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DNA Vaccines
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Superantigens
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Salmonella, Assessment of Infection
Risk
Wim H de Jong
Johan Garssen
H Havelaar
. Rob de Jonge .
. Katsuhisa Takumi . Arie
Laboratory for Toxicology, Pathology and Genetics
National Institute for Public Health and the
Environment (RIVM)
PO Box 1
3720 BA Bilthoven
The Netherlands
Synonyms
Salmonella enterica, Salmonella enterica serovar Enteritidis, Salmonella Enteritidis, salmonellosis, Salmonella food poisoning, Salmonella food-borne disease,
gastroenteritis
Definition
Salmonella spp. can cause severe enteric infections.
Especially the typhoid (S. typhi) and paratyphoid bacteria result in septic conditions with typhoid-like fever
and nausea, vomiting, abdominal cramps, and diarrhea. Nontyphoid salmonellae generally induce less
severe symptoms restricted to the gastrointestinal
tract—designated salmonellosis. Infection usually
occurs by food poisoning of which raw meats and
poultry, or poultry products (eggs), are common infection sources.
Characteristics
General Aspects
Contamination of food and water by pathogenic microorganisms such as Salmonella spp. is a risk for the
population, especially for specific subgroups with a
much higher risk profile. Microbiological risk assessment is an emerging tool for the evaluation of the
microbiological safety of food and water. In this process, hazardous microorganisms are identified and exposure of the consumer to these organisms is estimated
by a combination of observational data and mathematical modeling. The health risk arising from exposure
is then estimated by use of a dose-response model, that
gives a quantitative description for the relationship
between the exposure to a certain number of pathogens (the dose) and the probability of an effect, such as
infection or disease (1).
Experimental salmonellosis in rodents has been extensively studied and detailed information is available on
the host-pathogen interaction from in vivo as well as in
vitro experiments. Whereas in humans nontyphoid salmonellae induce self-limiting gastroenteritis with only
occasional bacteremia, systemic disease develops in
rats and mice. After oral exposure of rats to salmonellae the intestine is rapidly colonized, and salmonellae
can be detected in the small intestine and cecum within
2 h. Intestinal colonization is concentrated in the distal
ileum and cecum, and may be detected by fecal excretion. Invasion is quickly established via the M cells in
Peyer’s patches, causing salmonellae to be detectable
in mesenteric lymph nodes within 8 h (2,3). Systemic
infection results in high numbers of pathogens present
in spleen and liver, and is associated with an increase
in organ weights.
A major line of defence against invasion of salmonellae is uptake by macrophages. Later, these macrophages play a crucial role in the induction of T celldependent immunity that can be detected by the development of a delayed-type hypersensitivity reaction
(3–5). However, virulent salmonellae have adapted to
survive and can grow inside macrophages and may
induce cell death by apoptosis (6). Polymorphonuclear
leucocytes (mainly neutrophils) play a key role in the
clearance of Salmonella infections (7). Despite the
available insight in the pathogenesis and host-response, the published literature is of limited use for
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dose-response modeling of food-borne and waterborne pathogens. Usually only high doses were administered, often inoculated via the intraperitoneal or intravenous route, instead of orally. Most papers only
considered a few endpoints to detect infection, and it
is not known whether colonization of the intestine and
invasion occur in a dose-dependent manner.
Animal Model
Young adult Wistar Unilever male rats age 6–8 weeks
were infected by gavage with various doses of Salmonella enterica serovar Enteritidis 97–198, a patient’s
isolate origin RIVM, or for control with a nonpathogenic nalidixic acid-resistant Escherichia coli WG5
(3). In order to promote the survival of Salmonella
so that they could reach the target sites for colonization, the standard model included overnight fasting of
the animals and neutralization of gastric acid by suspending the inoculum in a sodium bicarbonate solution. This method also allows the investigation of the
colonization potency of low doses of Salmonella. The
dose-response for bacterial counts in feces is shown in
Figure 1, and colonization of the spleen is presented in
Table 1
Although in general no significant indications for
overt clinical disease were noticed, bacterial counts
in mesenteric lymph nodes and spleen indicated systemic infection. This was reflected by the time-related
increase in the number of neutrophilic granulocytes
(Figure 2). Indicative for the induction of a systemic
immune response was the delayed-type hypersensitivity reaction after challenge of the animals with heat
killed Salmonella antigens (Figure 3).
Mathematical Modeling
Dose-infection models as recommended by WHO and
FAO (8) are based on several assumptions:
* the single hit hypothesis: each inoculated organism
has a (possibly very small but not zero) probability
to cause infection; each surviving organism grows
to produce a clone of cells
* the hypothesis of independent action: the mean
probability per inoculated pathogen to cause a
Salmonella, Assessment of Infection Risk. Table 1 Colonization of the spleen after oral administration of
various doses of Salmonella Enteritidis
Dose (cfu/animal)
Animals exposed
Animals infected
% Infected
10
4
0
0
150
4
1
25
300
5
2
40
910
4
2
50
18 000 +
4
4
100
Salmonella, Assessment of Infection Risk. Figure 1
Salmonella, Assessment of Infection Risk
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Salmonella, Assessment of Infection Risk. Figure 2
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Salmonella, Assessment of Infection Risk. Figure 3
*
*
(symptomatic or fatal) infection is independent of
their number
microorganisms behave as discrete particles and
cannot be divided in units smaller than one
microorganisms are randomly distributed in the inoculum; this assumption is made for mathematical
convenience but is not necessary; models for nonrandom distributions have been described (9).
These assumptions led to the family of single-hit models that are routinely used to describe microbial doseresponse relationships. The best known models are the
exponential model, the Beta-Poisson model and the
hypergeometric model (10). Using this model, doseresponse parameters can be estimated. Salmonella infection occurred at doses as low as 500 colony-forming units (cfu) and occasionally even lower. Marked
histopathological alterations were mainly observed in
the large intestine at doses above 10E6 cfu.
When prepared under laboratory conditions, repeatedly using the same kind of Salmonella preparations
originating from the same deep-frozen stock, it appears reasonable to assume that the probability of
any single organism to establish itself in the animal
and cause infection is constant. Indeed, data for infection of the spleen could be fitted with the exponential
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Salmonella, Assessment of Infection Risk
model and indicated that on average, 1 per 860 cells in
the inoculum infected the spleen (Figure 4). In contrast, the reproducibility of detecting infection by microbiological examination of mesenteric lymph nodes
was poor, both with respect to the fraction of positive
animals and the mean counts in positive animals.
Immune Responses to Salmonella
Rats are able to mount a vigorous cellular immune
response in relation to Salmonella infection. There is
a consistent dose-response relationship for most leucocyte subsets (3). The only exception was a lack of
response by eosinophils, which was not unexpected
because these cells are mainly related to parasitic infections. The strong increase in monocytes on day 5
can be interpreted as a consequence of the innate, nonspecific immune response in which tissue macrophages are recruited from a pool of blood monocytes
to engulf the invading salmonellae. Initial depletion of
the monocyte pool in blood by migration into tissues is
overcompensated by increased production in the bone
marrow (11). Survival and growth in macrophages was
found to be a major virulence mechanism for Salmonella (7). Macrophage death by apoptosis is a host
defence mechanism, which leaves the bacteria susceptible to subsequent phagocytosis and killing by neutrophils, of which the number increased significantly
in the blood of infected animals. Later, the nonspecific
immune response is succeeded by a specific response,
which is manifest by a strong increase in lymphocytes
on day 11.
Salmonella, Assessment of Infection Risk. Figure 4
Th1-Th2 Balance Influences Immune Response to
Salmonella
T helper 1-T helper 2 balance can be skewed. Doseresponse studies in two different rat strains skewed
towards either a T helper-type 1 (Th1) directed immune response (Lewis rat) or a Th2-type directed immune response (Brown Norway rat) showed that the
probability of infection per single Salmonella Enteritidis cell was approximately 100 times higher in Brown
Norway rats than in Lewis rats (12). The probability of
infection per Salmonella Enteritidis cell was 1:25 000
for Lewis rats, 1:800 for Wistar Unilever rats, and
1:200 for Brown Norway rats (Figure 5). So, the
Th2 animals (Brown Norway rats) were more prone
to infection than Th1 responding animals (Lewis rats);
the Brown Norway rats were even more sensitive to
infection compared to the originally used Wistar Unilever rats (12). The Wistar Unilever rat showed a kind
of intermediate response between the more Th1 and
Th2 type of responses shown by the Lewis and Brown
Norway rats, respectively. It is plausible to attribute
this difference in sensitivity to the fact that Lewis rats
are Th1 prone and Brown Norway rats are Th2 prone.
However, it should be taken into account that host
factors other than the Th1-Th2 balance might be involved also in the sensitivity for Salmonella Enteritidis
infection.
The delayed-type hypersensitivity (DTH) reaction was
also found to be a sensitive and reproducible parameter to detect an immune response to Salmonella infection. Positive reactions were observed at challenge
doses above 100 cfu, which is equivalent to doses
that lead to colonization of the spleen. Not surprisingly, Lewis rats showed a more vigorous DTH re-
Salmonella, Assessment of Infection Risk
577
Salmonella, Assessment of Infection Risk. Figure 5
sponse than the Brown Norway rats (12). Cellular immune responses were more pronounced in Lewis rats
but antibody responses were higher in the Brown Norway rats, thus indicating the influence of the Th1-Th2
balance on infection with Salmonella.
In both rat strains the neutrophilic response in the
blood remained a very sensitive read out system for
infection. Although initial infection levels are more
prone to occur in the Th2 background (Brown Norway
rats), when infected in animals prone to a Th1-type
response (Lewis), systemic infection was more intense (12).
Preclinical relevance
The dose-response relationship of exposure by intragastric gavage of adult, male WU rats to Salmonella
Enteritidis is well reproducible. In all experiments, the
animals were shedding the bacteria in their feces after
exposure to intermediate to high doses (> 104 cfu). At
lower doses, no fecal excretion was detected within
the 6-day period of observation but salmonellae
could be isolated from the spleen and mesenteric
lymph nodes. These findings are in accordance with
the fact that Salmonella Enteritidis is highly invasive
in rodents and that the intestinal tract may not be the
major site of multiplication. Pathological results partly
confirm these observations, indicating that the gastrointestinal tract (although portal of entry) shows relatively little abnormalities in animals that succumb to
severe systemic illness after oral inoculation with very
high doses of Salmonella Enteritidis. However, at
lower, nonlethal doses lesions typical for gastroenteritis were observed in the ileum, cecum and proximal
colon (3). No evidence of clinical illness was associated with these histological abnormalities.
Relevance to Humans
Dose-response models can be based on observational
or experimental data. Observational data (usually from
food-borne or water-borne outbreaks) have the advantage that they are based on actual situations, but generally provide limited information as the dose may be
unknown (rare undetectable contamination, source unknown, uncontrolled storage conditions, sample unavailable) and the size of the exposed population is
often not known. Experimental data, either from
human volunteers or animal studies, have the advantage that they are obtained under well-controlled conditions and can therefore be subjected to mathematical
analysis.
Limitations for experiments in human volunteers are
that they are performed in healthy volunteers, and that
the dose range and pathogenicity of the microorganism
investigated are restricted to an infection resulting in a
mild, self-limiting disease only. In animal models the
dose-response relationship for infection can be assessed at a much broader range. Such models should
enable risk assessors to evaluate the effect of single
factors related to the host, pathogen and food matrix,
and to make inferences about dose-response relations
in humans.
Typical questions are: what is the effect of factors such
as age, immunological status, nonspecific barriers (e.g.
gastric acid, innate immunity) on the susceptibility of
the host, and factors such as bacterial adaptation or
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Salmonella enterica
protection by fatty foods on the infectivity of the
pathogen.
As a next step kinetic models of the infection process
can be developed, that describe the dynamics of the
host-pathogen interaction in the alimentary tract, for
which the animal models should provide insight in
important mechanisms and should provide parameter
estimates (13).
Salmonella enterica serovar
Enteritidis
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Salmonella, Assessment of Infection Risk
Salmonella Enteritidis
References
Salmonella Enteritidis Rat Model
Animal model for estimation of dose response relationship after Salmonella infection. Parameters for infection include spleen counts, neutrophilic granulocyte
response, and delayed-type hypersensitivity reaction.
Low dosages already can induce systemic infection as
indicated by spleen colonization after oral infection.
Salmonella, Assessment of Infection Risk
Salmonella Food-Borne Disease
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Salmonella, Assessment of Infection Risk
Salmonella Food Poisoning
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Salmonella, Assessment of Infection Risk
Salmonella typhimurium Mitogen
This is a substance which stimulates non-specifically
the proliferation of rat B cells, but does not cause them
to differentiate. It is used in immunology to investigate
the function of rat B cells.
Lymphocyte Proliferation
3
Salmonella, Assessment of Infection Risk
Salmonella, Assessment of Infection Risk
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Salmonella enterica
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1. WHO (1999) Risk Assessment of Microbiological
Hazards in Foods. Geneva, World Health Organization
2. Naughton PJ, Grant G, Spencer RJ, Bardocz S, Pusztai A
(1996) A rat model of infection by Salmonella typhimurium or Salm. Enteritidis. J Appl Bacteriol 81:651–
656
3. Havelaar AH, Garssen J, Takumi K et al. (2001) A rat
model for dose response relationships of Salmonella
Enteritidis infection. J Appl Microbiol 91:442–452
4. Collins FM, Mackaness GB (1968) Delayed hypersensitivity and Arthus reactivity in relation to host resistance
in Salmonella-infected mice. J Immunol 101:830–845
5. Takumi K, Garssen J, Havelaar A (2002) A quantitative
model for neutrophil response and delayed-type hypersensitivity reaction in rats orally inoculated with various
doses of Salmonella Enteritidis. Int Immunol 14:111–
119
6. Jones BD, Falkow S (1996) Salmonellosis: host immune
responses and bacterial virulence determinants. Ann Rev
Immunol 14:533–561
7. Vassiloyanakopoulos AP, Okamoto S, Fierer J (1998)
The crucial role of polymorphonuclear leukocytes in
resistance to Salmonelladublin infections in genetically
susceptible and resistant mice. Proc Natl Acad Sci USA
95:7676–7681
8. WHO (still in press?) Guidelines for Hazard Characterization of Pathogens in Water and Food. Geneva, World
Health Organization
9. Haas CN (2002) Conditional dose-response relationships
for microorganisms: development and application. Risk
Analysis 22:455–463
10. Teunis PFM, Havelaar AH (2000) The Beta Poisson
dose-response model is not a single hit model. Risk
Analysis 20:513–520
11. Volkman A, Collins FM (1974) The cytokinetics of
monocytosis in acute Salmonella infection in the rat. J
Exper Med 139:264–277
12. Havelaar AH, Garssen J, Takumi K et al. (2003)
Intraspecies variability in the dose-response relationship
for Salmonella Enteritidis, associated with genetic
differences in cellular immune response. Submitted for
publication
13. Takumi K, De Jonge R, Havelaar A (2000) Modeling
inactivation of Escherichia coli by low pH: application
to passage through the stomach of young and elderly
people. J Appl Microbiol 89:935–943
Salmonellosis
Gastroenteric infection due to Salmonella spp., usually
caused by consumption of inadequately heated contaminated food such as (raw) meats, poultry or poultry
products. Symptoms after infection can include fever,
severe nausea, vomiting, abdominal cramps and diarrhea.
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Secondary Neoplasms
Salmonella, Assessment of Infection Risk
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SAPS
Cytokine Polymorphisms and Immunotoxicology
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Secondary Antibody Response
The immune response that is induced following a second exposure to antigen and mediated by memory
cells and largely by IgG antibody, which allows for
a more rapid and stronger response than the primary
response.
B Lymphocytes
Assays for Antibody Production
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3
3
SARS
Secondary Cytokines
Respiratory Infections
Severe combined immunodeficient mouse.
Animal Models of Immunodeficiency
3
Scleroderma
Hardening of the skin. Systemic scleroderma has hardening of skin as well as involvement of other organs,
especially the lungs, esophagus, kidneys and heart.
Systemic Autoimmunity
Secondary Humoral Immune
Response
Assays for Antibody Production
Secondary Immune Response
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SCID Mouse
3
These are fatty acids which contain no double bonds.
Fatty Acids and the Immune System
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Saturated Fatty Acids
In immune responses like an inflammation master cytokines (e.g. interleukin-1, tumor necrosis factor α) are
rapidly released which organize the immune reaction
by stimulating tissue cells and leukocytes to produce
further mediators of the immune system like Interleukin-6 or chemokines. These cytokine-induced cytokines are called secondary cytokines.
Cytokine Receptors
Memory, Immunological
Secondary Lymphoid Organs
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SEB
Polyclonal Activators
3
Sodium dodecylsulfate (SDS)–polyacrylamide gel
electrophoresis (PAGE) separates proteins in an electric field based on their molecular size.
Western Blot Analysis
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SDS-PAGE
Organs and tissues in which mature immunocompetent
lymphocytes encounter trapped antigens and are activated into effector cells. In mammals, the lymph
nodes, spleen, and mucosal-associated lymphoid tissue
(MALT) like Peyer's plaques constitute the secondary
lymphoid organs.
Flow Cytometry
Antigen Presentation via MHC Class II Molecules
Secondary Neoplasms
Secondary neoplasms are cancers that arise in an individual as a result of previous chemotherapy and/or
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Secondary PLNA
radiation therapy. These new cancers may surface
months or years after the initial treatment for a variety
of cancers.
Leukemia
Lymphoma
Self-Renewal
The ability of a hematopoietic stem cell to produce at
least one other stem cell after cell division, and thus to
maintain the stem cell status.
Bone Marrow and Hematopoiesis
Hematopoietic Stem Cells
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3
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3
Secondary Prevention
Strategies employed to prevent reoccurrence of a lifethreatening medical event.
