a comparatively large
number of animals. Using a sensitization route that
mimics human exposure may require repeated treatment of animals over weeks or months.
3
526
Predictivity
The predictivity of the secondary PLNA has not been
formally evaluated. It has often been employed to investigate mechanisms of action of chemicals known to
cause hypersensitivity or autoimmunity in humans. In
general, involvement of chemical- or metabolite-specific T lymphocytes could be shown in cases where
the direct PLNA or other sensitization tests gave falsenegative results.
Relevance to Humans
While the secondary PLNA indicates the presence of
Popliteal Lymph Node Assay, Secondary Reaction
527
Popliteal Lymph Node Assay, Secondary Reaction. Table 1 Examples of chemicals studied in secondary
popliteal lymph node assays
Chemical
[reference]
Use, adverse effects
in humans, immunogenicity
Treatment of
animals during
sensitization
phase
Secondary PLNA
method and test
chemical used
Outcome of PLNA
p-BenzoquiChemical (e.g. in photonone (BQ) [5] graphic developers) causing allergic contact dermatitis
Positive in direct PLNA
C57BL/6 mice
Single subcutaneous injection of
100 nmol BQ into
hind footpad in a
direct PLNA
Secondary PLNA performed in sensitized
animals after primary
PLN reaction had subsided completely
(13 weeks)
Test chemicals were
0.1 nmol BQ or
0.1 nmol benzene
No reaction occurred in
mice that had received
only solvent during
priming; a specific
secondary reaction occurred in BQ-primed
mice to BQ and to
benzene, indicating
that enough benzene
was metabolized locally to BQ to elicit a
secondary T cell
reaction
Gold(I) disodi- Antirheumatoid drug
um thiomalate Induces dermatitis, hyper(Au(I)TM) [6] gammaglobulinemia, and/
or immune glomerulonephritis in high percentage
of patients after prolonged
treatment
Negative in direct PLNA
C57BL/6 mice
Weekly intramuscular injections of
22.5 mg/kg Au(I)
TM for 6–12 weeks
Adoptive transfer of
splenic T cells (MACS
removal of B cells)
Test chemicals were
Au(I)TM, gold(III) tetrachloride, homogenated
peritoneal phagocytes
from Au(I)TM-treated
mice
Homogenated peritoneal phagocytes incubated with Au(I)TM in
vitro
Transferred T cells
from Au(I)TM-treated
mice showed positive
response to gold(III),
not to Au(I)TM
They also reacted to
phagocytes from Au(I)
TM-treated mice and to
phagocytes incubated
with Au(I)TM in vitro,
indicating that T cells
were sensitized to gold
(III), a reactive metabolite of gold(I) formed
in phagocytes
Mercuric chlo- Potential to induce autoimmunity in humans unride (HgCl2)
[7]
clear
Induces antinuclear and
antinucleolar autoantibodies in susceptible mouse
and rat strains
Positive in direct PLNA
B10.S mice
Subcutaneous injections of 0.5 mg/
kg HgCl2 three
times a week for 1
or 8 weeks
Adoptive transfer of
splenic T cells (MACS
removal of B cells)
Test chemicals were
HgCl2, nuclei from
spleen cells of HgCl2treated or buffer-treated animals, and isolated nuclear protein
fibrillarin, either preincubated with HgCl2 or
buffer
Transferred T cells
from mice treated with
HgCl2 for 1 or 8 weeks
showed positive responses to HgCl2 and
also to nuclei of HgCl2treated mice and to
HgCl2-treated fibrillarin
In addition, after 8
weeks of HgCl2 treatment, T cells showed
responses to untreated
nuclei and fibrillarin,
indicating a shift from
mercury-specific to autoimmune T cells over
time
T cell sensitization against the test chemical, it does
not indicate whether this sensitization would cause
(clinically relevant) disease. For linking of chemical
exposure to induction of disease and for establishing
dose-response relationships more sophisticated animal
models are required. For some autoimmunogenic chemicals, e.g. mercuric chloride and gold(I) salts, prolonged treatment with the test chemical allowed detec-
P
528
Popliteal Lymph Node Assay, Secondary Reaction
Popliteal Lymph Node Assay, Secondary Reaction. Table 1 Examples of chemicals studied in secondary
popliteal lymph node assays (Continued)
Chemical
[reference]
Use, adverse effects
in humans, immunogenicity
Procainamide Antiarrhythmic drug
(PA) [8]
Induces lupus-like syndrome in high percentage
of patients after prolonged
treatment
Negative in direct PLNA
Treatment of
animals during
sensitization
phase
Secondary PLNA
method and test
chemical used
Outcome of PLNA
A/J strain (slow
acetylator) and
C57BL/6 (fast
acetylator) mice
Subcutaneous injections of 8 and
16 µmol PA, respectively, three
times a week for
16 weeks
Additional weekly
intraperitoneal injections of 600 ng
PMA in some
groups of C57BL/6
mice
Adoptive transfer of
splenic T cells (MACS
removal of B cells)
Test chemicals were
PA, N-hydroxy-PA, Nacetyl-PA, and homogenated peritoneal
phagocytes from PAtreated mice
Transferred T cells
from A/J mice showed
positive response to Nhydroxy-PA and to
phagocytes from PAtreated mice, but not to
PA or N-acetyl-PA
T cells from C57BL/6
mice treated with PA
+PMA, but not T cells
from mice treated only
with PA, reacted to Nhydroxy-PA and to
phagocytes from PA
+PMA-treated mice,
but not to PA, N-acetylPA or phagocytes from
mice treated only with
PA, indicating that T
cells were sensitized to
N-hydroxy-PA, a reactive metabolite of PA
While slow acetylator
mice formed enough
metabolite for sensitization, fast acetylator
mice were only sensitized after additional
stimulation of phagocytes
MACS, magnetic-activated cell sorting; PLNA, popliteal lymph node assay; PMA, phorbol myristate acetate.
tion of antinuclear autoantibodies and immune glomerulonephritis. Secondary PLNAs can help elucidating the mechanisms underlying allergic and autoimmune reactions to chemicals (especially drugs because
of the relatively high exposure dose).
Regulatory Environment
While validated skin sensitization tests are available,
there is currently no validated or widely applied standard toxicity test for the identification of compounds
with the potential to induce systemic allergic or autoimmune reactions. Validation of the direct PLNA is
currently considered (4). For regulatory purposes, results from secondary PLNA may currently only be
used as supplementary (mechanistic) information.
References
1. Pieters R, Ezendam J, Bleumink R, Bol M, Nierkens S
(2002) Predictive testing for autoimmunity. Toxicol Lett
127:83–91
2. Pieters R, Albers R (1999) Screening tests for autoimmune-related immunotoxicity. Environ Health Perspect
107 (Suppl 5):673–677
3. Goebel C, Griem P, Sachs B, Bloksma N, Gleichmann E
(1996) The popliteal lymph node assay in mice: screening
of drugs and other chemicals for immunotoxic hazard.