Fatty Acids and the Immune System
Semiquantitative PCR
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Popliteal Lymph Node Assay, Secondary Reaction
3
Secondary PLNA
Polymerase Chain Reaction (PCR)
Sensitization
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Secretory Immune System
Induction of IgE antibodies by an allergen. In general
also used for the induction of allergic reactions.
Food Allergy
Hapten and Carrier
Mucosa-Associated Lymphoid Tissue
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Sensitizer
Secretory immunoglobulin A (sIgA) is a complex of
two IgA molecules joined by a additional J-chain and
a secretory component (Sc). Sc is made by epithelial
cells of intestines and lungs as part of an IgA receptor
and is involved in the transportation of IgA across the
epithelial cells to mucosal surfaces where it can react
with pathogens.
Immunotoxic Agents into the Body, Entry of
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Selection
Cell Separation Techniques
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Self Antigen
A chemical that causes a substantial proportion of exposed people or animals to develop an allergic reaction
in normal tissue after repeated exposure to the chemical.
Three-Dimensional Human Skin/Epidermal Models
and Organotypic Human and Murine Skin Explant
Systems
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Secretory Immunoglobulin A
Septic Shock
Jutta Liebau
Fachklinik Hornheide
Dorbaumstrasse 300
D-48157 Münster
Germany
Synonyms
bacteremic shock, endotoxin shock, septicemic shock
Many structures of a healthy organism could be recognized by the adaptive immune system leading to an
autoimmune response. A number of mechanisms retain tolerance of the immune system against these
structures.
Antigen Presentation via MHC Class II Molecules
Autoantigens
Definition
Septic shock is a clinical syndrome of acute circulatory failure resulting from acute invasion of the bloodstream by microorganisms or their toxic products (1).
In adults, it is characterized by persistent arterial hypotension unexplained by other causes. Hypotension is
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Septic Shock
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Septic shock occurs if pathogens and/or their toxins
from a septic focus enter the blood and the tissues.
Microbial factors important in septic shock include,
among others, polysaccharides (LPS) from Gram-negative bacteria, enzymes, and exotoxins of Gram-positive bacteria. A variety of mediators are active in the
pathogenesis of septic shock, among them active metabolites of the complement system (with impaired
function of polymorphonuclear cells (PMNs), the coagulation system and factors released from stimulated
cells like tumor necrosis factor (TNF-α) and interleukins IL-1, IL-6, and others. These important mediators
are produced by LPS-stimulated monocytes and macrophages. There are proinflammatory cytokines (IL-1,
TNF-α and IL-6) and antiinflammatory cytokines (IL4, IL-10) which modulate the proinflammatory immune response. Cytokines induce secondary mediators
which cause damage to cell function and structure.
Activated tissue macrophages and monocytes are the
cellular promotors of inflammation (1,4,5).
Gram-negative bacteremia is associated with shock in
40% of patients (1).
Patients with septic shock suffer of hemodynamic instability with decreased systemic vascular resistance
caused predominantly by high levels of nitric oxide.
Initially an increased cardiac output is seen associated
with hypovolemia and low blood pressure. About
50% of the patients suffer of impaired myocardial
function (septic cardiomyopathy) in the course of the
disease. About the same amount of all patients experience some form of end-organ damage caused by
altered distribution of cardiac output, impaired microcirculation, and capillary leak syndrome associated
with deterioration of the complement system. Complications include renal and liver failure, adult respiratory
distress syndrome (ARDS) with respiratory failure,
and disseminated intravascular coagulation (1).
MODS (multiple organ dysfunction syndrome) means
failure or dysfunction of two or more organs and is
responsible for the high mortality of sepsis.
Clinical manifestations of septic shock are fever of
more than 38.3° C (some patients have hypothermia
less than 36.0° C), chills, tachycardia, tachypnea of
more than 30 breaths per minute, encephalopathy
with mental state changes, hypotension, acrocyanosis, and gastrointestinal manifestations.
Laboratory data show leukocytosis of more than
12,000 cells/μl or leukopenia of fewer than 4000
cells/μl, elevated C reactive protein of more than
2 standard deviations over normal value, toxic anemia,
low angiotensin III levels, pathologic urine analysis,
and elevated lactate levels (1,2).
Hemodynamic parameters are arterial hypotension
(see definition above), cardiac index of more than
3.5 L/min/m2 and mixed venous saturation of more
than 70%.
Organ dysfunction parameters are arterial hypoxemia,
acute oliguria, hyperbilirubinemia, thrombocytopenia, ileus, creatinine increase of more than 0.5 mg/dL
and coagulation abnormalities (2).
Blood cultures are positive in about 60% of patients.
Cultures of wounds, urine, and tracheal secret may be
positive (1,2).
Basic treatment must be started rapidly and consists of
removal of the source of infection (e.g. wounds, cathe3
3
Characteristics
Septic shock is caused by:
* Gram-negative bacteria (60%–70%) such as Escherichia coli, Klebsiella, Enterobacter, Proteus,
Pseudomonas, Serratia)
* Gram-positive bacteria (20%–40%) including staphylococci, pneumococci, and streptococci
* opportunistic fungi (2%–3%)
* (rarely) other agents like Mycobacteria, protozoans
(Plasmodium falciparum) or viruses such as dengue
fever.
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defined by a systolic arterial pressure less than
90 mmHg, mean arterial pressure less than
60 mmHg or a reduction in systolic pressure of more
than 40 mmHg. In children, septic shock is defined as
tachycardia with signs of decreased organ perfusion
including decreased peripheral pulses compared to
central pulses, altered alertness, flash capillary refill
or capillary refill longer than 2 seconds, mottled or
cool extremities, or decreased urine output (2).
Terms with close relation to "septic shock" are sepsis,
severe sepsis, and SIRS (systemic inflammatory response syndrome). SIRS is present if patients have
more than one of the following findings: body temperature of more than 38.0° C or less than 36.0° C, heart
rate of more than 90/min, respiratory rate of more than
20/min or PaCO2 lower than 32 mmHg, white blood
cell count of more than 12 000 cells/μl or less than
4000 cells/μl. In this concept "sepsis" is defined as
SIRS plus infection, "severe sepsis" as sepsis with
organ dysfunction, hypoperfusion, or hypotension,
and "septic shock" as sepsis with arterial hypotension
despite adequate fluid resuscitation (2,3).
In septic shock, there are signs of inadequate organ
perfusion. Common symptoms are fever, chills, tachycardia, tachypnea, and altered mental state. Circulatory insufficiency with low systemic vascular resistance and decreased myocardial function causes diffuse cell and tissue injury and organ failure (1).
Different scores related to sepsis have been used.
PIRO is a new classification scheme for sepsis
(where the acronym stands for: predisposition, insult
(infection), response, organ dysfunction) that has recently been proposed, which is now being tested (2).
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Septicemia
In industrialized countries most cases of septic shock
are seen in hospitals. Measures to reduce the risk of
septicemia in the population include proper vaccination (i.e. for pneumococci and meningococci), proper
surgical wound treatment, and calculated antibiotic
treatment of community-acquired infections like pneumonia and pyelonephritis (1).
Relevance to Humans
The overall mortality of septic shock is 50%–80%.
The prognosis of septic shock is worse in patients
with rapidly fatal diseases (4). Mortality of patients
with Gram-positive bacteremia is higher than with
Gram-negative bacteremia (5). Severe sepsis is the
most common cause of death in noncoronary critical
care units (more than 200 000 patients annually in the
USA) (2).
About 50% of cases of septic shock are seen in already
hospitalized patients (nosocomial infection) (5). Most
patients with septic shock are elderly, chronically ill,
or suffer of underlying diseases or procedures that
make them susceptible to bloodstream invasion.
Patients at risk for septic shock include those with
cancer (cytostatic therapy), malnutrition, chronic infection, renal failure, diabetes mellitus, immunosuppression, polytrauma, cardiac shock, burns, and
organ perforation. Thus, for all invasive medical procedures, the general condition of the patient and his
chronic diseases must be taken into account to allow a
reasonable risk-benefit calculation. This concerns not
only operations but also invasive diagnostic procedures, intravenous lines, and catheters (1).
In the last decades, mortality of septic shock has remained high in spite of multiple improvements in supportive therapy (4). Prevention of shock requires repeated physical examination, swabs, blood cultures,
and intensive use of imaging techniques for the detection and treatment of the septic focus. Early and precise antibiotic therapy for endangered patients with
infections is strongly encouraged (1,4,5).
The International Sepsis Forum (2001) Guidelines
for the management of severe sepsis and septic
shock. Intens Care Med 27[Suppl 1]:51–134
References
1. Braunwald E, Fauci A, Kasper DL, Hanser SL, Longo
DL, Jameson JL (eds) (2001) Harrison's principles of
internal medicine. McGraw Hill, New York
2. Levy MM, Fink MP, Marshall JC et al. (2003) International Sepsis Definitions Conference 2001 SCCM/
ESICM/ACCP/ATS/SIS. Intens Care Med 29:530–538
3. American College of Chest Physicians/Society of Critical
Care Medicine (ACCP/SCCM) Consensus Conference
Committee (1992) Definitions for sepsis and organ failure
and guidelines for the use of innovative therapies in
sepsis. Crit Care Med 20:864–874
4. Eckart J, Forst H, Burchardi H (2002) Intensivmedizin.
Ecomed, Landsberg
5. van Aken H, Reinhart K, Zimpfer M (2000) Intensivmedizin. Thieme, Stuttgart
Septicemia
A systemic disease associated with the presence and
persistence of pathogenic microorganisms or their toxins in the blood.
Streptococcus Infection and Immunity
Septicemic Shock
Septic Shock
SEREX
Serological expression cloning (SEREX) of tumor
antigens is a molecular cloning technique based on
the screening of cDNA expression libraries from
tumor specimens using sera of cancer patients. Cloning strategies usually aim at tumor antigens recognized
by high-titer antibodies of the IgG class, thus requiring
T cell recognition and help for immunoglobulin class
switch. For this reason SEREX frequently picks up
genes coding for antigens recognized both by antibodies and by T cells.
Tumor, Immune Response to
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Preclinical Relevance
*
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ters), support of respiration, hemodynamic support (e.
g. monitored volume replacement, noradrenaline (norepinephrine)), parenteral nutrition, treatment of acidosis, and bactericide antibiotics. New treatment concepts include administration of hydrocortisone, activated protein C and intensive insulin therapy. Monitoring in an intensive care unit is necessary (4).
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Regulatory Environment
The national vaccination guidelines are:
* Control and Prevention of Meningococcal Disease:
Recommendations of the Advisory Committee on
Immunization Practices (ACIP) (2001). MMWR
Recommendations and Reports
Serine Protease Inhibitors
Circulating plasma proteins—including antithrombin
or protein C inhibitor, which serve as pseudo-sub-
Serotonin
In addition to the gastric mucosa and the pineal
gland, synthesis of serotonin occurs in the brain, spinal
cord, bronchi, thyroid, pancreas and thymus. Circulating serotonin does not enter the brain by crossing the
blood-brain barrier. Depots of serotonin in mammals
are the enterochromaffin cells of the gastrointestinal
tract (accounting for approximately 80% of total
body serotonin), serotonergic neurons of the brain,
the pineal gland, and platelets. Serotonin can be
released from cells by stimulation with acetylcholine,
noradrenergic nerve stimulation, increased intraluminal pressure and a decline of intestinal pH (1).
* Serotonin is a potent
vasoactive amine. In the
circulation it is almost entirely confined to platelets,
thereby rendered functionally inactive. Clearance
mechanisms have evolved to decrease plasma serotonin concentrations: platelets possess an active serotonin uptake system
* the liver catabolizes serotonin
* pulmonary endothelial cells take up serotonin
* specific macromolecules bind free serotonin.
3
3
strates for coagulation proteases such as thrombin, factor Xa or activated protein C—form covalent inactive
complexes with the enzyme and thereby block its action. Heparin, or other glycosaminoglycans to which
these inhibitors and the proteases can bind simultaneously, catalyse the enzyme inhibition several fold
and thereby are effective anticoagulants.
Blood Coagulation
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Serotonin
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Helen V Ratajczak
Boehringer Ingelheim Pharmaceuticals
900 Ridgebury Road
Ridgefield, CT 06877
USA
Synonyms
serotonin, 5-hydroxytryptamine, 5-HT, enteramine
Definition
Serotonin (5-hydroxytryptamine, 5-HT) is classed as a
hormone and has been found to be the most diverse
physiological substance in the body. Serotonin is a
neurotransmitter in the central nervous system, regulating functions such as sleep, mood, and appetite.
Serotonin influences production of other hormones
and interacts with the immune system.
Serotonin was first isolated from enterochromaffin
cells originating from the gastric and intestinal mucosa
by Ersparmer and Vialli in 1937. It was characterized
by its ability to cause smooth muscle contraction and
was named enteramine. About the same time Rapport
isolated a substance from serum that caused vasoconstriction, and named it serotonin. After purification,
structure elucidation and chemical synthesis, enteramine was found to be identical to serotonin (1).
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Characteristics
The synthesis of serotonin takes place primarily in
gastric and intestinal mucosa and in the pineal
gland. It is synthesized from the essential amino acid
tryptophan which is also a precursor of the vitamin
niacin (nicotinamide) and of another hormone,
melatonin. Under normal conditions biosynthesis
accounts for only 2% of ingested tryptophan, leading
to a daily production of about 10 mg serotonin. The
major part of tryptophan is utilized for protein synthesis (1). Tryptophan and niacin are found in milk, beef,
whole eggs, salt pork, wheat flour, corn, lean meats,
poultry, fish, peanuts, organ meats, and brewer’s yeast,
with lower amounts in beans, peas, other legumes,
most nuts, whole grains, and enriched cereals (2).
Circulating plasma serotonin is taken up by platelets
mainly by an active transport mechanism. Platelet serotonin content is elevated in people with serotoninsecreting carcinoid tumors and during long-term serotonin ingestion. Platelet serotonin half-life is about
4.2 days, which approximates to that of platelets. In
platelets, serotonin is stored in dense granules. The
platelet membrane contains two types of serotonin
binding sites. One site mediates uptake, and the
other causes platelet aggregation. In addition to the
active uptake, a passive uptake process occurs at
high extracellular serotonin concentrations and is proportional to serotonin levels (1).
There is interaction between the central nervous and
the immune systems. Serotonin binding sites have
been demonstrated on lymphocytes, eosinophils, and
macrophages. Serotonin and its receptors are present
in the immune system in three types of molecular
structures:
* guanine nucleotide-binding protein-coupled receptors
* ligand-gated ion channels
* transporters.
Conversely, lymphokines and their respective receptors are present in the nervous and neuroendocrine
systems. Another major pathway of interaction between the two systems is direct neural connections
through the innervation of lymphoid organs. Serotonin
is synthesized in epithelial cells, peptigergic neurons,
and in leukocytes themselves in lymphoid organs, and
is released in active form in the periphery (1).
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Serotonin
Serotonin. Figure 1
Preclinical Relevance
The pharmacology of serotonin is particularly complex, with several receptor subtypes mediating responses at the different affinities identified. Brain serotonin influences the immune response via the hypothalamic-pituitary axis. An increase of brain serotonin
has been shown to be immunosuppressive (3).
In peripheral blood, serotonin is localized primarily in
platelets and is released at sites of injury. It has been
shown to affect immune function both in vivo and in
vitro.
In vivo studies include complex effects of serotonin
on:
* lymphocyte subpopulation numbers
* peripheral leukocyte function
* suppression of immune response (decreased immunoglobulin IgM and IgG plaque-forming responses
to sheep red blood cells in mice), and
* a permissive role in delayed-type hypersensitivity.
In vitro effects include:
a 50% decrease in mitogen-induced lymphocyte
proliferation and nearly complete inhibition of the
production of interferon(IFN)-γ and other lymphokines
* suppression of IFN-γ-induced Ia expression by
macrophages and phagocytes
* release of certain lymphokines and a polymorphonuclear cell chemotactic factor,
*
*
up to 50% augmentation of natural killer (NK) cell
cytotoxicity.
The serotonin receptor subtype 5-HT1a mediates the
effect of serotonin on the activation of NK cells by
monocytes and participates in the release of adrenocorticotropic hormone (ACTH) from the hypothalamus or pituitary. Serotonin has also been shown to
affect potassium channels in a transformed lymphocyte cell line. Because the 5-HT1a receptor subtype
is a target for newly developed anxiolytic medications,
these drugs may have effects on immune function (4).
An inverse relationship exists between brain serotonin
levels and antibody production: reduction of brain serotonin levels stimulates antibody production (during
the primary immune response), increases longevity,
and delays onset of tumor growth. Interactions between tumor growth and the pineal are likely the consequence of nutrient and metabolic changes in tissues
supporting the tumor cells, which in turn are due to an
altered endocrine balance. The inhibitory effects of 5hydroxytryptophan (5-HTP) can be reversed with exogenous luteinizing hormone, follicle-stimulating hormone, and ACTH, all of which are influenced by serotonergic pathways. In contrast, injection of 5-hydroxy tryptophane—the immediate precursor of serotonin—has been found to suppress the immune response, with increased latent period of antibody for-
Serotonin
mation and decreased intensity of both the primary and
secondary immune responses (2).
Relevance to Humans
Physiology
Serotonin is involved in a variety of physiological
processes, including smooth muscle contraction,
blood pressure regulation and both peripheral and central nervous system neurotransmission. In the central
nervous system serotonin acts as a neurotransmitterneuromodulator that is implicated in sleep pattern regulation, appetite control, sexual activity, aggression
and drive. Central nervous system serotonin exerts
its actions in concert with other neurotransmitters. In
the periphery serotonin acts as a vasoconstrictor and
proaggregator when released from aggregating platelets, as a neurotransmitter in the enteric plexuses of the
gut and as an autocrine hormone when released from
enterochromaffin cells from the gut, pancreas and elsewhere (1).
A basic knowledge of immune neuroendocrine interactions may be important to understanding certain disease mechanisms. Abnormalities of serotonin-related
processes give rise to various pathological conditions;
aberrations in central nervous system function are implicated in anorexia, anxiety, depression and schizophrenia, whereas degeneration of serotonergic neurons
has been noted in Alzheimer disease and Parkinson
disease, peripheral aberrations in drug-induced emesis,
hypertension, migraine, genesis of cardiac arrhythmias, Raynaud’s disease, fibrotic syndromes and
some symptoms of the carcinoid syndrome.
The quantitatively most pronounced aberration in serotonin production and metabolism is in people with
carcinoid tumors. Midgut carcinoid tumors produce
and secrete serotonin. Carcinoid patients may convert
as much as 60% of dietary tryptophan to serotonin.
Long-term augmentation of the serotonin biosynthetic
pathway may result in serious reduction of the freetryptophan body pool, causing niacin deficiency and
subsequent development of pellagra-like symptoms (1).
Platelet serotonin content is age-dependent—but not
gender-dependent. Platelet serotonin concentration in
elderly subjects is significantly lower than in adults
and children and significantly higher than in newborns. There is no significant variation in platelet serotonin content over a period of 24 hours, nor is there
any difference in levels in different seasons of the year.
Human in vivo concentration is 168 ± 13 ng/ml whole
blood and 341 ng/109 platelets. In contrast, both free
and total plasma tryptophan (the serotonin precursor)
have a circadian rhythm, with maximum values observed in the afternoon and minimum values at
night (1).
Platelet serotonin and other related compounds are
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increased in the presence of serotonin-producing carcinoid tumors. Platelet serotonin is a more sensitive
marker for increased serotonin production by carcinoid tumors than urinary 5-hydroxyindoleacetic acid
(5-HIAA). In cases with high serotonin secretion rate,
platelet serotonin reaches a maximum at approximately 50 nmol/109 platelets, whereas urinary 5HIAA does not. Other neuroendocrine tumors and celiac disease give moderately increased platelet and
plasma serotonin content, urinary serotonin and 5HIAA excretion. Increased concentrations of plateletpoor plasma serotonin have been found in several disease states such as preeclampsia and type I diabetes.
Menstrual cycle dependency was found, with higher
periovulatory and premenstrual concentration of serotonin found in platelet-poor plasma (1).
Significantly reduced platelet serotonin can be found
in subjects using selective serotonin reuptake inhibitors. Plasma tryptophan levels are dependent on dietary intake and have been found reduced in malabsorption syndromes, in several psychiatric disease states,
and in carcinoid disease. Cerebrospinal fluid (CSF)
levels of indoles are also dependent on dietary intake
of tryptophan and are reduced in several neurodegenerative and psychiatric disease states (1).
Blood platelets play a major role in normal hemostasis
and in the formation of occlusive thrombotic disorders.
Acquired platelet dysfunction after coronary clot formation likely affects short-term and long-term outcomes in patients after acute coronary events. Therefore, inhibiting platelet function is an important therapeutic goal in patients with acute coronary artery disease (5).
Clinical depression has been identified as an independent risk factor for increased mortality in patients after
an acute myocardial infarction, with increased platelet
activity suggested as the mechanism for this adverse
association. The prevalence of depression after an
acute myocardial infarction is 15%–20%. Studies suggest that serotonin not only has a role in the neuropathophysiology of depression but also promotes
thrombogenesis directly by enhancing platelet aggregation. Depressed patients exhibited 41% more platelet activation and higher procoagulant properties than
healthy controls (5).
Excessive transcardiac accumulation of serotonin has
been demonstrated in patients when chronic stable angina is converted to unstable coronary syndromes. Serotonin has been shown to be an important mediator of
intermittent coronary obstruction caused by platelet
aggregation and dynamic vasoconstriction. Recent
clinical evidence showed there were reduced restenosis rates in people after angioplasty treated with selective serotonin reuptake inhibitors (5).
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Serum Sickness
References
1. Kema IP, deVries EGE, Muskiet FAJ (2000) Clinical
chemistry of serotonin and metabolites. J Chromatog B
747:33–48
2. Mahan LK, Arlin M (eds) (1992) Krause’s Food,
Nutrition and Diet Therapy, 8th edn. WB Saunders,
Philadelphia
3. Ader R (ed) (1981) Psychoneuroimmunology. Academic
Press, New York
4. Plotnikoff N, Murgo A, Faith R, Wybran J (eds) (1991)
Stress and Immunity. CRC Press, London
5. Nair GV, Gurbel PA, O’Connor CM, Gattis WA,
Murugesan SR, Serebruany VL (1999) Depression,
coronary events, platelet inhibition, and serotonin
reuptake inhibitors. Am J Cardiol 84:321–323
Serum Sickness
histologic types. Opposed to unique tumor antigens
that are ideally present in single individual tumors
without cross-reactivity with other tumors of the
same or of other histotypes.
Tumor, Immune Response to
Sheep Red Blood Cell Receptor
These bind to the receptor CD2 on T cells.
Leukocyte Culture: Considerations for In Vitro Culture of T cells in Immunotoxicological Studies
Sheep Red Blood Cells (SRBC)
The most common antigen used by immunotoxicologists to induce a T cell-dependent antibody response.
Glucocorticoids
Immunoassays
Plaque-forming Cell Assay
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Serotonin is not a focused concern of the regulatory
environment.
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Regulatory Environment
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Serum sickness is caused by i.v. injection of foreign
proteins (antigen) which then lead to formation of immune complexes (antibody-antigen complexes). This
type III hyperreaction can be interpreted as a generalized Arthus reaction. It was first described in 1905 by
Piquet and Schick. The host's immunological hyperreaction is characterized by rash, fever, lymphadenopathy, athralgia, and/or nephritis.
Serum Sickness
Shingles
Caused by reactivation of latent varicella virus in sensory root ganglia in patients previously infected with
chickenpox. The lesions are vesicular on erythematous
base and usually appear along the line of one or two
dermatomes.
Dermatological Infections
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Seveso-Dioxin
Dioxins and the Immune System
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Seveso-Poison
Dioxins and the Immune System
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SFC
Signal Transduction During
Lymphocyte Activation
Kathleen M Brundage
Dept of Microbiology, Immunology and Cell Biology
West Virginia University
P.O.Box 9177
Morgantown, WV 26506-9177
USA
Synonyms
Plaque-Forming Cell Assays
Signaling through antigen receptors.
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Definition
Shared Tumor Antigens
Tumor antigens shared by many tumors of various
Signal transduction is by definition the conversion of a
signal from one form to another. For lymphocytes,
signal transduction begins at the plasma membrane
and is initiated by the binding of antigen to the
Signal Transduction During Lymphocyte Activation
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Activation of signal transduction pathways within
lymphocytes occurs when the antigen specific receptors (TCR for T cells and BCR for B cells) bind their
specific antigen. Receptor engagement results in the
clustering and reorganization of the plasma membrane
into large lipid rafts. It is hypothesized that these
lipid rafts allow for the quick and efficient activation
of signaling cascades upon antigen binding by the receptor. In T cells, these rafts are enriched with hyperphosphorylated CD3ζ chain and most of the T cell
receptor complex (TCR complex)-associated phosphorylated and activated Zap70. In B cells, these
rafts are enriched with B cell receptor complexes
(BCR complexes) containing phosphorylated immunoglobulin (Ig)α and Lyn.
Engagement of the TCR by peptide-MHC (major histocompatability complex) presented on antigen-presenting cells (APCs) results in the hyperphosphorylation of ITAM motifs ( immunoreceptor tyrosinebased activation motifs) on the CD3ε, γ and δ chains
by the Src family kinase members Lck and Fyn
(Figure 1). Phosphorylated ITAMs recruit the tyrosine
kinase Zap70 to the receptor complex. Lck activates
Zap70 by phosphorylation. Activated Zap70 recruits
and activates two adaptors, LAT (linker of activation
in T cells) and Slp76. These two adaptors recruit other
proteins that bind through their SH2 domains. One of
the proteins recruited in this manner is the Tec kinase
Itk. Itk is phosphorylated by Lck and phosphorylates
PLCγ (phospholipase C gamma) thereby activating
PLCγ. PLCγ is responsible for the cleavage of the
plasma membrane phospholipid phosphatidylinositol
bisphosphate (PIP2) into inositol trisphosphate (IP3)
and diacylglycerol (DAG). IP3 binds its receptor on
the endoplasmic reticulum resulting in the release of
calcium ions (Ca2+) from intracellular stores.
Release of the intracellular Ca2+ stores triggers the
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Characteristics
Signaling Through the TCR
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T cell receptor (TCR) or the B cell receptor (BCR). As
a result of this binding the activation of several signaling cascades occurs, resulting in the propagation and
expansion of the initial signal. For lymphocytes, ultimately the response to extracellular signals is the induction of a new gene transcription pattern.
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Signal Transduction During Lymphocyte Activation. Figure 1 Signaling through the T cell receptor.
Signal Transduction During Lymphocyte Activation
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The other product of PIP2 cleavage is DAG. DAG
remains associated with the plasma membrane and
activates members of the protein kinase C (PKC) family in particular PKCθ. PKCθ is a serine/threonine
protein kinase that is responsible for phosphorylating the serine/threonine kinase Raf-1, which interacts
with the small G protein Ras. The Ras-Raf-1 complex is responsible for activating several MAP kinase cascade pathways. These pathways eventually
result in the activation of several transcription factors including c-fos, Elk and c-jun.
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In T-cells there are at least two pathways that can activate Ras. One of these pathways involves the adaptor
protein LAT, which as discussed above, is activated by
Zap70. Activated LAT binds to the adaptor Gads,
which in turn, interacts with and activates the guanine exchange factor (GEF) SOS. Once activated SOS
activates Ras, which as previously discussed, goes on
to activate the MAP kinase pathways. Ras is also
activated when the co-stimulatory molecule CD28,
which is expressed on the surface of T cells, interacts
with its ligand B7.1 or B7.2 (CD80) expressed on
APCs. In addition to activating Ras, CD28 engagement also activates phosphatidylinositol 3-OH kinase
(PI-3 kinase). PI-3 kinase can activate several other
proteins including the adaptor VAV. Besides being an
adaptor, VAV is also a GEF similar to SOS. In addition, VAV can also be activated by Zap70. VAV activates Rac, a small G protein, and this results in the
cytoskeletal rearrangements necessary for T cell activation. VAV also activates PKCθ leading to the activation of Raf-1 and the activation of the MAP kinase
pathways as described above. In addition PKCθ directly activates the Iκ kinases (IKK) α/β. These kinases are also activated by AKT, another kinase that is
activated by PI-3 kinase. Activated IKK phosphorylates IκB, the inhibitory protein responsible for retaining the transcription factor NF-κB in the cytoplasm.
For NF-κB to be released from IκB, IκB has to be
phosphorylated, ubiquitinated and degraded. Once released from IκB, NF-κB translocates into the nucleus
where it binds to its target DNA binding site located in
the promoters of many genes.
Signaling Through the BCR
The initial steps in B cell activation involve the crosslinking of the cell surface immunoglobulin receptor
(BCR), by antigen. Prior to antigen binding, the
Src family kinases Blk and Lyn are only weakly
associated with the unphosphorylated ITAMs of the
invariant Igα and Igβ chains of the BCR complex.
Upon crosslinking and clustering of the BCR, Blk
and Lyn phosphorylate the ITAMs on the Igα and
Igβ chains (Figure 2). The phosphorylated ITAMs
recruit the tyrosine kinase Syk, which is activated by
phosphorylation by other Syk proteins as well as by
Blk and Lyn. Activated Syk phosphorylates the adaptor BLNK (B-cell linker adaptor protein) also known
as Slp-65. BLNK activates the Tec family kinase Btk.
Btk acts in a manner similar to Itk in T-cells, activating
PLCγ the protein responsible for cleaving PIP2 into
DAG and IP3. As previously described, DAG and IP3
are responsible for the activation of RAS, which activates the MAP kinase signaling cascade, and the calcium-dependent pathways that lead to the activation of
NFAT. BLNK also interacts with the adaptor Shc,
which binds the adaptor Grb2. The Shc-Grb2 complex
binds the GEF SOS, which in turn activates Ras as
previously described. In B cells, Ras is also activated
when the B cell co-receptor CD19 binds its ligand.
CD19-induced Ras activation is through activation of
VAV, which is activated by PI-3 kinase as previously
described for T cells. Activated VAV activates the
small G protein Rac which results in cytoskeletal rearrangements. As previously described for T cell activation, PI-3 kinase will also activate the kinase AKT
as part of the signaling cascade, resulting in the activation of the transcription factor NF-κB.
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opening of calcium channels in the plasma membrane,
allowing more Ca2+ into the cell. This increase in intracellular Ca2+ activates the calcium-dependent serine/threonine phosphatase calcineurin. Dephosphorylation of the transcription factor NFAT (nuclear factor of activated T cells) by calcineurin releases NFAT
from its cytoplasmic binding partner 14-3-3. Once released from 14-3-3, NFAT translocates into the nucleus where it binds to its target DNA binding sites located in the promoters of some genes, including cytokines such as the interleukin (IL)-2.
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Signaling Through Co-Receptors
In addition to the signaling pathways activated by
TCR and BCR complexes described above, there are
other co-stimulatory receptors on the surface of T cells
and B cells that have a role in cell activation. These
other receptors include CD19 (previously described),
CD21 (complement 2 receptor) and CD81 (TAPA-1)
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Signal Transduction During Lymphocyte Activation
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Signal Transduction During Lymphocyte Activation. Figure 2
ing protein tyrosine phosphatase-1), SHP-2 (src homology 2 (SH2) domain-containing protein tyrosine
phosphatase-2) and SHIP (SH2-containing inositol
phosphatase). These phosphatases are responsible for
the in activation of many protein kinases including Btk
and Itk as well as IP3 (an initiator of the calcium activation pathway).
A universal mechanism for the activation of signaling
pathways is phosphorylation by protein kinases. Once
these pathways have been activated they must be inactivated in order to prevent unregulated growth. In
many cases to inactivate these pathways, phosphatases
which can be activated by protein kinases, dephosphorylation the protein kinases thereby inactivating
them. In addition, some transcription factors initiate
transcription of their own inhibitors. For example
NF-κB initiates transcription of its inhibitor IκB, the
protein responsible for retaining NF-κB in an inactive
state in the cytoplasm. The activation of lymphocytes
is a tightly regulated process. During the lifespan of an
individual, the lymphocytes and the immune system
must maintain a delicate balance between both the
activation and inhibitory pathways in order to function
properly and protect an individual from infection or
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on B cells, and CD28 (previously described) and
CD40L on T cells, as well as cytokine receptors and
adhesion molecules. Thus, the activation of a lymphocyte is a complex process involving the interplay of
many proteins and the activation of many interconnected signaling cascades to expand and propagate
the initial signals. As stated previously, the ultimate
goal of the activation of these different signaling cascades is the initiation of new gene expression patterns.
Besides activation signaling cascades there are also
inhibitory signaling cascades. For lymphocytes inhibitory signals usually block the response by raising the
threshold at which signal transduction can occur. On
B cells, receptors such as Fc-receptor gamma IIB-1
(binds Fc portion of IgG), CD22 and PIR-B (paired
immunoglobulin-like receptor) are involved in inhibiting activation. On T cells, receptors such as CTLA-4
(cytotoxic T-lymphocyte-associated) and KIRs (killer
inhibitory receptors) inhibit the activation of T cells. A
common motif found in the cytoplasmic tail of
many inhibitory receptors is the ITIMs (immunoreceptor tyrosine-based inhibitory motifs). This motif, upon
phosphorylation, recruits the inhibitory phosphatases SHP-1 (Src homology 2 (SH2) domain-contain-
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Signaling Through Antigen Receptors
the development of autoimmune diseases.
3. Gauld SB, Dal Porto J, Cambier JC (2002) B cell antigen
receptor signaling: roles in cell development and disease.
Science 296:1641–1642
4. Dong C, Davis RJ, Flavell RA (2002) MAP kinases in the
immune response. Ann Rev Immunol 20:55–72
5. Jordan MS, Singer AL, Koretzky GA (2003) Adaptors as
central mediators of signal transduction in immune cells.
Nature Immunol 4:110–116
Preclinical Relevance
Signaling Through Antigen Receptors
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The investigations into the effect of chemicals on the
signaling pathways that lead to lymphocyte activation
are not regulated under special guidelines. However,
there are several examples in the literature of chemicals (e.g. cannabinol and the herbicide propanil) which
have been shown to alter the signaling pathways that
lead to the activation of lymphocytes.
Signal Transduction During Lymphocyte Activation
Single Amino Acid Polymorphisms
Relevance to Humans
1. Luster MI, Munson AE, Thomas PT et al. (1988)
Development of a testing battery to assess chemicalinduced Immunotoxicology: National Toxicology Program’s guidelines for immunotoxicity evaluation in mice.
Fund Appl Toxicol 10:2–19
2. Lewis RS (2001) Calcium signaling mechanisms in
T lymphocytes. Ann Rev Immunol 19:497–521
Cytokine Polymorphisms and Immunotoxicology
Sjögren Syndrome
A chronic systematic inflammatory disorder characterized by dryness of mucus membranes.
Systemic Autoimmunity
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References
Cytokine Polymorphisms and Immunotoxicology
Single Nucleotide Polymorphisms
3
At this time there are no specific guidelines for determining effects of chemicals on the signaling pathways
that lead to the activation of lymphocytes. However
there are National Toxicology Program’s guidelines
for Immunotoxicology evaluation in mice which recommend examining the effect of a chemical on lymphocyte blastogenesis in response to a mitogen and a
mixed lymphocyte response assay to allogenic lymphocytes (1). These assays measure the proliferative
and activation capacity of lymphocytes. In order for
proliferation or activation to occur a lymphocyte must
turn on new or alter gene transcription. It is clear that
alteration of these responses by chemical exposure
could be the result of alteration in the activation of
one or more of the signaling cascades described here.