Inflamm Res 45 (Suppl 2):S85–90
4. Vial T, Carleer J, Legrain B, Verdier F, Descotes J (1997)
The popliteal lymph node assay: results of a preliminary
interlaboratory validation study. Toxicology 122:213–218
5. Ewens S, Wulferink M, Goebel C, Gleichmann E (1999)
T cell-dependent immune reactions to reactive benzene
metabolites in mice. Arch Toxicol 73:159–167
6. Goebel C, Kubicka-Muranyi M, Tonn T, Gonzalez J,
Gleichmann E (1995) Phagocytes render chemicals
Porcine Immune System
immunogenic: oxidation of gold(I) to the T cell-sensitizing gold(III) metabolite generated by mononuclear
phagocytes. Arch Toxicol 69:450–459
7. Kubicka-Muranyi M, Kremer J, Rottmann N et al. (1996)
Murine systemic autoimmune disease induced by mercuric chloride: T helper cells reacting to self proteins. Int
Arch Allergy Immunol 109:11–20
8. Kubicka-Muranyi M, Goebels R, Goebel C, Uetrecht J,
Gleichmann E (1993) T lymphocytes ignore procainamide, but respond to its reactive metabolites in peritoneal
cells: demonstration by the adoptive transfer popliteal
lymph node assay. Toxicol Appl Pharmacol 122:88–94
Popliteal Plymph Node Assay (PLNA)
Reporter Antigen Popliteal Lymph Node Assay
3
Population Studies
Epidemiological Investigations
3
Porcine Immune System
Ricki M Helm
Arkansas Children's Hospital Research Institute,
Arkansas Children's Nutrition Center
University of Arkansas for Medical Sciences
1120 Marshall Street
Little Rock, AR 72202
USA
Synonyms
swine, minipig, pig
Short Description
The physiological and immunological similarities of
swine and humans have become important features
in large-animal models for biomedical research as evidenced in both veterinary and human literature citations (1). Swine are suitable for studies of developmental immunology, xenotransplantation, wound healing, immunization schemes, allergy and human asthma. The physiological relevance of the swine as an
intended target species directly affects the laboratory
“proof of concept” that can be translated to successful
clinical treatments in the lieu of human trials. The
different strains of pigs and miniature pigs available,
crucial natural disease models that occur in outbred
populations, combined with the rapidly growing
swine immune reagent repertoire, will provide cost-
529
effective studies that will reveal the importance of
the swine as valid animal model systems that will
more closely extrapolate to the human situation
under investigation.
Characteristics
For comparative studies on immunoglobulins, Butler
and Howard (2) summarized the immunogloblulins
and Fc receptors from the Comparative Immunoglobulin Workshop. Interestingly, the sequences of Cμ,
Cα, Cγ , Cκ, Cλ and Vh of swine are most similar
to their human counterparts. Based upon flow cytometric analysis of T cells, γδ T cells, B cells, myeloid
cells, activation/maturation markers, and CD45-specific antibodies, the Third International Swine CD Workshop listed a total of 38 pig leukocyte CD/SWC determinants for pig leukocytes (3).
Data from CD3, CD4, CD8 immunophenotyping suggests the porcine αβ thymocytes require 15 days to
fully differentiate, while γδ thymocytes differentiate
in less than 3 days and migrate asynchronously from
the thymus to the periphery. A summary of the flow
cytometry and immunohistochemistry data, which
identified 38 monoclonal antibodies to cluster group
ligands, was presented in the second-round analysis of
the B-cell sections at the Third International Swine CD
Workshop (4).
Cloned pig cytokines include tumor necrosis factor
(TNF)- α, interleukins IL-1α, IL-1β, IL-2, IL-4, IL6, IL-8, IL-12, IL-15, tumor growth factor(TGF)-β,
granulocyte-colony stimulating factor (G-CSF), monocyte chemoattractant protein-1, and the recent addition
of IL-5. Stimulation of porcine peripheral blood
mononuclear cells (PBMC) with CpG oligodeoxynucleotide (ODN) activates and upregulates proinflammatory cytokines IL-6, TNF-α and the T-helper 1-associated cytokine IL-12. The addition of swine IL-3
and c-kit ligand (KL) to fresh cultures of human and
swine bone marrow cells enhanced swine hematopoietic chimersim.
A Th1-biased immune response—activation/stimulation/secretion of swine PBMC results in proinflammatory cytokines IL-6, TNF-α and IL-12—that correlates
well with human PBMC responses. Direct skin tests
and passive cutaneous anaphylaxis serum results in the
swine closely mimic antigen/allergen responses with
respect to delayed and immediate hypersensitivity responses characterized in human biological assessments.
Similar gastrointestinal mucosal architecture, maturation and immune responses in swine will provide a
significant understanding to the immune mechanisms,
antigen handling, and innate immune defenses and
disease prevention that could be applied to human
neonates. For example, neonatal piglets sensitized to
food allergens followed by oral challenge mimic the
P
530
Porcine Immune System
physical and immunologic characteristics of food allergy in humans (5). The model should prove to be
useful in investigations to determine IgE-mediated mechanisms, immunotherapeutic intervention strategies,
and provide a predictive model for assessing novel
proteins as potential allergens. A caveat is that commercial anti-swine IgE is still not available and heatinactivated passive cutaneous anaphylaxis reactions
are still needed to indicate IgE-mediated diseases. In
related investigations, genetically modified maize (Btmaize) was shown to be substantially equivalent (a
characteristic required by regulatory agencies) to parental maize with respect to nutrition. Porcine models
of wound healing and therapeutic options are offering
significant contributions to treatment of human burn
victims. Airway hyperreactivity, eosinophil infiltration, treatments with agonists on airway mechanics
and tryptase inhibitors are providing mechanistic applications to the pathophysiology of asthma.
Because of the growing shortage of human organs and
tissues for transplantation, the swine has become a
major candidate for xenogenic organ donation. Problems with transmission of zoonoses, donor organ
anatomy and function are still areas of concern; however, immunological rejections can be over come with
remarkable progress being made in organ transplantation. With respect to immunological organization and
anatomy, the porcine larynx is similar to that of the
human larynx, suggesting a clearcut case for laryngeal
transplants. The mechanical properties, antithrombogeniety and tissue compatibility of decellularized, heparinized carotid swine arteries may also be suitable
for patients in need of grafts. The gene for hyperacute
rejection was the first knockout gene identified in
swine when organs from swine were transferred to
primates. Recently, genetic manipulation of porcine
B7 molecules, such as in the CD80- knockout swine
or the soluble CD80 transgenic swine, may potentially
provide the basis for therapeutic strategies to regulate
the human response to graft organs. Continued improvements in gene knockout swine models, homologous recombination of fetal somatic cells, and nuclear
transfer in swine suggest that specific modifications
made to the swine genome may be extrapolated, in
time, to humans.
Depending upon the balance between effector and regulatory function, and disturbances in this balance in
swine and humans, the similarities of fetal placentation
during gestation and the development of immunocompetence during ontogeny of the neonate, swine provide
distinct advantages for studies related to human diseases. Differences of extrathymic CD4+CD8+ doublepositive T cells, porcine blood T cells with high proportions of γδ T cells and features of lymphoid tissue
structure/lymphocyte transport, B cell differentiation
that follows different pattern, maternal/fetal and neo-
natal interaction that differ from other experimental
animals, the swine is by far the more realistic animal
model that will allow a better extrapolation of disease
models to the human.
Pros and Cons
The swine has a similar immune physiology to that of
the human and its use as an animal model will more
directly allow an extrapolation that can be translated to
clinical outcomes. Similarities include fetal placentation, duration of gestation and development of immunocompetence during ontogeny of the neonate. Swine
offer natural disease models that can be used to analyze host-pathogen interactions, infectious agents, xenotransplants and allergy that occur in outbred populations. Sequence similarity of MHC and immunoglobulins should provide distinct advantages in xenotransplantation and humanization of porcine proteins
in transgenic systems. Intensive work has advanced
the use of swine as large animal models with respect
to immunological reagents. Cost, convenience, ease of
handling, experience, and the available research tools
—monoclonal antibodies, gene probes, knockouts—
are significant problems; however, the benefits and
advances in swine immunological reagents are fast
becoming a reality.
Predictivity
The neonatal swine model of food allergy is being
investigated as a model to predict allergenicity of genetically modified foods (5). In this model, novel proteins introduced into crops destined for the food marketplace are being systematically compared to a profile
of known food allergens and food tolerant sources for
IgE-mediated immune mechanisms. In other models,
the risk of environmental hazards for developmental
immunotoxicology studies in large animals is becoming prominent. With reference to transcutaneous immunizations, epidermal thickness, relative proportions
of stratum epithelium and uncornified epidermis, and
the distribution of Langerhans cells and their dendrites, swine appear to be more appropriate models
than mice for extrapolation to the human system.
Relevance to Humans
Notable is the development of transgenic pigs for studies of organ transplantation. Because of its size,
availability and limited risk of zoonosis, use of the
pig as a donor for xenotransplantation investigations
has been reached as a consensus in transplantation
medicine.
The neonatal swine food allergy model closely resembles gastrointestinal food allergy in humans and
should offer significant contributions into mechanisms
of IgE-mediated allergy and therapeutic options for
treatment of allergy.
Primary Antibody Response
Studies that are performed in vitro or in vivo in animal
models to establish the pharmacokinetics, metabolic
profile, tolerability, and safety of a drug candidate formulation. These studies are performed to set safe dose
levels prior to initial human clinical trials.
Therapeutic Cytokines, Immunotoxicological Evaluation of
Prednisone
A synthetic steroid with antiinflammatory and immunosuppressive activity.
Cyclosporin A
Prenatal Immunotoxicology
3
1. Tumbleson ME, Schook LB (eds) (1996) Advances in
Swine in Biomedical Research, Volumes 1,2. Plenum
Press, New York
2. Butler JE, Howard C (2002) Summary of the Comparative Immunoglobulin Workshop (CIgW) on immunoglobulins and Fc receptors. Vet Immunol Immunopathol
87:481–484
3. Haverson K et al. (2001) Overview of the Third
International Workshop on Swine Leukocyte Differentiation Antigens. Vet Immunol Immunopathol 80:5–32
4. Boersma WJ, Zwarat RJ, Sinkora J, Rehakova Z,
Haverson K, Bianchi AT (2001) Summary of workshop
findings for porcine B-cell markers. Vet Immunol
Immunopathol 80:63–78
5. Helm RM, Furuta GT, Stanley JS et al. (2002) A neonatal
swine model for peanut allergy. J Allergy Clin Immunol
109:136–142
Preclinical Safety Assessment
3
References
Nonhuman Primates, Immunotoxicity Assessment
of Pharmaceuticals in
3
Similar agencies are in place worldwide to provide the
investigator with the appropriate guidelines in the use
of animals for research purposes. These agencies safeguard both the animal and the investigator from improper use of any animal in laboratory investigations.
Preclinical Immunotoxicity Evaluation
in the Nonhuman Primate
3
When proposing to use swine as an animal model in
the United States, investigators must stringently adhere to the guidelines established by two significant
agencies:
* Association or Assessment and Accreditation of
Laboratory Animal Care or similar agency
* the respective Institutional Care and Use Committee reviews.
The process in the thymus of selecting thymocytes
which recognize self peptides presented by self
major histocompatibility complex proteins.
Thymus: A Mediator of T Cell Development and
Potential Target of Toxicological Agents
Antigen-Specific Cell Enrichment
3
Regulatory Environment
Positive Selection
3
Skin parameters of epidermal thickness, proportion of
the epidermal layers and the distribution of Langerhans cells in swine appear to be appropriate models
for skin testing, transcutaneous immunization studies,
and human wound healing studies.
Investigations such as these provide a clinical basis for
future studies using pigs as large animal models; however, as in any animal model, extrapolation to human
studies must be critically evaluated.
531
Developmental Immunotoxicology
Prevention of Infection
This threshold level has to be exceeded—independent
of statistical significance—to classify a reaction as
positive. In case of lymph nodes assays these levels
are empiric values generated from historical control
values.
Local Lymph Node Assay (IMDS), Modifications
3
Positive Level
Attenuated Organisms as Vaccines
Primary Antibody Response
The immune response that is induced by initial exposure to an antigen. It is mediated largely by IgM anti-
P
3
532
Primary Humoral Immune Response
body and develops more slowly and to a lesser extent
than a secondary response. Antibody responses to the
antigen are measured as an indication of immune competence.
B Lymphocytes
Assays for Antibody Production
Canine Immune System
3
3
3
Primary Humoral Immune Response
Assays for Antibody Production
3
Primary Immune Response
The immune response that is induced by initial exposure to an antigen, which activates naive lymphocytes.
It is mediated largely by IgM antibody and sensitized
T cells. It develops more slowly and to a lesser extent
than a secondary immune response.
Flow Cytometry
3
Primary Lymphoid Organs
Organs in which lymphocyte precursors mature into
antigenically committed, immunocompetent cells. In
mammals, the bone marrow and thymus are the primary lymphoid organs in which B cell and T cell maturation occur, respectively.