Cytokine Polymorphisms and Immunotoxicology
Single Base Transition
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Regulatory Environment
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Any chemical or compound that inhibits the ability of
lymphocytes to respond to antigenic stimulation and
become activated will ultimately have an affect on the
ability of an individual to fight an infection. Paradoxically, a chemical or compound which enhances the
immune response and the ability of lymphocytes to
become activated can also be detrimental. Individuals
who are predisposed to develop autoimmune disease
or who have a pre-existing autoimmune disease an
enhanced immune response can increase both the potential to develop an autoimmune disease and/or the
severity of the disease.
Skin, Contribution to Immunity
Emanuela Corsini
Dapartment of Pharmacological Sciences
University of Milan
Via Balzaretti 9
20133 Milan
Italy
Synonyms
Local immune system
Skin, Contribution to Immunity
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Characteristics
The skin represents the interface between the environment and internal organs and it is essential for survival. The skin protects the body against external insults
and prevents water loss. The skin participates directly
in thermal, electrolyte, hormonal, metabolic, and immune regulation. Skin is constantly exposed to many
antigens. Rather than merely repelling them, the skin
has a rapid response immune system of its own to
ward off invasion by infectious agents, and to react
to noxious chemicals applied to the skin.
Skin Structure
In order to understand the defensive capacities of the
skin, it is important to define its structural basis. The
skin consists of two major components: the outer epidermis and the underlying dermis, which are separated
by a basal membrane. The epidermis is a multilayered
epithelium composed of several different cell types.
Keratinocytes (KC) are the major epidermal cell
type, and represent about 95% of epidermal cell
mass. They are responsible for the biochemical and
physical integrity of the skin via the formation of the
stratum corneum. Langerhans cells (LC) comprise the
second most prominet cell type in the epidermis. LC
are bone-marrow derived dendritic cells and represent
only 2%–5% of epidermal cell population. Melanocytes represent 3% of epidermal cells and by generating melanin they protect the skin against ultraviolet
radiation. The blood supply to the epidermis is guaranteed by the capillaries located in the rete ridges at
the dermal-epidermal junction. The dermis is separated from underlying tissues by a layer of adipocytes
(hypodermis). The dermis makes up approximately
90% of the skin and has mainly a supportive function.
In addition, epidermal appendages (hair follicles, sebaceous glands and eccrine glands) span the epidermis and are embedded in the dermis.
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Atopy is the clinical manifestation of type I hypersensitivity reactions including eczema, asthma and rhinitis. General predisposition toward development of
IgE-mediated hypersensitivity reactions toward common environmental antigens. Atopic dermatitis is a
chronic, itching, inflammation of the skin in atopic
individuals. Contact hypersensitivity is a delayed inflammatory reaction on the skin seen in type IV hypersensitivity, resulting from allergic sensitization.
Dermatitis is an inflammatory skin disease showing
redness, swelling, infiltration, scaling and sometimes
vescicles and blisters. Percutaneous absorption represents the passage of compounds across the skin. The
composition and structure of the stratum corneum determine the pathways for diffusion as well as the solubility and diffusivity of compounds within the skin.
The skin is a physical barrier between an organism and
its environment. It can be divided into four different
regions: the stratum corneum, the viable epidermis, the
dermis and the hypodermis. Skin irritation is a form
of skin inflammation induced by primary contact with
chemicals and is thought not to be mediated by lymphocytes. Stratum corneum is the outermost layer of
the skin, the primary barrier to percutaneous absorption. The organization of the stratum corneum can be
viewed as a brick wall with the bricks representing the
corneocytes and the mortar representing the intercellular lipids. It contains approximately 15% water, 70%
protein and 15% lipid.
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Definitions
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Skin Immune System
Besides its barrier function, the skin has been recognized as an immunologically active tissue. The immune system, associated with mucosal surfaces,
evolved mechanisms discriminating between harmless
antigens and commensal microorganisms and dangerous pathogens (1). LC are the principal antigen-presenting cells in the skin, and the KC act as signal
transducers, converting nonspecific exogenous stimuli
into the production of cytokines, chemotactic factors
and adhesion molecules. Cells in the dermis and epidermis, including dermal dendritic cells (DC), epidermal LC, melanocytes, and migrating lymphocytes, are
all important in skin immune reaction and are know to
produce a great variety of cytokines.
The skin innate immune response is rapid, provides
the initial line of defense against microorganisms, is
antigen nonspecific, and lacks immunological memory. Cellular constituents of the skin innate immune
system include keratinocytes, DC, macrophages, natural killer cells and neutrophils. The innate immune
system is able to recognize the conserved pathogenic
patterns on microbes by pattern recognition receptors,
such as the Toll-like receptor and others. Adaptive
immunity, in contrast, is delayed in time, is antigen
specific, and involves a recall response. A proper balance between innate and adaptive immunity is essential, because if the innate response is inadequate septic
shock may result from a cutaneous bacterial infection,
or the exaggerated innate response may yield a chronic
inflammatory response.
Epidermal Cytokines
The major mechanisms used by epidermal cells to
participate in immune and inflammatory skin reactions
are the production of cytokines and responses to cytokines (2). Within the epidermis, the KC are the major
source of cytokines, along with the LC and melanocytes (3). Epidermal cells can produce constitutive or
following activation an arsenal of cytokines, strongly
supporting the idea that the skin functions as an immune organ and that an important role of the skin is to
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Skin, Contribution to Immunity
provide an immune barrier between the external environment and internal tissues. Table 1 gives a list of the
cytokines produced by epidermal cells.
The histopathological pattern of nearly every inflammatory skin disease can be accounted for by the appropriate cytokine or combination of cytokines (4). It
is important to remember that multiple mechanisms
and cell types may be involved in the induction of
skin toxic responses. Determining the source, the kinetics of production, and the regulation of inflammatory mediators in the skin will be of value in predicting
the various toxicities arising from exposure to environmental agents. Differences in skin toxic responses
may be the result of early differences in the epidermal
response to cell injury, and in the production of inflammatory signals.
logically active IL-1α, in addition to inactive pro-IL1β. Damage to the KC releases IL-1α, which essentially is a primary event in skin defense. IL-1α stimulates further release of IL-1α and the production and
release of other cytokines such as IL-8, IL-6, and GMCSF. Thus, by cytokine cascades and networks, an
inflammatory response can be rapidly generated. In
this scenario, KC act as proinflammatory signal transducers, responding to nonspecific external stimuli with
the production of inflammatory cytokines, adhesion
molecules, and chemotactic factors, preparing the dermal stroma for specific immunological activity.
On the other hand, in the skin, TNF-α is stored in
dermal mast cells, but following stimulation it may
be produced by KC and LC. Antibody to TNF-α
abolishes many inflammatory skin reactions, including
allergic and irritant contact dermatitis (6). An important mechanism by which TNF-α influences the development of an inflammatory reaction is induction of
the expression of cutaneous and endothelial adhesion
molecules.
3
Contribution of Keratinocytes to Skin Immunity
In the last two decades it has become clear that the KC
play an important role in the initiation and perpetuation of skin inflammatory and immunological reactions. While resting KC produce some cytokines constitutively, a variety of environmental stimuli, such as
tumor promoters, ultraviolet light and chemical agents,
induce epidermal KC to release inflammatory cytokines (IL-1, TNF-α), chemotactic cytokines (IL-8,
IP-10), growth promoting cytokines (IL-6, IL-7, IL15, GM-CSF, TGF-α) and cytokines regulating humoral versus cellular immunity (IL-10, IL-12, IL-18)
(3). Cytokine production by KC can affect migration
of inflammatory cells, can have systemic effects on the
immune system, can influence KC proliferation and
differentiation processes; and regulate the production
of other cytokines.
Of all the cytokines produced by KC, only IL-1α, IL1β and TNF-α activate a sufficient number of effector
mechanisms to independently trigger cutaneous inflammation (5). Unstimulated KC contain large
amount —in biological terms—of preformed and bio-
Contribution of Langerhans cells to Skin Immunity
DC and LC are unique cell types, in that they function
in both innate and specific immunity, depending on
their state of maturation and local microenvironmental
conditions. It has been recently reported that LC have
the potential to reduce inflammatory responses in skin
(reviewed by Nickoloff (7)). There is evidence that LC
in normal skin may actually function as antiinflammatory agents to counter balance the proinflammatory
tendencies of keratinocytes. When LC are decreased
in number or function within the epidermis an enhanced inflammatory reaction in skin is observed.
LC are intraepidermal antigen-presenting cells whose
dendrites intercalate between KC. In normal skin, immature LC monitor the environment as sentinel cells,
on guard to detect foreign intruders. An antigen when
absorbed through the epidemis will likely encounter a
Skin, Contribution to Immunity. Table 1 Cytokines expressed by epidermal cells
Epidermal
Cells
Cytokine (Constitutive or Inducible Expression)
Keratinocytes
IL-1α, IL-1β, IL-1RA, IL-3 (mouse), IL-6, IL-7, IL-8 (human), IL-10, IL-12, IL-15, IL-18, IL-20,
TNF-α, G-CSF, GM-CSF, M-CSF, GRO, MIP-2 (mouse), IP-10, RANTES, MCP-1, TGF-α,
TGF-β
Langerhans
cells
IL-1α, IL-1β, IL-6, IL-15, IL-18, TNF-α, Gro, MIP-2, MIP-1α, TGF-β
Melanocytes
IL-1α, IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12, IL-24, TNF-α, G-CSF, GM-CSF, M-CSF, GRO, MIP-2
(mouse), RANTES, MCP-1, TGF-α, TGF-β
G-CSF=granulocyte-colony stimulating factor; GRO=Growth Related Oncogene, IL=interleukin; MCP=monocyte chemotactic
protein; MIP=macrophage inflammatory protein; RANTES=regulated on activation, T-cell expressed and secreted;
TGF=transforming growth factor; TNF=tumor necrosis factor.
Skin, Contribution to Immunity
Preclinical Relevance
Whereas irritant contact dermatitis is a form of skin
inflammation induced by primary contact with chemicals and is thought not to be mediated by lymphocytes,
allergic contact dermatitis represents a lymphocytemediated delayed-type hypersensitivity reaction that
requires previous sensitization by the same chemicals.
The biochemical mechanisms involved in skin irritation are complex and not fully understood. Different
skin irritants can trigger different inflammatory process. In addition to destroying tissue directly, chemicals can alter cell functions and/or trigger the release,
formation, or activation of autocoids, such as histamine, arachidonic acid metabolites, kinins, complement, reactive oxygen species and cytokines. Substances that are keratin solvents, dehydrating agents,
oxidizing or reducing agents, and others, may be irritants. Due to the eterogenicity of skin irritants, there is
no reliable method for assessing irritancy based on
chemical structure. Virtually any chemical substance
may be an irritant under conditions of exposure that
predispose to the occurrence of an irritant response.
The biological processes necessary for producing hypersensitivity are grouped into two phases: an induction phase and an elicitation phase. Induction has been
referred to as the afferent phase, the initial exposure
through clonal expansion and the release of memory
cells. Elicitation, the efferent phase, consists of local
recognition of the antigen by the memory cells, the
release of cytokines, and the activity of inflammatory
mediators which are generated locally and produce the
dermatitis.
3
LC. The antigen is then captured and processed by LC.
Activated LC move out of the epidermis into the dermis, and into the regional lymphatic system, eventually finding their way to the regional draining
lymph node. In the lymph node, LC differentiate
into mature dendritic cells and present antigen to specific T lymphocytes, using major histocompatibility
complex (MHC) class II molecules to hold the processed antigen in place. Adhesion molecules on both
the antigen-presenting cell (i.e. B7) and the T cell (i.e.
CD28) ensure appropriate contact and costimulation.
Following appropriate stimulus, a clone of T cells is
produced with the ability to react to the antigen which
caused their production. The activation and clonal expansion of allergen-reactive T cells is the pivotal event
in the acquisition of skin sensitization.
If the antigen persists or if the antigen is re-encountered, again LC in the epidermis take up the antigen
and migrate into the dermis. In theory, there is contact
with specific antigen-responsive T cells in the dermis.
This is because clones of lymphocytes stimulated by
skin-encountered antigens “home” to the skin using
special receptors on the cell surface that attach only
the skin microvasculature. Antigen is presented to
these antigen specific T cells. The lymphocytes divide
and release a large amount of proinflammatory cytokines that mediate the ensuing inflammatory response.
These T cell products also activate KC, which in turn
produce cytokines. Inflammation eventually eliminates the antigen, either by killing the viable organisms, washing out the antigen with edema fluid, or
engulfing particulates in activated macrophages, neutrophils, or other phagocytic cells.
At least in experimental animals it has been shown that
there are two major populations of DC in afferent
lymph draining the skin that differ in their capacity
to stimulate CD4 and CD8 T cells. While expression
of the costimulatory molecules CD80, CD86 and
CD40 appear similar for both DC populations, differences in expression of cytokine transcripts have been
shown: the CD11a+/SIRPalpha−/CD26+ population
synthesizes more IL-12, whilst the CD11a−/SIRPalpha+ population produces more IL-10 and IL-1α.
This is likely to affect the bias of the immune response
following presentation of antigen to T cells by one DC
subpopulation or the other. It is proposed that SIRPalpha− DC would promote a T helper type 1(Th1)-biased
response. No definitive data are available relatively to
the lymphoid or myeloid origin of the two DC populations: SIRPalpha− cells are propably myeloid, while
SIRP+ cells are probably lymphoid (9).
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Relevance to Humans
The two most frequent manifestations of skin toxicity
are irritant contact dermatitis and allergic contact dermatitis. Hypersensitivity reactions to drugs and industrial chemicals are relatively common in man. Depending on the country, dermatoses account for 20%–70%
of all occupational diseases, and 20%–90% of these
are irritant contact dermatitis; most of the remaining
contact dermatitis is allergic contact dermatitis.
Hypersensitivity reactions are often considered to be
increased at such a rate to become a major health
problem in relation to environmental chemical exposure. Contact hypersensitivity is characterized by an
eczematous reaction at the point of contact with an
allergen. It develops normally in two temporally distinguished phases: the induction phase and the elicitation phase. Exposure to allergen will induce in susceptible individuals the immune response necessary for
sensitization (the induction phase). Sensitization
takes 10–14 days in humans. Re-exposure to the
same antigen will result in elicitation of the inflammatory reaction after a characteristic delay of usually 12–
48 h (the elicitation phase). The nature of the immune
responses induced by chemical allergens is essentially
no different from that which characterizes protective
immunity. Allergic contact dermatitis is a multifactorial disease, the onset of which depend on the nature of
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Skin Prick Test
Skin Irritation Testing in Animals
Predictive animal tests are routinely employed to assess topical irritation responses, such as acute or cumulative irritation, corrosion or photoirritation. Despite multitudinous objections, the test most widely
used for prediction potential skin irritation is the
Draize test (10) and its modifications, in which the
tested material is topically applied on abraded or intact
skin of the albino rabbit, clipped free of hair, under an
occluded patch for 4–24 h. Other species have been
used as well, such as albino guinea pigs, hairless mice,
swine and beagle dogs, but albino rabbits are still used
for most skin irritation testing, with the recognition
that rabbits are more responsive than other species
(including humans) to mild or moderate irritant insults.
Recently, alternative in vitro tests have been validated
and accepted by regulatory authorities in the EU and
OECD member countries to characterize the corrosive
and phototoxic potential of xenobiotics.
Skin Allergy Testing in Animals
Several standardized predictive tests in guinea pigs (cf.
“Guinea Pig Assays for Sensitization Testing”) and
some tests in mice use the response in the efferent
phase as an indication of immune reactivity to the
chemical. Some newer predictive assays in mice involve stimulation of lymphocytes in local draining
lymph nodes during the afferent phase as the endpoint
(cf. “Local Lymph Nodes Assay (LLNA)” and
“IMDS”).
References
1. Tlaskalova-Hogenova H, Tuckova L, Lodinova-Zadnikova R et al. (2002) Mucosal immunity: its role in
defense and allergy. Int Arch Allergy Immunol 128:77–
89
Skin Prick Test
The skin prick test is the most common test for atopy.
It involves injection of small amounts of allergen into
the skin, leading to an immediate wheal-and-flare reaction in allergic individuals.
Mast Cells
Food Allergy
Skin Sensitization Assay
Guinea Pig Assays for Sensitization Testing
Skin Sensitization Potency
The inherent potency with which a contact allergen
will induce skin sensitization. Activity is considered
usually as a function of the amount of chemical required for the acquisition of sensitization.
Local Lymph Node Assay
3
As a wider varieties of xenobiotics can cause cutaneous damage, it is requested by legislative agencies to
characterize this potential when developing, registering, or certifying new materials (see also “Contact
Hypersensitivity”).
3
Regulatory Environment
2. Corsini E, Galli CL (2000) Epidermal cytokines in
experimental contact dermatitis. Toxicology 142:203–
211
3. William IR, Kupper TS (1996) Immunity at the surface:
homeostatic mechanisms of the skin immune system.
Life Sci 58:1485–1507
4. Luster MI, Wilmer JL, Germolec D et al. (1995) Role of
keratinocyte-derived cytokines in chemical toxicity.
Toxicol Lett 82/83:471–476
5. Kupper TS (1990) Immune and inflammatory process in
cutaneous tissues. Mechanisms and speculations. J Clin
Invest 86:1783–1789
6. Piguet PF, Grau GE, Houser C, Vassalli P (1991) Tumor
necrosis factor is a critical mediators in hapten-induced
irritant and contact hypersensitivity reactions. J Exp Med
173:673–679
7. Nickoloff BJ (2002) Cutaneous dendritic cells in the
crossfire between innate and adaptive immunity. J
Dermatol Sci 29:159–165
8. Kimber I, Dearman RJ (2002) Allergic contact dermatitis: the cellular effectors. Contact Dermat 46:1–5
9. Howard CJ, Hope JC, Stephens SA, Gliddon DR,
Brooke GP (2002) Co-stimulation and modulation of the
ensuing immune response. Vet Immunol Immunopath
87:123–130
10. Draize JH, Woodward G, Calvery HO (1944) Methods
for the study of irritation and toxicity of substances
applied topically to the skin and mucous membranes. J
Pharm Exp Therap 82:377–389
3
the chemical, concentration, type of exposure, age,
sex, genetic susceptibility, and not genetic idiosyncrasies.
Cutaneous antigens are generally of low molecular
weight (hapten). It is clear that some allergens (urushiol, dinitrocholorobenzene, oxazolone, diphenylcyclopropenone) are very potent sensitizers and appear
able to induce sensitization in all normal people,
whereas some others (metallic salts of nickel, cobalt
and chromium, and a huge variety of organic compounds) are weak antigens and appear to sensitize
only susceptible individuals.
3
594
Southern
Autoimmune Disease, Animal Models
3
Sleeping Sickness
Sleeping Sickness is a disease in humans caused by
the protozoan parasites Trypanosoma brucei rhodesiense and T .brucei gambiense which are transmitted
by tsetse flies (Glossinidae). Sleeping Sickness occurs
in sub-Saharan Africa. The disease is untreated 100%
fatal.
Trypanosomes, Infection and Immunity
SNPs
3
SLE
595
Cytokine Polymorphisms and Immunotoxicology
Somatic Hypermutation
Somatic hypermutations occur during the immune response to thymus-dependent (TD) antigens and are
inserted in the variable regions of antibody H chains
and L chains. Somatic hypermutations cause an increase in the affinity of antibodies during the primary
as well as following immune responses and are thus
responsible for the phenomenon of immune maturation.
Idiotype Network
3
3
Slp 76 (SH2 Domain-Containing
Leukocyte Protein of 76 kDa)
Southern
Slp 76 is an adaptor protein found in T cells. It is
phosphorylated by the tyrosine kinase Zap70 and is
involved in the activation of the pathway that leads
to activation of PLCγ and the release of intracellular
Ca2+ as well as the activation of the Ras pathway.
Signal Transduction During Lymphocyte Activation
The transfer of DNA molecules from an agarose gel to
a membrane by capillarity or an electric field. The
cells or tissue
native RNA or
digested DNA
3
Smad
Members of the Smad family of signaling proteins
serve as substrates for the TGF-β receptor type I kinase, and function to transduce signals from the membrane into the nucleus and regulate ligand-specific
gene transcription. A genetic screen in Drosophila designed to identify mutations that would modify the
dpp mutant phenotype isolated a gene that was
named Mothers against Dpp (mad). A genetic screen
in Cunninghamella elegans designed to identify components of TGF-β1 isolated a gene that was named
sma. It was later shown that Sma proteins are homologous to the Mad proteins, and thus the Drosophila
and C. elegans nomenclature was combined to Smads.
Transforming Growth Factor β1; Control of T cell
Responses to Antigens
3
Small Secreted Cytokines
Chemokines
isolate nucleic
acid
transfer and blotting
(see figure 2)
gel
electrophoresis
membrane
immobilization
hybridization with
a specific DNA
or RNA probe
signal detection
via autoradiography
Southern and Northern Blotting. Figure 1 The
southern and northern blotting techniques. Enzymatically digested DNA or denatured RNA is fractionated by
gel electrophoresis. After transfer and fixation of the
DNA fragments or RNA to a membrane support, the
membrane is incubated with a labeled DNA or RNA
probe that is specific for the target of interest. Because
the probe hybridizes to only the target fragment/
molecule, a specific band is visualized following
detection.
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3
596
Southern and Northern Blotting
immobilized DNA can be detected at high sensitivity
by hybridization to a sequence specific probe.
Southern and Northern Blotting
3
Southern and Northern Blotting
inserted transgenes. It also is used to study RNA splicing and antibody and T cell receptor formation, and
has aided in the identification of gene rearrangements
associated with a variety of human genetic disorders
and cancers.
Northern blotting is the transfer of RNA from an agarose gel to a membrane similar to that used in Southern blotting (2). Northern blotting is used to evaluate
gene expression in a manner that is both qualitative
(in which tissues or cells, or under which physiological conditions a gene is expressed) and quantitative
(level of gene expression). This method also is used
to detect the expression of foreign genes in transgenic
organisms and is a useful adjunct to complementary
DNA (cDNA) cloning because mRNA molecular
weights can be compared with those of cloned DNAs.
The basic principles of Southern and northern blotting
are similar; however, there are a few notable exceptions. RNA is more labile than DNA and is very sensitive to degradation by enzymes called RNases. RNA
degradation reduces the quality of data and the ability
to quantify message expression. RNA molecules often
form secondary structures that significantly alter their
mobility, requiring gel electrophoresis to be performed under denaturing conditions (in the presence
of glyoxal or formaldehyde). With Southern blotting,
genomic DNA is digested with restriction enzyme (s) to produce small fragments that can be easily
fractionated, whereas RNA is analyzed as an intact
molecule. Genomic DNA also does not require many
of the special handling precautions necessary with
RNA.
The general schemes used in Southern and northern
blotting are depicted in Figures 1 and 2. DNA or RNA
is isolated from cells or tissues and then fractionated in
a solid support (gel) in an electric field (electrophoresis). Following size separation, the DNA or RNA is
transferred onto a membrane (generally nylon or nitrocellulose) and immobilized by exposure to ultraviolet
radiation or heat.
Membrane-bound DNA or RNA undergoes hybridization to a sequence homologous, labeled nucleic acid
probe (usually prepared using cDNA or RNA). Detection of the probe depends on the labeling method employed. For example, with 32P-labeled probes the signal is detected and quantified using X-ray film (autoradiography). Probes are generated by several methods, including random-priming, nick-translation, and
polymerase chain reaction (PCR). Sequences with
only partial homology (e.g. species-specific cDNA
or genomic DNA fragments) are used to prepare
probes as well.
3
Synonyms
DNA and RNA blotting, nucleic acid blotting.
Short Description
Nucleic acid detection can be performed using a technique known as blotting, which is the transfer of
DNA (deoxyribonucleic acid) or RNA (ribonucleic
acid) onto a membrane support. When this procedure
is used to examine DNA or RNA it is referred to as
Southern or northern blotting, respectively.
3
Characteristics
Southern blotting is the transfer of DNA from an agarose gel to a membrane support (1). Since its inception, Southern blotting has been used to determine
gene copy number, detect restriction fragment length
polymorphisms, determine modifications to DNA (e.g.
methylation), and detect the presence of genetically
3
NIEHS Mail Drop C1-04, Envionmental Immunology
Laboratory
111 Alexander Drive
P.O.Box 12233
Research Triangle Park, NC 27709
USA
3
Kevin Trouba
3
Southern and Northern Blotting. Figure 2 Capillary
transfer of DNA or RNA from agarose gels. Buffer is
drawn from a reservoir via a paper wick and passes
through the gel into a blotting material (e.g. paper
towels, blotting paper). The DNA or RNA is eluted from
the gel by the flow of buffer and is deposited onto a
membrane. A weight applied to the top of the blotting
apparatus ensures a tight junction between the layers
of material used in the transfer system.
Pros and Cons
Northern blotting remains the standard for detection
and quantification of mRNA despite the development
Spherocytes
of more sensitive molecular techniques such as the
reverse transcription-polymerase chain reaction (RTPCR). It presents several advantages over newer techniques because it is a useful method for determining
mRNA size and for identifying alternatively spliced
transcripts and multigene family members. One advantage of Southern blotting is that it can be used to
examine stretches of DNA such as the multikilobase
restriction fragments produced by some restriction enzymes. For both Southern and northern blotting, the
analysis of different genes via membrane stripping and
reprobing also is an advantage over traditional PCR
gel analysis techniques.
A disadvantage of northern blotting is that it is the
least sensitive of newer RNA analysis methods like
PCR, gene microarrays, and ribonuclease protection
assays (RPA). One disadvantage of Southern blotting
is that it usually requires a relatively large quantity of
high quality DNA. For this reason, PCR techniques
are becoming widely applied to tasks for which Southern blotting was traditionally used (e.g. DNA fingerprinting and identification) because much less DNA is
required and, in many cases, PCR techniques can be
performed using partially degraded DNA. The use of
radioisotopes in Southern and northern blotting also is
considered a disadvantage from a health perspective,
and the short half-life of phosphorus 32P (14 days)
necessitates that the probe be prepared and used in
an expeditious manner. Although it can be an advantage over traditional PCR gel analysis, reprobing of a
Southern or northern membrane usually requires removal of the initial probe before hybridizing with a
second probe. This process can be time consuming
and can reduce subsequent DNA and RNA detection,
a distinct disadvantage.
597
quired infectious diseases and neoplastic disease, as
well as their prediction, prognosis, and therapeutic
monitoring, will be aided by these technologies.
Obtaining a detailed picture of how genes and other
DNA sequences function together and interact with
environmental factors ultimately will lead to the discovery of pathways involved in normal processes and
in disease. Such knowledge will have a profound impact on the way disorders are diagnosed, treated, and
prevented, and will bring about changes in clinical and
public health practices. The characterization of (southern) and expression profiling (northern) of genes in
normal and diseased human tissues is an important
first step to understanding the potential role of genes
in human disease.
Regulatory Environment
Southern and northern blotting technology is applicable to any study where the detection, characterization,
or quantification of a nucleic acid in biological material is required. These methods are often used in basic
research, in the analysis and diagnosis of human inherited disease, as well as in a forensic capacity.
References
1. Southern EM (1975) Detection of specific sequences
among DNA fragments separated by gel electrophoresis.
J Mol Biol 98:503–517
2. Alwine JC, Kemp DJ, Stark GR (1977) Method for
detection of specific RNAs in agarose gels by transfer to
diazobenzyloxymethyl paper and hybridization with
DNA probes. Proc Natl Acad Sci USA 74:5350–5354
Predictivity
DNA and RNA underlie many aspects of human
health, both in function and dysfunction. Southern
and northern blotting will continue to aid in the identification of genetic events involved in neoplastic and
non-neoplastic diseases, inherited disorders, genetic
susceptibility, and predisposition to multigenic diseases. The accurate diagnosis and classification of ac-
Abnormal erythrocyte characterized by its smaller,
round size, and darker color without central pallor. It
is seen on Romanofski-stained peripheral blood
smears of patients with immune-mediated hemolytic
anemia (IMHA).
Antiglobulin (Coombs) Test
Hemolytic Anemia, Autoimmune
3
Relevance to Humans
Spherocytes
3
Southern and northern blotting is used to better understand the molecular basis of disease. For example,
northern blotting, which displays a high degree of correlation with RT-PCR, is used to monitor target gene
expression or to identify biomarkers in isolated tissues
and organs that results from a potential toxicological
event or challenge. Genes of toxicological interest include proinflammatory cytokines, growth factors, cytochrome P450s, glutathione-S-transferases, cyclin dependent kinases, and cell cycle inhibitors.
S
598
Spleen
Spleen
C Frieke Kuper
Toxicology and Applied Pharmacology
TNO Food and Nutrition Research
Zeist
The Netherlands
Synonyms
Definition
A large vascular, lymphoid organ in the upper part of
the abdominal cavity of vertebrates near the stomach,
with two main compartments designated red pulp
and white pulp. Its has two main functions: as a
blood-filtering organ (clearance of particulates, aged
or defective erythrocytes and platelets) and in the production of immune responses against blood-borne
antigens. In some species like the dog, the spleen
functions as a storage organ of red blood cells.
3
3
Characteristics
General
The spleen is a large blood-filtering and blood-forming organ, consisting of two main compartments that
are named the red pulp and the white pulp. The white
pulp is visible grossly on cross-section of the spleen as
white patches within the deep red (red pulp) organ,
and represents the lymphoid compartment of the
spleen. The spleen contains about a quarter of the
body's total lymphocyte population; during lymphocyte recirculation more cells pass through the spleen
than through all the lymph nodes. The main immunological function of the spleen is to guard the blood
compartment of the body. It does so, among other
ways, by generating T cell-independent antibody responses to bacterial polysaccharides and by exerting
enormous phagocytic power. The antibody response
may not be completely T cell-independent in all
cases, since T cell-derived factors enhance the response to some of these antigens. Antibodies generated are mainly of IgM class. The marginal zone
plays a key role in these antibody responses and retains B lymphocyte memory.
3
Red Pulp
The red pulp consists of venous sinuses lined by reticuloendothelial cells and splenic cords (cords of Billroth), which contain macrophages, lymphocytes, and
plasma cells. The red pulp is also rich in natural killer
cells. Macrophages perform a major function in blood
cell clearance (for example, of old red blood cells) and
in phagocytosis, especially of nonopsonized particles.
This high volume blood-filtering function is made pos-
sible by two factors: the direct contact, unobstructed
by blood-vessel walls, between phagocytic cells and
blood-borne particles and the large blood supply. The
phagocytic function is especially important in the case
of intravascular pathogenic microorganisms, before
formation of antibody and subsequent opsonization
occur (early bacteremia). The splenic macrophages together with the hepatic phagocytic system synthesize
the majority of complement components involved in
the classical complement cascade. In the case of systemic septicemia, the red pulp increases and contains
large proportions of (immature) granulocytes.
In the fetus, the spleen is an important site of hematopoiesis. This function is maintained in certain animal
species (rats and mice) throughout adulthood, whereas
in others extramedullary hematopoiesis is resumed
only in certain diseases.
White Pulp
The white pulp is composed of lymphocytes, macrophages and plasma cells within a mesenchymal reticular network. Description of the histology of the
spleen varies according to the animal species (1). Generally, the white pulp consists of a central arteriole
surrounded by the periarteriolar lymphocyte sheath
(PALS), the inner part being a T lymphocyte area (Figure 1).
The outer PALS contains B lymphocytes and plasma
cells. Adjacent follicles contain mainly B cells.
Around the PALS and follicles is a zone containing
B cells and called the marginal zone; this region is
easily distinguished, especially in rats, but is less prominent in dogs, and may even be absent in the human
spleen (2). The PALS has a microenvironment and
passenger leukocyte content similar to that of the
lymph node paracortex. The follicles are comparable
in structure and function with those of lymph follicles.
In contrast, the marginal zone has a microenvironment
that is unique to the spleen. Histologically, the B cells
at this site are of medium size; they are larger and
paler than B cells in primary follicles and the follicular
mantle of secondary follicles. In addition, they do not
show the morphology of centrocytes or centroblasts
found in germinal centers, and the phenotypic expression indicates that the marginal zone B cells are a
separate population of B cells. Also there are characteristic macrophages, namely the marginal zone macrophages, which are dispersed throughout the marginal
zone, and the marginal metallophilic macrophages,
which border the marginal sinus. Hematogenically
spreading infectious pathogens are controlled initially
by being trapped to marginal zone macrophages, a
process that is enhanced by binding the pathogens to
natural antibodies (3). Subsequently, marginal zone
B cells proliferate, B cell foci are formed and IgM
antibodies against the pathogens are produced.
Spleen
599
Spleen. Figure 1 Schematic presentation of the spleen compartments. Marginal zone, sinus, periarterial
lymphocyte sheath (PALS) and follicle are depicted in a cross-section of the white pulp. The white pulp contains
marginal zone macrophages (MZM), marginal metallophillic macrophages (MM), tingible body macrophages
(TBM), interdigitating dendritic cells (IDC), follicular dendritic cells (FDC), B lymphocytes (B) and T lymphocytes (T).
The splenic cords (cords of Billroth) in the red pulp contain predominantly macrophages (M), and several
lymphocytes (T and B).
The white pulp reaches peak development in rats, as in
man, at puberty, which coincides with peak thymus
function, followed by a gradual involution with age.
A loss of functional splenic lymphocytes does occur
with age, however, with a corresponding increase in
the number of reticular cells and macrophages. It is
probable therefore that—at least in rodents—the
spleen (unlike the thymus) continues to increase in
weight with age, maintaining a fairly consistent ratio
of organ weight to body weight (4).
Preclinical Relevance
The spleen has both lymphoid and nonlymphoid functions. Primary follicles are characteristic of germ-free
rodents and are frequently found in the spleen of laboratory rodents, reflecting the relative protection of
this lymphoid organ from exogenous antigen. Immunotoxicity that is manifested in the splenic lymphoid
compartment can be a direct effect of a compound or
occurs indirectly via effects on thymus and/or bone
marrow. The reaction to antigenic substances involves
germinal centre development. The enormous phagocytic system is affected by macromolecules and
other “reticuloendothelial system expanders” (5) and
by increased hemosiderin accumulation due to enhanced erythrocyte degradation. Hemodynamic de-
rangements and various types of anemia lead to changes in the spleen, in the red pulp as well as in the white
pulp.
Relevance to Humans
Spleen histology varies somewhat between laboratory
animal species and between some laboratory species
and humans, the major difference being the distinction
of the boundaries between PALS, the follicles and
marginal zone, and the prominence of the marginal
zone. In the human spleen, the white pulp architecture
and the distribution of lymphocyte subpopulations is
comparable to those in the rat and mouse, although the
marginal zone is less prominent or may even be absent. Also splenic function is comparable between experimental animals and humans, as is exemplified in
humans by splenectomy, after which reduced nonspecific phagocytosis of nonopsonized particles, lowered
serum IgM levels, and increased susceptibility to infections by encapsulated bacteria have been documented. Hemopoiesis is common in adult rats, but in humans only present under pathological conditions. Differences in immunotoxicity between laboratory animals and humans appear to depend predominantly
on differences in toxicokinetics and metabolism of
substances, and these aspects should therefore be con-
S
Split-Plot or Split-Unit Design
sidered when results from animal studies are extrapolated to humans.
Regulatory Environment
Most guidelines require that the spleen is weighed and
examined histopathologically. Moreover, functional
tests as required by guidelines often include the measurement of antibody responses in spleen cells, and
analysis of surface markers on immune cells using
blood and spleen cells.