Flow Cytometry
3
Primate Immune System (Nonhuman)
and Environmental Contaminants
Helen Tryphonas
Toxicology Research Division
Food Directorate, Health Products and Food Branch
Frederick G Banting Research Center, Tunney’s
Pasture
Ont. K1A 0L2 Ottawa
Canada
Synonyms
Symian, ape
Definition
The subject of chemically induced alterations on structural and functional components of the immune system
is rapidly becoming a major part of research in toxicology. Such research is required to support the evaluation of potentially adverse health effects of chemicals. While a few examples of direct exposure of humans to potentially harmful chemicals exist, the majority of data supporting evaluation is currently generated in experimental animal models such as rodents
and canine species. However, it is known that considerable differences exist in the structure and function of
the immune systems of human and experimental animal models. Therefore, it is recommended that the
extrapolation of data from experimental animals to
human be made with caution. To maximize the degree
of relevance of experimental data to the human situation, scientists in established nonhuman primate centers continue to develop and validate methodologies in
several monkey species. The need for the development
of a nonhuman primate model in immunotoxicology is
becoming increasingly important in safety evaluations
as new and potentially immunotoxic pharmacologic
agents and biotechnology products are added to the
market.
Characteristics
The use of nonhuman primate species in immunotoxicology offers distinct advantages over rodent species.
These are especially useful in situations where established guidelines for toxicity testing of chemicals require, in addition to a rodent model, the use of a nonrodent experimental animal model (1). The advantages
include the following:
1. Phylogenetic proximity to man: As a result of their
phylogenetic proximity to man nonhuman primates
share a number of physiologic, metabolic and behavioural similarities with humans. For example,
the ovarian cycles of the nonhuman primate are
similar to those of humans. Absorption, biotransformation and excretion of several drugs and chemicals are similar in monkeys and man. In view of the
evolution of the brain, which is the last stage before
man, social behaviour in monkeys is similar in
many respects to that of humans.
2. The anatomy and function of the immune system of
nonhuman primates is similar in many respects to
that of humans: This allows the application of reagents available for use in human immunology to
be used to study corresponding immune parameters
in monkeys. For example, cross-reactivity between
mouse antihuman monoclonal antibodies (mAbs)
and monkey leukocyte surface antigens has been
demonstrated for several human mAbs using the
whole blood lysis technique in two-color, fluorescein isothiocyanate (FITC) and phycoerythrin (PE),
flow cytometric analysis (1). Reference values have
been established for infants and adult Macaque fascicularis (cynomolgus), the Macaque mulatta (rhe-
Primate Immune System (Nonhuman) and Environmental Contaminants
Preclinical Relevance
Immune Parameters Available for Use in Nonhuman
Primates
A number of immunologic parameters have been developed and validated for application to nonhuman
primates. In addition to hematologic profiles (total
white blood cell counts and differentials) and immunohistopathology techniques, there are a number of
other assays which are typically used in a clinical laboratory and have been successfully applied to monkeys (2). They are grouped as follows:
* Assays to study effects on humoral immunity
– total serum immunoglobulin (IgG, IgA and IgM)
levels using the enzyme-linked immunosorbent
assay (ELISA)
– challenge with specific antigens: sheep red blood
cells (SRBC), tetanus toxoid (tt), pneumococcal
(pneu) antigens and determination of antigenspecific antibody levels in serum using the hemmaglutination and ELISA techniques
Assays to study effects on cell-mediated immunity
(CMI)
– lymphocyte transformation (3H-thymidine incorporation) (LT) in response to the mitogens
phytohemagglutinin-P (PHA-P), concanavalin
A (Con A) and poke weed mitogen (PWM) or
specific antigens such as tetanus toxoid
– mixed lymphocyte cultures using allogeneic lymphocytes: delayed-type hypersensitivity (DTH)
using either dinitrochlorobenzene (DNCB) as
the sensitizing agent or the multitest kit (Multitest-CMI) which contains a group of recall antigens [Candida albicans, Trichophyton mentagrophytes, Proteus mirabilis, tuberculin purified
protein derivative (PPD), streptococcus group C,
diphtheria, tetanus toxoid, and a glycerine control
Assays to study effects on nonspecific immunity
– monocyte function (activation, phagocytosis and
respiratory burst activity) using latex particles,
SRBC in flow cytometric techniques and activating agents such as phorbol myristate acetate or
zymosan
– natural killer cell activity using the cell line K562
as target cells in a 4-h chromium-51 release
assay
Assays used for mechanistic studies
– cell surface marker analysis (CSMA) using crossreacting mouse anti-human monoclonal antibodies or monkey-specific monoclonal antibodies
when available
– serum complement levels
– cytokine levels—basal and in lectin-activated cultures
– hydrocortisone levels.
3
In general, members of the New World monkeys (such
as the marmosets) and of the Old World monkeys
(including the cynomolgus) are preferred over other
species such as the Papio (baboons) and rhesus monkeys. Firstly, they are easy to breed. This allows the
establishment and maintenance of a large colony thus
avoiding the problems associated with wild-caught
monkeys. Secondly, they are relatively small in size
with short pregnancy periods thus making breeding
and husbandry relatively easy and, thirdly, they are
easy to hand tame. This minimizes the need of restraint methods in administering the experimental
dose or in performing other procedures such as
blood or milk collection and fat biopsies.
The use of nonhuman primates as experimental animal
models is not without drawbacks. These, although not
unsurmountable, include the potential for anthropozoonoses especially to B virus infection, and the increased cost over rodent and canine species for procuring and maintaining especially large monkeys such as
the rhesus monkey or baboons.
Furthermore, ethical issues raised against the use of
animals in research are also applicable to the use of
nonhuman primates in immunotoxicology. Such ethical issues often impose restrictions on the numbers of
monkeys used in research. This can present a problem
especially in cases where multiple doses of an agent
must be studied and in cases where there is a need to
increase the number of monkeys/test dose so as to
enhance the power of statistics.
*
3
sus), the Macacca nemestrina (pig-tailed macaque),
the Cercocebus atys (sooty mangabeys), the Callithrix jacchus (marmoset) and the Pan troglodytes
(chimpanzees) monkeys.
533
*
*
The majority of these assays have been developed or
adapted for use in the cynomolgus, rhesus, the pigtailed macaque and the marmoset monkeys. In comparison, fewer assays have been developed or adapted
for use in the baboons, squirrels and chimpanzee monkeys. While validation of these assays across laboratories is an issue that needs to be addressed, assays for
CMI, challenge with foreign antigens, NK cell assay
and CSMA have been reproduced in several laboratories. Of these assays, challenge with foreign antigens, CSMA and NK have repeatedly proven to be
the most sensitive for detecting chemical-induced immunotoxicity.
Examples of Immunotoxicity Studies in which
Nonhuman Primates were Used
Nonhuman primates have been used extensively in
studies designed to investigate the potential immunotoxic effects of chemicals particularly those which are
of environmental concern such as polychlorinated bi-
P
534
Primate Immune System (Nonhuman) and Environmental Contaminants
phenyls (PCBs) (2), 2,3,7,8-TCDD (dioxins) (3) and
toxaphene (4). Following is a brief discussion of data
available for each of these chemicals.
PCBs
The majority of the immune parameters listed above
have been applied to studies concerning the potential
immunotoxic effects of the PCB mixture known as
Aroclor 1254, in adult and infant rhesus monkeys.
This chronic, multidose, two-generation toxicity/reproductive/immunotoxicity study generated a great
deal of data on effects of PCBs not only in adults
but also in infant monkeys. Table 1 lists the number
of parameters investigated and their outcome. A large
number of immune parameters were affected by PCB
treatment (2). In particular the response to SRBC antigens was significantly affected at levels of Aroclor
1254 as low as 5 μg/kg body weight/day.
TCDD
Immunotoxicity data regarding the effects of TCDD in
nonhuman primates are scarce. Changes in the
CD4+ : CD8+ cell ratio similar to that observed in
the rhesus PCB studies have been reported in rhesus
monkeys exposed to 5 or 25 p.p.t. of TCDD in the diet
(3). These changes were not associated with T-cell
function as measured by the lymphocyte transformation assay in response to mitogens, alloantigens, or
xenoantigens. Also, NK activity and antibody production following immunization with tetanus toxoid were
not affected by treatment. Offspring of the exposed
dams had increased levels of antitetanus antibodies
which correlated with TCDD levels in tissues. Changes in T-cell subsets characterized by a decrease in
CD4+ and an increase in CD8+ cells were also reported
in marmosets treated with 10 ng/kg TCDD (1).
Toxaphene
Recent studies have shown that the pesticide toxaphene, a complex mixture of chlorinated bornanes
with more than 13 000 individual isomers, is also immunotoxic in cynomolgus monkeys (Table 2). In this
study young adult female monkeys, with ten monkeys
per group, were administered doses of 0.00, 0.1, 0.4 or
0.8 mg/kg body weight/day for 75 weeks while five
male monkeys/group were administered toxaphene at
a dose of 0.8 or 0.0 mg/kg body weight/day (4). A
striking feature of this study was the statistically significant reduction in antibody titers in response to immunization with SRBC and tetanus toxoid antigens
without any significant effect on the antibody response
to pneumococcal antigens indicating that the T-cell
dependent humoral immune response was compromised. The effect was highly significant at the
0.8 mg/kg dose. Studies on the infants of the same
monkeys indicated that there were no effects on the
humoral immune response but there was a statistically
highly significant increase in the CD4+ cell number
with a concurrent highly significant decrease in the
CD8+ cell population, suggesting that the regulatory
cells of the immune system were affected by treatment.
Relevance to Humans
The relevance of immunotoxicity data generated in
monkeys in relation to the human population remains
unresolved. However, the available data in humans
accidentally or occupationally exposed to various
agents of environmental concern strongly indicate
that the human immune system is a target for chemical-induced immunotoxic effects (5). Examples of
these include the populations exposed to PCBs, polychlorodibenzofurans (PCDFs) and quaterphenyls
(PCQ) via contaminated rice oil (The Yusho and YuCheng episodes), humans consuming fatty fish species
from the Baltic sea, and studies on the Inuit (Northern
Quebec) populations consuming large amounts of fish
fat (5). Studies in newborn and children exposed in
utero to ambient levels of PCBs and dioxins suggest
that this population of humans may be particularly
sensitive to the immunotoxic effects of environmental
chemicals. This is due to the fact that certain chemicals
including the PCBs are known to cross the human
placenta and to be secreted in large amounts in the
mother’s milk. Examples of such studies include
women working in a capacitor manufacturing factory
in the Shiga Prefacture, Japan, studies on fish-eating
populations from the Great Lakes, and the Netherlands
studies (5). All these studies report effects on several
parameters of the immune system. Many of the affected parameters are similar to those for which effects
were shown in nonhuman primates exposed to PCBs
or dioxins (2).
Regulatory Environment
The ultimate purpose of conducting immunotoxicity
studies is to enable the regulatory agencies to determine “safe” levels of unwanted chemicals in the environment and in the food chain. To facilitate this process, several countries have issued guidelines for immunotoxicity testing in rodents and the majority of the
studies performed during the last decade followed
these guidelines. Although no such guidelines exist
for nonhuman primate models, several of the assays
used in monkeys correspond to those listed in the
proposed guidelines for rodents making the process
of across species comparisons possible (5).
Studies in experimental animals such as guinea pigs,
rabbits and rodents have been helpful in identifying a
No Observed Adverse Effect Level (NOAEL) for several of the chemicals which are of environmental concern. The best example of such studies would be those
Primate Immune System (Nonhuman) and Environmental Contaminants
535
Primate Immune System (Nonhuman) and Environmental Contaminants. Table 1 PCB-induced immunotoxic
effects in adult and infant rhesus monkeys (modified from (2))
Adults
Parameter
22 weeks
Antibody titers to:
SRBC primary IgM and IgG at 23 months
SRBC secondary IgM and IgG at 55 months
Pneumococcal antigens
55 weeks
Infants
9
NS
9
ND
ND
9
Cell surface markers:
T lymphocytes
T helper/inducer cells (Th/i)
T suppressor/cytotoxic cells (Ts/c)
Th/I :Ts/c ratio
B lymphocytes
NS
9
8
9
NS
9
NS
NS
NS
NS
ND
ND
ND
ND
ND
Lymphocyte transformation
H-thymidine incorporation
Phytohemagglutinin
Concanavalin A
Poke weed mitogen
NS
NS
NS
9
9
NS
NS
NS
NS
Mixed lymphocyte culture
NS
NS
ND
Total serum IgG, IgM and IgA
NS
ND
ND
ND
ND
9
NS
ND
ND
ND
ND
9
8
ND
ND
Interleukin-1
ND
9
ND
Tumor necrosis factor
ND
NS
ND
Natural killer cell activity
ND
8
NS
Serum complement (CH50) activity
ND
8
ND
Thymosin alpha-1
Thymosin beta-4
ND
ND
8
8
ND
Interferon levels (Con A-stimulated peripheral blood)
ND
8
ND
Serum hydrocortisone
8
ND
ND
3
Monocyte function
Stimulant: Zymosan:
Peak reading
Time to peak reading
Stimulant: phorbol myristate acetate:
Peak reading
Time to peak reading
Ig, immunoglobulin; SRBC, sheep red blood cells
NS: not significantly different from control P ∃ 0.05; ND: not done
* Statistically significant increase at P # 0.05.