Src Family Kinases
A group of protein kinases anchored to the cell membrane by a lipid moiety attached to their amino terminal region. Src family kinases are responsible for phosphorylating other proteins. They are activated by phosphorylation of their tyrosine residue in the kinase domain and inhibited by phosphorylation of the tyrosine
residue in their carboxy terminus.
Signal Transduction During Lymphocyte Activation
3
600
References
An experimental design structure in which there are
variance components which must be estimated between subjects as well as within subjects.
Statistics in Immunotoxicology
A protein domain found in many signaling proteins
that binds to phosphotyrosine residues on other proteins. SH2 domains are used to recruit intracellular
signaling molecules to the activated receptors on lymphocytes.
Signal Transduction During Lymphocyte Activation
Src Homology 3 (SH3) Domain
A protein domain found in many signaling proteins.
SH3 domains bind proline-rich regions in other proteins recruiting these other proteins to the signaling
complexes (see Adaptors).
Signal Transduction During Lymphocyte Activation
3
Split-Plot or Split-Unit Design
Src Homology 2 (SH2) Domain
3
1. Zapata AG, Cooper EL (1990) The Immune System:
Comparative Histophysiology. John Wiley, Chichester
2. Young B, Heath JW (2000) Immune system. In: Functional Histology. Churchill Livingstone, Edinburgh,
pp 193–222
3. Ochsenbein AF, Zinkernagel RM (2000) Natural antibodies and complement link innate and acquired immunity. Immunol Today 21:624–630
4. Losco P (1992) Normal development, growth, and aging
of the spleen. In: Mohr U, Dungworth DL, Capen CC
(eds) Pathobiology of the Aging Rat, Volume I. ILSI
Press, Washington, DC, pp 75–95
5. Gopinath C, Prentice DE, Lewis DJ (eds) (1987) The
lymphoid system. In: Atlas of Experimental Toxicological Pathology. MTP Press, Lancaster, pp 122–137
3
Statistics in Immunotoxicology
Split-Well Analysis
Michael L Kashon
To confirm the specificity of cytotoxic analyses, multiple target cells are used.
Limiting Dilution Analysis
3
Spot-Forming Cells
Biostatistics Branch
National Institute for Occupational Safety and Health
Morgantown, WV 26505
USA
Synonyms
Quantitative analysis, experimental design, modeling,
risk assessment
Plaque-Forming Cell Assays
3
Definition
SRBC ELISA
Plaque Versus ELISA Assays. Evaluation of Humoral Immune Responses to T-Dependent Antigens
Statistical methodologies are powerful and indispensable tools in risk assessment and the experimental analysis of the immune system. As such, they are utilized
at all levels including hazard identification, dose-response assessment, exposure assessment, and risk
characterization. Statistical methods are utilized to
3
Statistics in Immunotoxicology
Experimental Design Issues
The process in which the data are collected, irrespective of the specific laboratory techniques used, is a
direct reference to the experimental design. Experimental design can be parceled into two parts including
the treatment structure and the design structure.
* Treatment structure appropriately belongs in the
realm of the scientist and reflects such things as
the choice of treatment groups, dosing regimens,
time courses, and the selection of appropriate control groups.
* The design structure is in the realm of the statistician and reflects issues including randomization of
experimental units, appropriate degree of replication for sufficient power, prevention of confounding, and generally striving to obtain the maximum
amount of information from a finite pool of resources. Some common design structures include
the completely randomized design, randomized
complete blocks design, split-plot or split-unit designs, crossover designs, and repeated measures designs.
3
3
3
It is important to note that for a given treatment structure, any number of design structures are appropriate,
and the selection of a particular design structure is
determined by factors not limited to the treatment
structure.
Analysis Issues
With the exception of the completely randomized de-
3
A detailed description of the variety of statistical techniques that are utilized in immunotoxicology and toxicology in general is well beyond the scope of this
essay. A well written and practical guide for the appropriate use of various statistics in the field of toxicology
is presented by Gad and Weil (1). This essay will
emphasize the inseparable link between sound experimental design and valid statistical tests, and give an
overview of some of the fundamental statistical tests
used in immunotoxicology experiments. In all experiments, the process in which data are collected, and the
nature of the data (that is, continuous, discrete, categorical) will dictate the type of statistical analysis that
is performed. It is critical that effective communication
between statisticians and research scientists occur at
the time of study design to ensure that the experimental design is appropriate and that the data are collected
in a manner that allows the hypothesis under investigation to be answered using the appropriate statistical
methods.
3
Characteristics
sign (the simplest design structure) all of these experimental designs fall into the category of analytical procedures known as mixed models, in that they have
more than one random variance component that must
be estimated.
The simplest of these is the randomized complete
blocks design in which groups of experimental subjects, representing one from each treatment combination, are processed simultaneously for many or all
parts of the experiment. The number of these groups
or blocks determines the sample size. This design is
particularly useful when labor intensive laboratory
procedures limit the number of samples that can be
processed simultaneously. Ultimately, any variation
which occurs from block to block is accounted for in
the analysis and removed from the error term that is
utilized for the statistical test. This reduction in the
magnitude of the experimental error term increases
the likelihood of finding significant treatment effects.
Mixed models can be complex and the analyses require that the covariance structure of the random effects (blocking factors, correlation structure of repeated measures) be fit prior to estimating the fixed
effects of the experiment (dose, sex, time). It is often
the case that an investigator has utilized one of the
above designs without realizing it and proceeds with
data analysis as though it were a completely randomized design. Unfortunately the incorrect—usually inflated—error term is utilized in tests of significance,
causing a type II error and significant treatment effects
are missed. Some very powerful mixed model statistical procedures are available in SAS (2) and the analysis of such data should be performed by those who
are familiar with the program to ensure proper coding.
Types of data subject to statistical analysis include
numerical, ordinal and nominal. Numerical data can
be either continuous as in the case of organ weights,
or discrete as in the case of tumor incidence. Ordinal
data are essentially ranks and might include a scale
indicating the severity of histopathological changes.
Finally, nominal data are categories and might include
the presence or absence of a particular attribute.
Data generated by many immune function assays are
continuous numerical data such as organ weights, or
burden of infectious agent in host-resistance assays.
Other types include discrete count data such as the
number of antibody-producing cells which are in
such high numbers that they can be treated as though
they are continuous numerical data. Many of these
variables follow a normal or Gaussian distribution
and can be analyzed using normality based statistical
methods, such as analysis of variance, analysis of
covariance, and regression analysis.
However, many biological parameters follow a lognormal distribution and thus may violate the assumptions
of the above-mentioned statistical tests, particularly
3
summarize, model, and draw inferences based on empirically collected data.
601
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Statistics in Immunotoxicology
the assumption regarding homogeneity of variance
(thus rendering inaccurate P values). Traditionally,
these data have been analyzed using normality based
statistical tests following a transformation of the data
which serves to reign in extreme values and make the
variances comparable between groups. Another analytical approach which allows for the original data to be
analyzed in the case of heterogeneous error variance is
to model each group variance separately. This can be
performed using the SAS procedure Proc Mixed (2,3).
Additional options include using nonparametric statistical methods which make no assumptions regarding
the underlying distribution of the data (4). These tests
can often be as robust and as powerful as their parametric counterparts.
All of the normality based statistical analyses described above fall into the category of linear models.
Linear models assume that the response variable is a
linear combination of the independent variables, and
that the model is linear in all of its parameters. There
are many cases in which linear models are inadequate
to describe the data appropriately. When this occurs,
one can use nonlinear models which do not force the
data to fit a given linear structure when in fact a linear
model is inadequate.
A good example of the use of nonlinear models is in
the case of the local lymph node assay (LLNA). This
assay is often used in hazard identification to determine the sensitizing potential of a given compound.
Typically, if application of a chemical to the skin results in a three-fold or greater increase in cell proliferation in the local draining lymph nodes, then the
chemical is marked as a potential sensitizer. Estimation of the concentration which yields a three-fold increase in cell proliferation (EC3) was initially performed by interpolation between two doses; one
above the point in which a three-fold stimulation relative to control animals occurs, and one below. Nonlinear regression models allow for a better fit of the
available dose-response relationship, and allow for
more precise estimates of the EC3 (5). Additional
analysis of these data utilizing bootstrap resampling
methods allows for confidence intervals to be calculated around this point estimate and thus yields an
uncertainty margin for sensitizing concentrations of a
given chemical.
Many host-resistance assays generate nominal data,
such as whether the animal survives a specified length
of time or not. These data are not amenable to the
statistical analyses under linear or nonlinear models.
The data are often analyzed in a contingency table
using a Chi-squared statistic, or exact probabilities
are generated using Fisher’s exact test to assess significance of the treatments. If dose-response studies
are utilized in the host-resistance assay, tests for
trend can be performed using the Cochran-Armitage
linear trend test. It is also feasible to record the length
of time taken for an animal to show clinical signs of
disease. When time to an event is the outcome of an
experiment, then survival analysis is the appropriate
statistical analysis to use.
Screening procedures for evaluating the potential immunotoxicity of compounds will utilize dose-response
experiments and often attempt to determine at which
dose a compound is toxic. Answers to these questions
require that multiple comparisons between treatment
groups be made, and how these are made should be
determined prior to the experiment. It is common practice to compare all treatment groups with a single control group; this results in a series of statistical tests
which are not independent of one another, and thus
influences the overall error rate, specifically increasing
the likelihood of a type I error. Adjustments such as
Dunnett’s test will make appropriate changes to the
critical value of the t test and reduce the family-wise
error rate.
When pair-wise comparisons between all treatment
groups are desired, several useful procedures for adjusting the overall error rate can be found in nearly all
textbooks on experimental design. Often the effect of a
compound on immune function is strongly suspected
or known to occur in a specific direction (it increases
or decreases). When this is the case, an increase in the
power of the statistical test can be achieved by using
an ordered alternative hypothesis. There are parametric
as well as nonparametric versions of these tests which
derive order-restricted means and calculate tests of
significance using the new means.
Immunotoxicity screening studies are often performed
using a tiered testing approach and the assessment of
multiple endpoints. In this regard, tests which assess
cell-mediated immunity, humoral-mediated immunity,
and various immunopathologic parameters are often
performed such that the likelihood of determining
whether a compound is immunotoxic is increased at
the expense of knowledge regarding specificity of the
immune defect. Positive indications of toxicity in the
first tier lead to more detailed tests of immune function
in a second tier, including host-resistance assays. The
result of this approach is that the identification of immunotoxic chemicals, and the organ systems affected,
will not be determined from a single immune function
test and reliance on its associated statistical analysis.
This strategy of utilizing converging lines of evidence
to conclude that a given compound is immunotoxic
appropriately places the emphasis on proper experimental design.
In the end, so long as an experiment is designed and
carried out appropriately, a legitimate statistical model
can be fit and the effects of a given compound can be
accurately assessed. However, there is often little that
3
Steroid Hormones and their Effect on the Immune System
While specific guidelines for immunotoxic testing of
compounds have been published by the Environmental
Protection Agency (EPA), the Food and Drug Administration (FDA), and the (EMEA), there are no specific
guidelines regarding the use of statistical methods in
immunotoxicological studies. There is however, a section from FDA/CFSAN with statistical considerations
for toxicity studies examining food ingredients.
Further, there are clear statistical guidelines put forth
by the FDA with respect to statistical methods used in
clinical trials which would cover investigations of immunotoxic potential of compounds for consumption or
medicinal purposes.
References
1. Gad SC, Weil CS (1994) Statistics for toxicologists. In:
Hayes AW (ed) Principles and Methods of Toxicology,
3rd edn. Raven Press, New York, pp 221–274
2. Littell RC, Milliken GA, Stroup WW, Wolfinger RD
(1996) SAS System for Mixed Models. SAS Institute,
Cary
3. Milliken GA, Johnson DE (2002) Analysis of Messy
Data, Volume III: Analysis of Covariance. Chapman and
Hall/CRC, New York
4. Hollander M, Wolfe DA (1999) Nonparametric Statistical
Methods, 2nd ed. John Wiley, New York
5. van Och FMM, Slob W, de Jong WH, Vandebriel RJ, van
Loveren H (2000) A quantitative method for assessing
the sensitizing potency of low molecular weight chemicals using a local lymph node assay: employment of a
regression method that includes determination of the
uncertainty margins. Toxicology 146:49–59
Attenuated Organisms as Vaccines
Steroid Hormones
Steroid hormones are lipophilic compounds derived
from cholesterol metabolism, which typically mediate
their biological activity by binding to a specific intracellular cytosolic receptor.
Steroid Hormones and their Effect on the Immune
System
3
Regulatory Environment
Sterilizing Immunity
3
can be done statistically to salvage a poorly designed
or poorly performed experiment.
603
Steroid Hormones and their Effect on
the Immune System
Michael Laiosa
NIAID/NIH
Center Dr., Room 111
Bethesda, MD 20892
USA
Synonyms
steroid hormones, gonadal hormones, estrogen, testosterone, androgens, glucocorticoids, glucortisol, environmental estrogens, endocrine disrupting chemicals
Definition
Steroid hormones, which include glucocorticoids
estrogen, progesterone,
and the sex hormones
and testosterone, possess immunomodulatory roles
that include effects on T and B cell development, lymphoid organ size, lymphocyte cell death, immune
function, and susceptibility to autoimmune disease.
Steroid hormones are lipophilic compounds derived
from cholesterol metabolism, which typically mediate
their biological activity by binding to an intracellular
cytosolic receptor. Hormone binding to its receptor
causes the receptor to translocate to the nucleus
where it forms a homodimer with another ligand-activated receptor. The steroid receptor homodimer will
then bind to specific DNA sequences in steroid responsive genes modulating their transcription (Figure
1) ( 1–4). Biological specificity of steroid hormone
activity is dependent on interaction between the hormone and its specific receptor—glucocorticoid receptor (GR), progesterone receptor (PR), estrogen receptor (ER), and androgen receptor (AR). All four
steroid receptors have been found in immune cells
3
3
3
The spatial relationship of atoms and groups in a molecule of a substance and its effect (s) on the properties
of the substance.
Chromium and the Immune System
3
3
Stereochemistry
3
A class of cells having two generative capabilities:
replication and formation of more differentiated offspring.
Colony-Forming Unit Assay: Methods and Implications
3
Stem Cells
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Steroid Hormones and their Effect on the Immune System
and tissues, making the immune system an important
target for steroid activity (1–4).
Environmental estrogens or endocrine disrupters are
chemicals found throughout the environment that have
estrogenic activity and include compounds in plastics
such as bisphenol-A, and phthalates, in detergents and
surfactants such as octylphenol and nonylphenol, in
pesticides such as methoxychlor, dichlorodiphenyl-trichloroethane (DDT), hexachlorobenzene, and dieldrin,
in industrial chemicals such as polychlorinated biphenyls (PCBs) and 2,3,7,8,tetrachlorodibenzo-p-dioxin
(TCDD), and in natural plant estrogens (genistein
and coumesterol) (5). The presence of steroid receptors in immune tissues and cells make the immune
system a potential target of environmental estrogens
because of the ability of these molecules to bind to
steroid receptors, mimic hormones, antagonize hormones, alter hormonal binding to receptors, and/or
alter metabolism of natural or endogenous hormones (3–5).
3
Characteristics
Glucocorticoids
Glucocorticoids mediate their biological activity by
binding to the intracellular cytosolic glucocorticoid receptor (GR). The GR exists in the cytosol in a complex with heat shock proteins and immunophilin proteins. Receptor binding by ligand leads to the GR
translocating to the nucleus where it forms a homodimer and binds to specific DNA sequences referred to
as glucocorticoid responsive elements (GREs). GR
binding to GREs may enhance or inhibit transcription
of the particular gene (1). Additional biological activ-
ity of glucocorticoids is conferred through cross-talk
between the ligand-activated GR and other transcription factors including AP-1, nuclear factor NFκB,
CREB, STAT3 and STAT5 (1).
One of the most profound effects of glucocorticoids on
the immune system is the rapid thymic atrophy that
occurs within hours after exposure to a pharmacological dose of the hormone. Moreover, thymic atrophy
has been observed following periods of psychological,
emotional, or physical stress when glucocorticoid levels are abnormally elevated. Glucocorticoid-induced
thymic atrophy has been attributed to selective loss of
CD4+CD8+ thymocytes due to apoptosis or programmed cell death (1). Moreover, the apoptosis in
CD4+CD8+ thymocytes induced by glucocorticoids
is due to the low levels of the antiapoptotic protein
Bcl-2 found in these cells compared with other thymocytes and T-cells (1).
Although CD4+CD8+ thymocytes are acutely sensitive
to glucocorticoid-induced apoptosis during drug administration or periods of stress, peripheral T-cells
are not induced to die. Nevertheless, it has been long
appreciated that glucocorticoids have potent immunosuppressive effects on peripheral T-cells and have been
used extensively for treating a variety of autoimmune
and inflammatory diseases. Recent studies have demonstrated that glucocorticoids mediate their immunosuppressive effects by inhibiting the production of Tcell growth factors and cytokines, including interleukins IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-13,
granulocyte-macrophage colony-stimulating factor
(GM-CSF), the tumor necrosis factor TNF-α, and
the interferon IFN-γ (1). Inhibition of cytokine pro-
Steroid Hormones and their Effect on the Immune System. Figure 1 General biological response to steroid
hormones: Steroids are lipophillic compounds that freely pass through the cellular membrane where they bind to a
specific steroid hormone receptor. Steroid receptors are typically localized in the cytosol in an inactive state,
complexed with heat shock and immunophilin proteins (GR, PR, AR), but may be found in the nucleus (ER). Once
activated by binding to their specific ligand, steroid receptors translocate to the nucleus and form homodimers
which then modulate expression of genes containing response elements specific to the ligand—steroid receptor
homodimer. In some instances, additional biological responses may be made by cross talk between the activated
steroid receptor and other nuclear transcription factors (GR can interact with AP-1, NF-kB, CREB, STAT3 and
STAT5 transcription factors).