** Statistically significant decrease at P # 0.05.
that were performed using several of the commercially
available PCBs. The calculated NOAELs in these animals were high in comparison to those calculated from
similar data generated in monkeys (2). This was attributed to the higher rate of PCBs eliminated in mice and
rats compared to the rate observed in monkeys. Increased sensitivity of monkeys has been reported not
only for commercially available PCBs but also for a
mixture of congeners representative of those commonly found in human milk. The PCB immunotoxi-
city data in monkeys has been used extensively by
regulatory and advisory agencies. The Agency for
Toxic Substances and Disease Registry (ATSDR) has
derived a Minimal Risk Level (MRL) of 0.02 μg/kg/
day for chronic-duration oral exposure to PCBs. The
chronic oral MRL is based on a Lowest Observed
Adverse Effect Level (LOAEL) of 0.005 mg/kg/day
for immunological effects in adult monkeys that were
evaluated after 23 and 55 months of exposure to Aroclor 1254 (6). Typically these calculations apply an
P
536
Primed Macrophages
Primate Immune System (Nonhuman) and Environmental Contaminants. Table 2 Immunotoxic effects of
toxaphene in adult cynomolgus monkeys
Parameter
Females
Males
Infants
9
9
9
NS
9
NS
ND
ND
NS
NS
NS
NS
9
NS
ND
ND
ND
ND
T-lymphocyte subsets
CD4
CD8
CD4/CD8 ratio
B lymphocyte numbers (absolute)
NS
NS
NS
9
NS
NS
NS
NS
8
9
9
NS
Lymphocyte transformation in response to mitogens
NS
ND
ND
Natural killer cell activity
Natural killer cell numbers
NS
NS
ND
NS
ND
8
Delayed-type hypersensitivity (DTH) to DNCB
NS
ND
ND
Serum hydrocortisone levels
NS
ND
ND
Response to: sheep red blood cells (SRBC)
Primary IgM
Primary IgG
Secondary IgM
Secondary IgG
Response to:
Tetanus toxoid IgG
pneumococcus IgG
Adult monkey data compiled from reference (4). Infant data compiled from a manuscript in preparation.
References
1. Neubert R, Helge H, Neubert D (1996) Nonhuman
primates as models for evaluating substance-induced
changes in the immune system with relevance for man.
In:.Smialowicz RJ, Holsapple MP (eds) Experimental
Immunotoxicology. CRC Press, Boca Raton, pp 63–117
2. Tryphonas H, Feeley M (2001) Polychlorinated biphenylinduced immunomodulation and human health effects. In:
Robertson LW, Hansen LG (eds) PCBs. Recent advances
3.
4.
5.
6.
in environmental toxicology and health effects. The
University Press of Kentucky, pp 194–209
Hong R, Taylor K, Abonour R (1989) Immune
abnormalities associated with chronic TCDD exposure
in Rhesus. Chemosphere 18:313–321
Tryphonas H, Arnold DL, Bryce F et al. (2001) Effects of
toxaphene on the immune system of cynomolgus (Macaca fascicularis) monkeys. Food Chem Toxicol 39:947–
958
Tryphonas H (2001) Approaches to detecting immunotoxic effects of environmental contaminants in humans.
Environ Health Persp 109 [Suppl 6]:877–884
US DoH. (2000) Toxicological Profile for Polychlorinated Biphenyls (Update). U.S. Department of Health &
Human Service Agency for Toxic Substances and
Disease Registry, November 2000
Primed Macrophages
Inflammatory macrophages exposed to interferon-γ or
elicited with certain polymers such as poly-IC become
primed what have further enhanced functions associated with killing.
Macrophage Activation
3
uncertainty factor of 300 (10 for extrapolating from a
LOAEL to a NOAEL, 3 for extrapolating from monkeys to humans, and 10 for compensating for the observed variability among humans). Similarly, the US
Environmental Protection Agency (EPA) has calculated an oral reference dose (RfD) of 0.02 μg/kg/day
for Aroclor 1254 (IRIS 2000) based on the evaluation
of dermal/ocular and immunologic effects in monkeys,
and an oral RfD of 0.07 μg/kg/day based on reduced
birth weight in monkeys (6).
An MRL of 0.03 μg/kg/day has been derived for intermediate-duration oral exposure to PCBs. The intermediate oral MRL is based on a LOAEL of 0.0075
mg/kg/day for neurobehavioral alterations in infant
monkeys that were exposed to a PCB congener mixture representing 80% of the congeners typically found
in human breast milk. This MRL was also supported
by immunologic effects reported for the same monkeys (6).
Prostaglandins
3
Progesterone
Progesterone is a steroid hormone produced in the
ovaries. Biological activity of progesterone is
mediated by interaction with the progesterone steroid
hormone receptor.
Steroid Hormones and their Effect on the Immune
System
3
Programmed Cell Death
Haploid nucleus resulting from meiosis. In animals the
female pronucleus is the nucleus of the unfertilized
ovum.
Transgenic Animals
Prostaglandins
Tina Sali
NIEHS Mail Drop E4–09, Laboratory of Molecular
Carcinogenesis
111 Alexander Drive
P.O.Box 12233
Research Triangle Park, NC 27709
USA
Synonyms
prostanoids,
3
Procainamide
(4-amino-N-(2-(diethylamino)ethyl)benzamide) is used to treat cardiac arrhythmia (abnormal heart rate).
Systemic Autoimmunity
Pronucleus
3
Procainamide
537
eicosanoids, cyclooxygenase, COX
3
Apoptosis
3
Definition
A cytokine that supports an inflammatory response by
stimulating leukocytes to an enhanced activity towards
microbial agents or other antigenic compounds. Usually associated with induction of other inflammatory
mediators, e.g. prostaglandins or leukotrienes. Principal proinflammatory cytokines are TNF-α and IL-1 or
IL-6. These cytokines activate multiple inflammatory
response pathways, including the lymphocyte response in delayed type IV hypersensitivity.
Cytokines
Metals and Autoimmune Disease
Chronic Beryllium Disease
3
Proinflammatory Cytokine
Prostaglandins are lipid metabolites that induce a diverse spectrum of biological effects. They are potent
modulators of signaling pathways that regulate pain,
inflammation, cell proliferation, transformation, angiogenesis, metastasis, and apoptosis. Production of prostaglandins from cis-unsaturated fatty acids, such as
arachidonic acid, requires the activity of the cyclooxygenase (COX) enzymes, of which there are two
isozymes (COX-1 and COX-2). Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, prevent
prostaglandin production through the inhibition of
COX.
3
Characteristics
3
Prostaglandins are secondary messengers that are produced in response to a variety of stimuli. They act as
short-lived, local hormones in order to alter activities
in the cells in which they are synthesized, as well as in
adjoining cells. Prostaglandins thereby regulate numerous pathways which are important both for normal
cellular function as well as the body's response to cell
injury and disease. For example, prostaglandins have
been shown to stimulate inflammation and induce endothelial cell growth, as well as relax and contract
various types of smooth muscle, modulate synaptic
transmission, regulate blood flow to certain organs,
control ion and water transport in the kidneys, and
induce sleep. Alterations in prostaglandin metabolism
have been implicated in the pathogenesis of hypertension, asthma, inflammation, pain, fever, swelling, redness, headache, and the formation of ulcers and tu-
3
Prolymphocytic Leukemia
Leukemia
3
Promoter
A regulatory region usually located 5’ of a gene’s
coding region which regulates where (i.e. which cell
types) and when genes are expressed.
B Lymphocytes
P
3
538
Prostaglandins
mors. Failure to regulate prostaglandin production can
also enhance tumor spread and metastasis.
On the molecular level, prostaglandins are polyunsaturated fatty acids which contain a cyclopentane ring
and two alkyl side chains. They are synthesized from
C-20 cis-unsaturated fatty acids: icosatrienoic acid (1series), eicosatetraenoic acid, which is also known as
arachidonic acid, (2-series), and eicosapentainoic acid
(3-series). Letters A–J designate the nature and position of substituents on the cyclopentane ring, as well
as the presence and position of double bonds within
the ring. Numerical subscripts (1, 2, or 3) indicate the
number of double bonds in the alkyl side chains,
which is a direct reflection of the fatty acid precursor
from which the prostaglandin is synthesized. The prostaglandin (PG) family includes PGA, PGB, PGC,
PGD, PGE, and PGF as well as the PGG and PGH
intermediates, PGI (prostacyclin) and PGJ. The PGG
and PGH intermediates are the direct products of COX
and they are quickly converted to other prostaglandins
by a second class of enzymes known as the synthases.
PGA, PGB, and PGC are believed not to occur naturally, but to be produced only artificially during extraction procedures.
Arachidonic acid is the most abundant fatty acid precursor in most mammals, including humans, making
the 2-series prostaglandins predominant in these organisms. The first step in the metabolism of prostaglandins from arachidonic acid is the release of arachidonic acid from membrane glycerophospholipids
by phospholipase A2 (PLA2) (Figure 1). COX (also
known as prostaglandin H synthase) has both cyclooxygenase and peroxidase activities. The cyclooxygenase activity of COX first converts arachidonic acid
into PGG2, which is followed by the subsequent conversion of PGG2 to PGH2 by the peroxidase activity of
COX. The PGH2 intermediate can then be further metabolized into other prostaglandins (PGD2, PGE2,
PGF2α), prostacyclin (PGI2), or thromboxane
(TXA2) by the corresponding synthases. Thromboxane is formed from PGH2 by thromboxane synthase,
but it is not formally considered a prostaglandin due to
the insertion of oxygen in the cyclopentane ring.
Thromboxane is a vasoconstrictor that induces platelet
aggregation, while prostacyclin serves the opposite
function of being a vasodilator that reduces platelet
aggregation. It is therefore believed that the balance
of these two lipid metabolites is important for maintaining vascular homeostasis. While PGD2 promotes
sleep, PGF2α is important for many aspects of reproduction. The primary metabolite of the arachidonic
acid pathway, however, is PGE2. Among other functions, PGE2 is an important mediator of the inflammatory response and the major prostaglandin found in
colonic polyps. It is formed from PGH2 by
prostaglandin E synthases (PGES) and its formation
appears to be coordinated by the co-regulation of
PGES and COX.
Once produced, prostaglandins activate cell-signaling
cascades which are linked to prostaglandin-specific
G protein-coupled receptors. A variety of receptors
have been identified which bind thromboxane,
PGD2, PGE2, PGF2α, and PGI2 as ligands (Figure 1).
These receptors show selective ligand-binding specificity, and vary in terms of their abundance and tissue
distribution. The effect elicited by any given prostaglandin is therefore dependent upon the location in the
body where it is synthesized, as well as the presence
and number of receptors to which it may bind.
The COX-1 and COX-2 isozymes are heme proteins
which are highly homologous in terms of their amino
acid sequence (61% identity), size and overall structure. COX-1 and COX-2 are encoded by separate
genes and differ in their tissue distribution and subcellular localization. COX-1 is constitutively expressed
and is responsible for various housekeeping duties
such as maintaining normal gastric and kidney function. Production of COX-2, however, is rapidly induced by a variety of inflammatory stimuli, tumor
promoters, and growth factors, and it is the isoform
found mainly in cancer cells. It is believed that increased activity of the arachidonic acid pathway due
Prostaglandins. Figure 1 The synthesis of prostaglandins and thromboxane from arachidonic acid is
shown, as well as the G protein-coupled receptors to
which they may bind.
Prostaglandins
to chronic irritation or inflammation results in an increased risk of cancer. In support of this, both in vitro
and in vivo data suggest that COX-1 and COX-2 may
play a role in human cancer of the prostate, colon, and
breast and other cancers. Excessive prostaglandin production due to overexpression of COX-2 is a commonly observed feature of neoplastic transformation,
and COX-2 expression also appears to be involved in
angiogenesis. A widespread role for COX-2 in carcinogenesis is evidenced by increased levels of COX-2
having been found in biopsies from tumors of the
breast, stomach, lung, esophagus and skin. Furthermore, expression of COX-2 is increased by phorbol
ester treatment, which is linked to tumor promotion,
while COX-2 inhibitors suppress tumor proliferation
and stimulate apoptosis in human colon cancer cells.
Dramatic reductions in tumor size and number were
observed upon independent knockout of the COX-1
and COX-2 genes in APC(adenomatous polyposis
coli)-deficient mice—like human FAP (familial adenomatous polyposis) patients, these spontaneously develop a large number of intestinal tumors. These results
suggest a role for both isoforms of COX in cancer.
NSAIDs reduce pain, inflammation, and other prostaglandin-induced symptoms by competitive inhibition of
the cyclooxygenase activity of COX. A variety of
drugs are currently available which vary in terms of
their potency and toxicity profiles. Three classes of
NSAIDs exist:
* non-selective COX inhibitors such as aspirin, ibuprofen, and naproxen
* drugs which selectively inhibit COX-1 such as the
experimental drug SC-560
* COX-2-specific inhibitors like celecoxib and rofecoxib.
Selective COX inhibition can be attributed, at least in
part, to positional amino acid differences between the
two isozymes. Superposition of the 3-D structures of
COX-1 and COX-2 isoforms reveals two non-conserved residues in the cyclooxygenase binding site
which are responsible for a difference in the size and
potential ligand interactions for the two active sites:
Ile523 and His513 in COX-1, which correspond to
Val523 and Arg513 in COX-2. However, molecular
modeling and mutagenesis experiments confirm the
importance of additional, conserved residues in ligand
selectivity as well. In recent years, there has been great
interest in the development of drugs which specifically
inhibit COX-2. This is because of the important role
COX-1 plays in the constitutive expression of prostaglandins for normal cellular function. For example,
prostaglandins in the gastrointestinal tract act to protect the stomach and intestinal mucosa from inflammation, inhibit gastric acid secretion, and promote the
healing of gastric and duodenal ulcers. The inhibition
539
of COX-1 by NSAIDs therefore often results in unwanted side effects, such as stomach ulcers. These
unwanted side effects can often be avoided by taking
NSAIDs which specifically target the inducible COX2 enzyme. On the other hand, studies in mice show
that COX-1 specific inhibitors hold promise for delaying the onset of premature labor during pregnancy,
without interfering with closure of the ductus arteriosus (a fetal blood vessel that joins the aorta and the
pulmonary artery)—a side effect which can occur with
the use of non-selective COX inhibitors.