Steroid Hormones and their Effect on the Immune System
duction appears to be at the level of gene expression
such that the ligand activated GR may form proteinprotein interactions with other transcription factors
thereby inhibiting DNA binding and function. One
such factor, AP-1, has been implicated in the GRmediated inhibition of IL-2 transcription (1).
Androgens
Biological activity of androgens is mediated through
their interaction with the intracellular androgen receptor. As with the glucocorticoid receptor, the AR is a
ligand-activated transcription factor that is involved in
modulation of gene expression. Castration and hormone replacement studies have revealed androgenic
effects on both B- and T-cell development (3). In numerous different organisms including cattle and rodents significant thymic enlargement has been observed following male castration. Moreover, thymic
enlargement following castration was even seen in
aged males (3). Using mice that possess a spontaneous
mutation in their AR and thus are androgen unresponsive, it has been shown that the effect of thymus enlargement following castration is AR dependent (3).
The AR protein has been found in all thymocyte subsets with the highest expression found in the immature
CD8+CD3lo thymocyte subset (3). AR expression has
also been found in the cortical and medullary stromal
elements of the thymus (3). To determine the cellular
target of androgen activity, a set of in vivo experiments
utilizing both AR defective, and C57Bl/6 mice were
used to generate hematopoietic chimeras such that the
stromal cells expressed the normal AR and the thymocytes expressed the mutant AR. In these AR chimeric mice it was found that the thymus size would
only enlarge following castration (and shrink with androgen replacement) when a functional AR was present in the stromal tissue (3). These data indicate that
AR expression in thymic stromal cells is necessary for
mediating androgen-induced thymic atrophy. It has
been proposed that the mechanism for androgen-induced thymic atrophy may be due to accelerated thymocyte apoptosis mediated by the thymic stromal cells
(3). Alternatively, the AR may increase transcription
of cytokines such as the transforming growth factor
TGF-β, which would inhibit T-cell development (3).
B-cell development in the bone marrow is also affected by androgens with significant expansion of immature B-cells occurring following male castration.
Furthermore, androgen replacement reverses the Bcell expansion in the bone marrow, but has no affect
on spleen size or B-cell number. The probable explanation for why B-cells in the bone marrow are androgen sensitive, but splenic B-cells are not, comes from
the finding that the AR is expressed only in immature
B lymphocytes but not in mature splenic B-cells (3).
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Progesterones
Unlike AR expression, which has been found in both
lymphocytes and stromal cells, the progesterone receptor (PR) has thus far only been found in stromal cells
in the thymus (4). Nevertheless, at doses of progesterone comparable to the levels present during pregnancy, progesterone has been shown to cause thymic atrophy, especially when administered simultaneously
with estrogen (4). Moreover, thymic atrophy following
simultaneous administration of both estrogen and progesterone was shown to depend on the presence of the
PR in the thymic stroma. These data indicate that progesterone inhibits T-cell development indirectly by affecting the stromal cells (4).
Estrogens
Classical estrogen signaling is mediated by the estrogen receptors ERα and ERβ. The inactive receptors
are localized to the nucleus where they remain in a
complex with other proteins until ligand activation results in the ER binding to and activating genes that
possess estrogen-responsive elements in their promoters. There is high homology between the DNA-binding domains of ERα and ERβ and, therefore, it has
been hypothesized that functional specificity is likely
to be conferred by the differential tissue expression
observed between the two receptors (2).
The list of immunological endpoints that are implicated as targets of estrogenic activity is considerable
and diverse. Estrogen has been shown to inhibit both
B- and T-cell development (5). Additionally, estrogen
has effects on B and T lymphocytes in the lymph node
and spleen (5). Both lymphocytes and non-lymphoid
stromal cells have been shown to express estrogen
receptors, indicating there may be multiple cellular
targets of estrogen in the immune system (5). Estrogen
also is suspected to play an important role in autoimmune diseases, especially systemic lupus erythematosus (SLE), because of the disproportionate number of
women who are affected by this disorder of the immune system (5-7). B-cells producing immunoglobulins directed against self antigens, anti-DNA antibodies, anti-actin, and anti-cardiolipin antibodies have all
been found to be elevated in mice exposed to estrogen
(5). Plasma B-cell numbers in the spleen (mature Bcells) were found to be nearly 10 times higher in these
same mice (5). Moreover, splenic B-cells from estrogen-treated mice had heightened expression of the
anti-apoptotic protein Bcl-2 and were consequently
more resistant to apoptosis following activation (5).
Immunological defects have also been observed following exposure to the broad class of chemicals collectively referred to as environmental estrogens or endocrine disruptors. Bottle-nosed dolphins found in the
Gulf of Mexico experienced decreased proliferative
responses to T-cell mitogens correlating with elevated
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3
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Steroid Hormones and their Effect on the Immune System
DDT and PCB levels (5). A similar inhibition of splenocyte and peripheral blood leukocyte T-cell proliferation was observed in DDT-exposed and PCB-exposed
Beluga whales (5). Endocrine disruptors have also
been shown to inhibit lymphocyte development in
chicks where embryos exposed to PCB 126 during
incubation had decreased numbers of B- and T-cells
(5). Furthermore, male rats exposed to the pesticide
methoxychlor during perinatal and prepubertal developmental periods had decreased antibody production
and thymic weights (5).
In addition to the effects of endocrine disruptors on Band T-lymphocytes, macrophages and antigen-presenting cells have also been shown to be affected by endocrine disruptors. For example, bisphenol-A has been
shown to inhibit plastic adherence of rat peritoneal
macrophages. The adherence deficiency observed in
vitro is therefore likely to affect the process of phagocytosis, which is dependent on macrophage adherence
to surfaces and particles (5).
Preclinical Relevance
One of the most striking aspects of steroid hormones is
their influence on the gender bias of autoimmune diseases, whereby women are afflicted far more and often
with much greater severity than men (6,7). Approximately 5% of the population in the USA has been
diagnosed with an autoimmune disorder and, depending on which specific disorder one is analyzing, 60%–
95% of the afflicted patients are women (6,7). Specifically, 60%–70% of people diagnosed with multiple
sclerosis (MS) and rheumatoid arthritis (RA) are
women (7). Women make up 85%–95% of all cases
of thyroid disease, SLE, and Sjrögren syndrome (7).
Evidence of the influence of steroid hormones comes
in part from observations made during pregnancy
when hormonal fluctuations, notably estrogen and progesterone, are the greatest. The severity of MS and RA
is lessened during the third trimester of pregnancy
when estrogen and progesterone levels are highest.
However, flare-ups in the disease state are often observed when hormone levels drop after birth (7). In
contrast to these diseases, SLE seems to worsen during
pregnancy. The differences in disease states have been
attributed to direct hormonal effects on T-cell-dependent responses known as T-helper type 1 (Th1) and Thelper type 2 (Th2) responses. Th2-dependent responses, which enhance antibody production, appear
to be favored during pregnancy (7).
In addition to the direct affects of sex steroids on the
Steroid Hormones and their Effect on the Immune System. Table 1 Effects of steroid hormones.
Steroid hormone
Receptor
Developmental effects
Peripheral immune effects
Glucocorticoids
Cortisol
Corticosterone
GR
Immunosuppressive
Thymic atrophy
Apoptosis of CD4+CD8+ thymocytes Inhibits T-cell growth factors IL-1, IL-2,
IL-3, IL-4, IL-6, IL-7, IL-10, IL-13, GMCSF, TNFα, IFNγ
Progesterone
PR
Thymic atrophy
Unknown
Androgens
AR
Dihydrotestosterone
Thymus size and bone marrow B-cell No effect on splenic B-cells due to loss
number increase following castration of AR expression as B-cells mature
and decrease with testosterone administration
Estrogens
17β-estradiol
ER
Both T-cell and B-cell development
are inhibited
Women tend to have higher incidence
and severity of autoimmune diseases
T-helper type 2-dependent responses
are enhanced during pregnancy
Animal models exposed to elevated
estrogen have higher titers of autoantibodies, and plasma B-cell numbers are increased
Environment disrupting chemicals
(EDCs)
Various
Thymic atrophy
Inhibition of B-cell and T-cell development
Decreased proliferative responses to
T-cell mitogens
Decreased antibody production
Decreased phagocytosis of macrophages
Regulation of cytokines and chemokines has been shown in vitro to be
affected by various EDCs
Steroid Hormones and their Effect on the Immune System
Steroid Hormones and their Effect on the Immune
System. Figure 2 Chemical Structure of four principle
steroid hormones, corticosterone, 17β-estradiol, progesterone and dihydrotestosterone.
immune system, which make women more susceptible
to autoimmune diseases, additional indirect neural-endocrine-immune interactions may also influence the
immune response (7). In a potential feedback loop
involving glucocorticoids, sex hormones are known
to modulate the hypothalamic-pituitary-adrenal axis,
possibly affecting stress responses. Interestingly, females tend to have higher circulating concentrations
of the glucocorticoid hormone corticosterone. Furthermore, glucocorticoids can suppress sex hormone production and action (7).
Relevance to Humans
The prevalence of autoimmune disease in women is
significantly higher than for men. However, the precise mechanism by which different hormones may
contribute to the type and severity of disease is still
unknown. Moreover, studies in animal models suggest
there are likely to be genetic differences, which make
certain individuals and/or populations more susceptible to environmental factors (including steroid hormones), which may affect disease onset and presentation (7).
In addition to the effects of steroids on autoimmune
disease, a growing number of studies on humans accidentally exposed to a variety of endocrine disruptors
have revealed numerous alterations to the immune
system. Men who ingested PCB-contaminated fish
have decreased activity of natural killer cells compared
to unexposed individuals (5). Workers exposed to pesticides have enhanced macrophage activity and reduced antibodies (5). Additionally, workers who
607
have been chronically exposed to pesticides have a
higher ratio of CD4+ to CD8+ lymphocytes (5).
Diethylstilbestrol (DES) is a model endocrine disruptor, originally used under the mistaken assumption that
it helped to prevent miscarriages. It has been shown to
have negative health effects in both the mothers and
children exposed prenatally. Exposure has been associated with an increased risk for clear cell adenocarcinoma (a rare vaginal/cervical cancer) in prenatally exposed daughters, as well as with significant increases
in autoimmune diseases (5).
Finally, a number of endocrine disruptors have been
shown to cause changes in cytokine and chemokine
regulation in vitro using human cell lines and human
estrogen receptor constructs (5). However, far more
research is needed to assess the mechanism of action
of this large class of compounds, the effects of these
compounds in vivo, and dosage ranges where deleterious effects may be seen.
Regulatory Environment
The understanding that there are compounds in the
environment possessing properties that can modulate
hormonal pathways and responses has captivated
worldwide attention. As a result, several government-sponsored agencies and committees have initiated research goals, guidelines, and rules for identifying endocrine disrupting chemicals (EDCs), and determining the risk of EDCs to the health of wildlife
and human populations. Specifically, in 1995 US Environmental Protection Agency held two international
workshops to identify research needs for risk assessment of EDCs. The workshops suggested the potential
effects of these chemicals on reproductive, neurological, and immunological tissues, as well as carcinogenesis, were especially important to understand and expand research (8). In 1996 the Food Quality Protection
Act and the Safe Drinking Water Act were amended
by US Congress to include testing of use of pesticides
in food and drinking water contaminants, for estrogenicity and other hormonal activity (8). In 1998 the
Endocrine Disruptor Screening and Testing Advisory
Committee (EDSTAC), under the auspices of the EPA,
defined 'other hormonal activity' to be androgens and
compounds affecting thyroid function (8). EDSTAC
also recommended an extensive process for prioritizing, screening, and testing the nearly 70 000 chemicals
regulated by the EPA for endocrine disrupting activity.
The screening battery involves a combination of in
vitro and in vivo assays involving several different
taxa (8).
In 1998 the EPA-sponsored Research Plan for Endocrine Disruptors posed a number of questions to stimulate research priorities. Some of the questions were:
* What effects are occurring in populations?
* What are the chemical classes and their potencies?
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*
*
Current progress on each of these questions can be
found in the excellent review by Daston et al (8).
More recently, the Global Assessment of the State of
the Science of Endocrine Disruptors was published in
2002 by the World Health Organization, United Nations Environmental Program, and International Labor
Organization. This report concluded that there is enough evidence from the known effects of endogenous
and exogenous hormones to suggest EDCs are capable
of harming certain human biological functions, including reproductive and developing systems. The report
goes on to emphasize that the possible effects on
human populations is a cause for concern and is an
area of high research priority (9).
References
1. Ashwell JD, Lu FW, Vacchio MS (2000) Glucocorticoids
in T cell development and function. Annu Rev Immunol
18:309–345
2. Hall JM, Couse JF, Korach KS (2001) The multifaceted
mechanisms of estradiol and estrogen receptor signaling.
J Biol Chem 276:36869–36872
3. Olsen NJ, Kovacs WJ (2001) Effects of androgens on T
and B lymphocyte development. Immunol Res 23:281–
288
4. Tibbetts TA, DeMayo F, Rich S, Conneely OM, O'Malley
BW (1999) Progesterone receptors in the thymus are
required for thymic involution during pregnancy and for
normal fertility. Proc Natl Acad Sci USA 96:12021–
12026
5. Ahmed SA (2000) The immune system as a potential
target for environmental estrogens (endocrine disrupters):
a new emerging field. Toxicology 150:191–206
6. Vidaver R (2002) Molecular and clinical evidence of the
role of estrogen in lupus. Trends Immunol 23:229–230
7. Whitacre CC (2001) Sex differences in autoimmune
disease. Nat Immunol 2:777–780
8. Daston GP, Cook JC, Kavlock RJ (2003) Uncertainties
for endocrine disrupters: our view on progress. Toxicol
Sci 74:245–252
9. Damstra T (2003) Endocrine disrupters: the need for a
refocused vision. Toxicol Sci 74:231–232
Steroid receptors are typically localized in the cytoplasm in an inactive state until activated by binding to
their specific steroid hormone ligand: glucocorticoids
with glucocorticoid receptors, estrogen with estrogen
receptors, testosterone with androgen receptors, and
progesterone with progesterone receptors. When the
hormone binds to its receptor it causes the receptor
to translocate to the nucleus, where it forms a homodimer with another ligand-activated receptor. The steroid receptor homodimer will then bind to specific
DNA sequences in steroid-responsive genes modulating their transcription.
Steroid Hormones and their Effect on the Immune
System
Stevens–Johnson Syndrome (SJS)
This syndrome is characterized by widely distributed
erythematous or purpuric macules and papules, with
fever and involvement of two or more mucus membrane surfaces. Although the term is frequently used as
a synonym for erythema multiforme major, this syndrome is more severe, with target-like lesions extending to the trunk and face, and it is though to be druginduced. According to the criteria proposed by Roujeau et al (1), this syndrome is defined as cases with
limited areas of epidermal detachment (< 10%).
Drugs, Allergy to
Stimulation Index
For each concentration of test chemical a stimulation
index (SI) is calculated relative to the concurrent vehicle control. By definition the SI of the control (vehicle) group is set to 1. The interpretation of results is
based upon derivation of these SI, thus an SI of 2
means a doubling of the values measured for the controls.
Local Lymph Node Assay (IMDS), Modifications
Stomatitis
3
*
Steroid Receptors
3
*
What are the dose-response characteristics in the
low-dose region?
Do our testing guidelines adequately evaluate potential endocrine-mediated effects?
What extrapolation tools are needed?
What are the effects of exposure to multiple EDCs
and will a toxic equivalency factor approach be
feasible?
How and to what degree are human and wildlife
populations exposed to EDCs?
What are the major sources and environmental fates
of EDCs?
How can unreasonable risks be managed?
3
*
Steroid Receptors
3
608
Oral Mucositis and Immunotoxicology
Streptococcus Infection and Immunity
A protein toxin produced by Streptococcus that acts as
a superantigen for mouse and human T lymphocytes.
Polyclonal Activators
3
Streptococcus Infection and
Immunity
S Gaylen Bradley
College of Medicine/Research Affairs
Penn State University
500 University Drive
Hershey, PA 17033
USA
cavity, intestine, or urinary track, leading to subacute
endocarditis. In streptococcal toxic shock syndrome,
the cell wall protein antigens act as superantigens,
stimulating T cells to release cytokines that mediate
shock and tissue injury. Several weeks after a streptococcal infection, especially a sore throat, the host may
react to bacterial cell membrane antigens that crossreact with human heart tissue antigens, leading to
rheumatic fever—the most serious sequela of Streptococcus infection. Alternatively, antibody complexes
involving Streptococcus may attach to the glomerular
basement membrane, leading to blood in the urine and
acute glomerulonephritis. The organism multiplies in
the tissues, and the pneumococcal cell wall activates
procoagulation activity at the surface of the endothelial
cell, leading to release of fibrin into the alveoli (1).
Enterococcus faecalis is a member of the normal intestinal floral, but it is among the more common
agents causing nosocomial infections, especially in
intensive care units of hospitals. The prevalence and
seriousness in the hospital setting reflects the high
level of resistance of E faecalis to most antibiotics.
The streptococci have a wide range of virulence factors and factors to protect the bacterium from host
defenses. Anticapsular antibody to one of the many
polysaccharide capsular antigenic types does not afford protection against infection by S. pneumoniae
with a different capsular type. In S. pyogenes, the
cell surface M protein protects the bacterium from
phagocytosis by polymorphonuclear leukocytes.