Studies reveal a 40–50% decrease in colorectal cancer
mortality due to the use of NSAIDs, as well as a
decrease in the number and size of colorectal polyps
in human and mouse models. The use of NSAIDs has
also been linked to chemoprevention of breast and
lung cancer, though to lesser extents. While there is
data demonstrating this anti-tumorigenic activity is
due to COX inhibition, other data suggest NSAIDs
have COX-independent effects as well. For example,
NSAIDs have been shown to induce cultured human
colorectal cancer cells devoid of COX to undergo apoptosis. In addition, the amount of COX inhibitor required to induce apoptosis is usually greater than that
needed to inhibit COX. One possible mechanism for
these COX-independent effects involves an additional
role for NSAIDs in the regulation of gene expression.
One gene recently found to be greatly upregulated by
NSAID treatment is NAG-1 (NSAID-activated gene).
NAG-1 is a divergent member of the transforming
growth factor-beta (TGF-β) superfamily, and expression of NAG-1 has been shown to result in apoptosis
and anti-tumorigenic activities in several model systems. The induction of NAG-1 expression has been
observed with a variety of NSAIDs and in several
cancer cell lines including colorectal, lung, breast,
and prostate. NAG-1 induction occurs independently
of COX inhibition and is not affected by prostaglandins. Therefore, NAG-1 appears to be a common link
between NSAIDs and their pro-apoptotic activity.
Preclinical Relevance
The regulation of prostaglandin metabolism has potential clinical relevance for the endocrine, reproductive,
nervous, digestive, respiratory, cardiovascular, and
renal systems. Daily intake of a low dose of aspirin
may reduce the risk of heart attack by inhibiting COX1 present in platelets (inhibition of COX-1 reduces the
formation of TXA2, thereby preventing the platelets
from clumping). The use of NSAIDs is also potentially
relevant to the prevention of cancer, though this effect
cannot be exclusively attributed to the inhibition of
prostaglandin production by COX. The development
of COX-specific inhibitors shows promise for prevention of premature labor (COX-1-specific) and treatment of rheumatoid arthritis and osteoarthritis (COX-
P
Prostaglandins
A subgroup of eicosanoids (lipid metabolites) that are
produced by the enzymatic activity of cyclooxygenase.
Prostaglandins
Protein kinases are enzymes that alter the activity or
confirmation of other proteins by adding a phosphate
group to specific tyrosine, serine or threonine residues.
Signal Transduction During Lymphocyte Activation
Protein-Modifying Compound
Hapten and Carrier
Proteins
Immunotoxicology of Biotechnology-Derived Pharmaceuticals
Pseudoallergic Reaction
A reaction that resembles allergy but is not due to the
interaction of antigen with specific antibody.
Complement, Classical Pathway/Alternative Pathway
Complement and Allergy
3
Prostaglandins
Protein Kinases
3
1. Hsi LC, Eling TE (2002) Carcinogenesis involving
cyclooxygenase and lipoxygenase. In: Harris RE (ed)
COX-2 Blockade in Cancer Prevention and Therapy.
Humana Press, Totowa, pp 245–255
2. Narumiya S, Sugimoto Y, Ushikubi F (1999) Prostanoid
receptors: structures, properties, and functions. Physiol
Rev 79:1193–1226
3. Filizola M, Perez JJ, Palomer A, Mauleon D (1997)
Comparative molecular modeling study of the threedimensional structures of prostaglandin endoperoxide H2
synthase 1 and 2 (COX-1 and COX-2). J Molec Graphics
Model 15:290–300
4. Steele VE, Hawk ET, Viner JE, Lubet RA (2003)
Mechanisms and applications of non-steroidal anti-inflammatory Drugs in the chemoprevention of cancer.
Mutat Res 523–524:137–144
5. Baek SJ, Kim KS, Nixon JB, Wilson, LC, Eling TE
(2001) Cyclooxygenase inhibitors regulate the expression
of a TGF-β superfamily member that has proapoptotic
and anti-tumorigenic activities. Molec Pharmacol
59:901–908
Protein blotting is a synonym for western blot analysis.
Western Blot Analysis
3
References
Protein Blotting
3
The only relevant regulations governing prostaglandin
and NSAID studies are the standard regulations for
human clinical trials and animal experiments.
Enzymes that catalyze the breakdown of peptide
bonds.
Mitogen-Stimulated Lymphocyte Response
Respiratory Infections
3
Regulatory Environment
Proteases
3
Prostaglandins have been found in nearly all human
tissues and fluids examined, though they usually occur
only in minute amounts and are degraded soon after
synthesis. This nearly ubiquitous production of shortlived prostaglandins at low concentrations underscores
both their biological importance and their potency.
Prostaglandins and related lipids regulate a wide
range of biological actions and alterations in their production are linked to a number of important human
diseases and cancer. A number of drugs are currently
on the market which are related to prostaglandins, the
most notable of which are the NSAIDs.
A subgroup of eicosanoids (lipid metabolites) consisting of the prostaglandins and thromboxanes.
Prostaglandins
3
Relevance to Humans
Prostanoids
3
2-specific). COX inhibitors are also potentially useful
in the treatment of a variety of ailments which include
cardiovascular disease, osteoporosis, cataract formation, diabetes and Alzheimer's disease.
3
540
3
Pyrogen
3
Psoriasis
Respiratory Infections
PWM
3
Polyclonal Activators
Pyrene
3
Psoriasis vulgaris is a frequently occurring skin disease in fair-skinned individuals. Disease starts often in
the second decade of life triggered by infections like
angina or measles. Pathogenesis involves hyperproliferation of keratinocytes, and accelerated migration of
keratinocytes from the basal to the horny layer in
4 days (normal 28 days). Symptoms are intense desquamation of the skin and itching.
Cyclosporin A
Exposure Route and Respiratory Hypersensitivity
Pulmonary Infections
3
Symptoms similar to an IgE-mediated allergy (urticaria, angioedema, anaphylaxis), but without sensitization
of the individual. Frequently caused by direct mast cell
stimulation and subsequent degranulation. Typically
caused by prostaglandin synthesis inhibitors, muscle
relaxants, or contrast media.
IgE-Mediated Allergies
Pulmonary Hypersensitivity
3
Pseudoallergy
541
Polycyclic Aromatic Hydrocarbons (PAHs) and the
Immune System
3
Pyrogen
Fatty Acids and the Immune System
Pyrogens are compounds that induce fever. Exogenous
pyrogens are bacterial constituents such as lipopolysaccharide (LPS). Endogenous pyrogens are proinflammatory cytokines such as TNF-α or IL-1.
Cytokines
3
PUFA
P
3