M protein preparations contain antigenic determinants
eliciting antibodies that cross-react with human cardiac sarcolemma. The streptococcal cell wall contains
many components contributing to the organism's virulence, including recruitment of leukocytes into the
lung and subarachnoid space, enhancement of permeability of cerebral endothelia and alveolar epithelia,
induction of cytokine production, initiation of the procoagulation cascade, and stimulation of platelet-activating factor production (2). Lipoteichoic acid is a cell
wall constituent that facilitates colonization of host
structures by forming a complex with the M protein
of the bacterium and fibronectin on epithelial cells of
the host. C-reactive protein, a serum protein that binds
the lipoteichoic acid of S. pneumoniae, has bactericidal activity and blocks the adherence of the bacterium
to the platelet- activating factor receptor on the epithelial surface (3). Streptokinase is an enzyme that converts human plasminogen into plasmin, an activated
enzyme that digests fibrin. Hyaluronidase is an enzyme that degrades connective tissue thereby allowing
S. pyogenes to spread more rapidly. Streptococci produce several hemolysins, enzymes that lyse erythrocytes. Two well-characterized hemolysins are
streptolysin O and streptolysin S. S. pneumoniae
does not produce a battery of toxins and enzymes
3
Streptococcal Enterotoxin B (SEB)
609
3
Definition
Streptococci are spherical Gram-positive bacteria that
lack the enzyme catalase and grow as facultative
anaerobes. They are members of the so-called "lactic
acid" bacteria because they share with Lactobacillus
the ability to grown in high concentrations of sodium
chloride and produce lactic acid as a primary endproduct of carbohydrate metabolism. Streptococci are
widely distributed in nature with respect to host species and site of colonization on or in a host. They are
members of the normal microbial flora of the mouth
and intestinal tract of humans, but the genus also includes agents of serious human disease, in part by
invasive processes and in part by autoimmune-like
processes. Streptococcus is a heterogeneous genus,
some members of which produce polysaccharide capsules, while others produce a variety of extracellular
virulence factors.
3
3
Characteristics
Streptococci are important agents in human infectious
disease throughout the lifespan, and therefore drugs
that alter host resistance may have serious consequences. Streptococcus pyogenes may invade the
skin leading to edema or enter the lymphatics and
ultimately the blood stream after trauma or surgical
wounds, or establish local infections in the throat
that may spread to the middle ear and the meninges.
In the course of streptococcal bacteremia, the bacteria may attach to heart valves, leading to fatal acute
endocarditis. Even members of the normal streptococcal flora may enter the circulatory system from the oral
3
host resistance assays, pneumococcal disease models,
in vivo immunotoxicology testing
3
Synonyms
S
3
610
Streptococcus Infection and Immunity
but relies on its polysaccharide capsule to avoid host
defenses.
The immune system is a complex interactive network
of cells and humoral products that protects the intact
animal from infectious agents and other foreign matter,
and removes certain damaged or markedly altered host
cells (4). Numerous arms of the immune system are
available to the host in its defense against
S. pneumoniae infections; and the sum of the integrated attack on the invader is more effective than
the sum of the parts. Complement, for example, enhances recruitment of polymorphonuclear leukocytes
to infected sites. The first line of defense against
S. pneumoniae consists of phagocytosis and killing
of the bacteria after opsonization by complement
C3 (5,6). A successful host defense must not only
stop the growth of the invading bacterium, but must
also foster repair of damage to host tissue. During the
recovery process, polymorphonuclear leukocytes recruited to the infected lung adhere to the pulmonary
endothelium leading to the release of proteases that
can digest the fibrotic deposits (1).
3
3
Preclinical Relevance
The clinical relevance of immunotoxicity detected
in preclinical assays should be interpreted in conjunction with the pharmacologic dose, the reversibility of
the immune alteration, and effects on other target organs. It is generally recognized that decreases in serum
immunoglobulin are an insensitive indicator of immune perturbation and more reliable indicators must
be used to detect immune alternation reliably. Several
in vitro assay have proven to be useful indicators of
immune impairment; for example, the T-dependent antibody response to sheep erythrocytes as detected in a
hemolytic plaque assay. There is an excellent correlation between in vitro assays and host resistance assay
in that impaired host resistance is invariably linked to
a depressed in vitro response (7). On rare occasions
statistically significant alterations have been measured
in vitro, but altered host resistance has not been detected, documenting the important role of compensatory mechanisms in the complex immune network (4).
The S. pneumoniae infectious model has been found to
be a useful, reproducible, and informative tool for
detecting altered host defense subsequent to exposure
to a candidate drug (8). By considering timing of morbidity and mortality as well as final results, insight into
the mechanism of immune impairment can be predicted. Signs of early morbidity and mortality usually
indicate a diminution of complement activity. Morbidity and mortality that develop 2–4 days after challenge
are indicative of an altered response of the polymorphonuclear leukocytes. Effects on this cell type may
reflect impaired migration, phagocytosis, and/or nitric
oxide production. Differential expression occurring
beyond the fifth day after challenge indicates an effect
on antibody production. The S. pneumoniae model is
one of the most sensitive host resistance assays that
has been used in evaluating immunotoxicity of drug
candidates and industrial and environmental chemicals
(9). One of the reasons for its predictability is that it is
one of the least complicated host resistance models,
and one that measures the capability of the host to
mount a T-independent antibody response to the polysaccharide capsule, the functional capacity of granulocytic and monocytic cells, and serum complement activity.
There are practical and social limitations to the use of
animal models as a screening tool to detect immunotoxicity. These assays use a large number of mice and
are expensive to conduct. The mice require special
facilities to isolate the infected animals and require
special facilities to grow and manipulate the pathogen
(9,10). Pathogenic strains of streptococci do not grow
well on most bacteriologic media unless enriched with
blood or tissue fluid, and may require an atmosphere
containing 10% carbon dioxide. Accordingly, most
streptococcal models rely on death as the endpoint
rather than counts of colony-forming units in infected
tissues and organs.
Relevance to Humans
An intact immune system is critical for good health.
Modest changes in immune function, although reproducible and statistically significant, do not necessarily
indicate immunotoxicity that precludes further development of a drug candidate. Experimental animal
models are the best surrogates for detecting the harmful effects of drugs and chemicals and for detecting
their mechanisms of action. Although the mouse is not
usually considered the species of choice for toxicologic studies, the mouse immune system is the model of
choice for immunotoxicologic evaluations because it
is well characterized and most critical reagents are
available. Animal models provide the means whereby
dose-response and mechanistic studies can be performed to provide a basis for hazard evaluation as
related to human risk (10).
S. pneumoniae is the causative agent of streptococcal
pneumonia and bacterial meningitis. It accounts for the
majority of bacterial pneumonia, in part because so
many people carry strains capable of causing disease.
At least 1 million children die of pneumococcal disease every year, mostly in developing countries.
S. pneumoniae is the most common cause of bacterial
meningitis in the USA (2). In the US and other developed countries, morbidity and mortality from pneumococcal diseases occur most often among the elderly
population. People whose spleens have been damaged
or removed, and individuals with sickle cell disease
are also at increased risk to S. pneumoniae infection.
3
Stress
611
action. In: Biologic markers in immunotoxicology.
National Academy of Science USA, Washington DC,
pp 83–98
Numerous studies have found that host resistance
models are the best available means to illustrate a
link between immunosuppression and clinical manifestations of disease endpoints (5,10).
Regulatory Environment
Streptokinase
Streptococcus pyogenes produces the enzyme streptokinase which activates the plasminogen activator. Plasminogen activator catalyses the conversion of plasminogen in plasmin. Plasmin degrades the fibrin layer
around pathogens.
Dermatological Infections
Streptozotocin-Induced Diabetes
Mellitus, STZ-Induced Diabetic Rat
3
Diabetes and Diabetes Combined with Hypertension, Experimental Models for
Streptozotocin-Induced
Spontaneously Hypertensive Rat,
3
Diabetes and Diabetes Combined with Hypertension, Experimental Models for
Stress
The sum of the biological reactions to any adverse
stimulus, physical, mental, or emotional, internal or
external, that tends to disturb the organism’s homeostasis.
Stress and the Immune System
3
1. Wang E, Simard, M, Ouellet N, Bergeron, Y, Beauchamp
D, Bergeron MG (2002) Pathogenesis of pneumococcal
pneumonia in cyclophosphamide-induced leukopenia in
mice. Infect Immun 70:4226–4238
2. Tuomanen EI, Austrian R, Masure HR (1995) Pathogenesis of pneumococcal infection. N Engl J Med
332:1280–1284
3. Gould JM, Weiser JN (2002) The inhibitory effect of Creactive protein on bacterial phosphorylcholine-platelet
activating factor receptor mediated adherence is blocked
by surfactant. J Infect Dis 186:361–371
4. Keil D, Luebke RW, Pruett SB (2001) Quantifying the
relationships between multiple immunological parameters and host resistance: Probing the limits of
reductionism. J Immunol 167:4543–4552
5. Bradley SG (1995) Streptococcus host resistance model.
In: Burleson GR, Dean JH, Munson AE (eds) Methods
in immunotoxicology, volume 2. New York: Wiley &
Sons; 159–168
6. Bradley SG, Munson AE, McCay JA et al. (1995)
Subchronic 10-day immunotoxicity of polydimethyl
siloxane (silicone) fluid, gel and elastomer and polyurethane disks in female B6C3F1 mice. Drug Chem
Toxicol 17:175–220
7. Luster MI, Portier C, Pait, DG et al. (1993) Risk
assessment in immunotoxicology. II. Relationships
between immune and host resistance tests. Fund Appl
Toxicol 21:71–82
8. Bradley SG (1995) Introduction to animal models in
immunotoxicology: Host resistance. In: Burleson GR,
Dean JH, Munson AE (eds) Methods in immunotoxicology, volume 2. Wiley & Sons, New York, pp 135–
141
9. Bradley SG (1985) Immunologic mechanisms of host
resistance to bacteria and parasites. In Dean J, Luster MI,
Munson AE, Amos (eds) Immunotoxicology and
immunopharmacology. Raven Press, New York,
pp 45–53
10. Talmadge D (Chair: Subcommittee on Immunotoxicology) (1992) Animal models for use in detecting
immunotoxic potential and determining mechanisms of
A bacterium that causes respiratory tract infection.
Host Resistance Assays
3
References
Streptococcus pneumoniae
3
The effects of new drugs on the immune system
should be assessed before the drug is approved for
clinical trials. Evidence of immunotoxicity may be
detected during standard nonclinical toxicology evaluations, but additional studies directed specifically to
immunotoxicologic assessment are often warranted.
Any follow up investigations might include in vitro
immune function or in vivo host resistance assays
(10). Guidelines are given by FDA (CDER), Guidance
for Industry: Immunotoxicology Evaluation of Investigational New Drugs. October 2002, p 35. (http://
www.fda.gov/cder/guidance/index.htm)
S
Stress and the Immune System
Stress and the Immune System
Helen G. Haggerty
Bristol-Myers Squibb Co.
6000 Thompson Road
East Syracuse, NY 13057
USA
Synonyms
neuroendocrine response
network of signals that result in the bidirectional interaction of the nervous, endocrine, and immune systems. The hypothalamic-pituitary-adrenal (HPA) axis
and the sympathetic-adrenal medullary (SAM) axis are
activated, triggering a cascade of events that results in
the down regulation of immune function. In addition,
the activation of the immune system due to an infection, allergic response, or inflammation, can also cause
an increase in the activity of the HPA axis, which in
turn leads to the dampening of that immune response.
Characteristics
3
3
3
3
Stress and the Immune System. Figure 1 In response to a stress or immune activation, the hypothalamic-pituitary-adrenal (HPA) axis and the sympatheticadrenal medullary (SAM) axis trigger a cascade of
events that results in the downregulation of immune
function. Stimulation of the hypothalamus by either
stress or immune activation via cytokines leads to the
release of corticotropin-releasing hormone (CRH). CRH
stimulates the release of adrenocorticotropic hormone
(ACTH) from the pituitary gland, which promotes the
release of glucocorticoids. Circulating glucocorticoids
dampen the immune response and inhibit further
release of ACTH. CRH also stimulates the release of
epinephrine (adrenaline) and norepinephrine (noradrenaline) which can also down modulate immune
responses.
3
Stress is any natural or experimentally contrived
circumstance that poses an actual or perceived threat
to an animal's well being. The stressor can be psychological, physical, environmental, immunological,
or chemical. When an animal encounters a stressor,
its "fight-or-flight" response is activated. Systems
needed to deal with an immediate threat are upregulated, while those that are not necessary, such as the
digestive, reproductive, and immune system are downregulated. In response to a stress, the central nervous
system modulates the immune system by a complex
Stress-induced activation of the HPA axis and the
SAM axis stimulates the release of corticotropin-releasing hormone (CRH) from the hypothalamus.
CRH promotes the release of adrenocorticotropic hormone (ACTH) from the pituitary gland, which in
turn stimulates the release of glucocorticoids (corticosterone in rats and mice and cortisol in humans)
from the adrenal cortex. Glucocorticoids have been
shown to have potent immunosuppressive as well as
antiinflammatory and antiallergic properties. By inhibiting the function and trafficking of many immune
cells such as lymphocytes, macrophages, monocytes,
and neutrophils, glucocorticoids can downregulate the
immune response and keep it from becoming excessive. Glucocorticoids inhibit the release of a number of
inflammatory mediators and cytokines, as well as diminish the effect of these mediators on their target
tissue. Circulating glucocorticoids also exert a negative feedback on the HPA axis downregulating its activity.
CRH can also stimulate the production and release of
norepinephrine (noradrenaline) and epinephrine
(adrenaline) from the adrenal medulla. Lymphoid
and myeloid cells have been shown to exhibit β-2
adrenergic receptors that allow them to respond to
signals from the SAM axis. Generally, these signals
have been found to downregulate the immune response. Furthermore, the primary and secondary lymphoid tissues are innervated by noradrenergic and
sympathetic nervous system, which release neurotransmitters that can subsequently affect the immune
system.
Activated immune cells can also release a number of
mediators, such as proinflammatory cytokines, that
can then influence the HPA axis and nervous system.
For example, immune cells can be stimulated to release proinflammatory cytokines such as IL-1β, which
can directly stimulate the release of CRH from the
hypothalmus, initiating the cascade of events described.
How stress affects the immune system can also be
dependent on the degree of stress and its duration,
acute or chronic. Mild stress for a short period of
time can have no effect on the immune system or
3
Definition
3
612
Stress and the Immune System
may increase the activity for some immune parameters. However, intense or long-term stress has
been found to significantly lower the immune response. It is hypothesized that following an initial
stress response, resting immune cells become activated
to deal with that insult. However, if that stress response is not removed, the cells become tolerant and
no longer respond to an activating signal.
Preclinical Relevance
There are a number of preclinical models that have
been developed to study stress such as restraint, heat
or cold, electric shock, auditory stimulation, and
crowding or isolation. All these stresses have been
shown to activate the HPA axis and effect various
immune parameters, such as B cell and T cell proliferation, cytokine production, antibody production, natural killer cell cytotoxicity, and chemotaxis of monocytes and neutrophils. Therefore, when assessing the
immunotoxicity of compounds in preclinical studies, it
is important that these types of conditions, along with
stress due to the handling of laboratory animals during
experimental testing, are carefully controlled so that
they do not complicate the interpretation of study
data assessing immune parameters.
Many chemicals and pharmaceuticals can induce a
neuroendocrine stress response that can be immunosuppressive. In addition, the toxicological testing of
chemicals and pharmaceuticals in animals is often
conducted at high doses at or near the maximum tolerated dose (MTD). The significant toxicity exhibited
at these high doses can often be a source of stress for
the animal inducing the activation of the HPA axis.
Thus, when a change is observed on an immune parameter at toxic doses, it can often be difficult to discern whether it is due to a direct effect of the chemical
on the immune system or a stress-induced immunological change. In some cases, adrenal hypertrophy
and/or thymic depletion can be observed suggesting
a stress-mediated effect. Therefore, when studying
the effects of chemicals on the immune system,
doses that are not overtly toxic should be tested so
that a stress response does not lead to the erroneous
identification of a test compound as an immunotoxicant.
Corticosterone can account for a significant portion of
the suppression of several immunological parameters
in rodents treated with potent chemical stressors. A
linear model has been developed which demonstrates
a clear relationship between the cumulative plasma
corticosterone exposure and suppression of several immunological parameters. It is hoped that this model
may allow the determination of the contribution of
stress to an immunosuppressive response by a chemical by measuring a single neuroendocrine mediator.
613
Relevance to Humans
In humans, chronic and repeated stress has been
shown to impair immune function to the extent that
it can impact human health. Psychological factors that
can cause stress have been found to be associated with
increased susceptibility to and/or progression of a variety of pathophysiologic processes which involve the
immune system such as infections, tumors, allergies,
and autoimmune diseases. For example, in a healthy
person the immune system plays an important role in
keeping viruses under control. Many viruses are present in healthy people, but remain latent due to the
host’s immune defenses. If the cellular immune response becomes compromised, viruses can become
reactivated and lead to active infection. Studies conducted in individuals undergoing psychological stress,
such as medical students during exams or caregivers of
people with Alzheimer’s disease, have demonstrated a
reduction in cellular immune responses and an increase in antiviral antibody titers, suggesting viral reactivation. They also demonstrate a reduced response
to viral vaccines, as well as a higher incidence of respiratory infections.
Regulatory Environment
The effect of stress on the immune system is addressed
in the FDA guidance document on Immunotoxicity
Testing of Investigational New Drugs. The FDA recognizes the role stress can play in contributing to an
immunosuppressive response and recommends that it
be carefully controlled. Even when there is a potential
indirect mechanism, such as stress, for alterations in
immune parameters, it is recommended that the pattern
of that response be carefully evaluated to determine if
immune function studies would be warranted. One approach that is cited for determining the contribution of
stress in an immunosuppressive response is the quantitation of stress-related blood hormones, such as corticosterone, and comparison with systemic drug exposure.
References
1. Black PH (1994) Central nervous system-immune system
interactions: Psychoneuroendocrinology of stress and its
immune consequences. Antimicrob Agents Chemother
38:1–6
2. Pruett SB, Collier S, Wu WJ, Fan R (1999) Quantitative
relationship between the suppression of selected immunological parameters and the area under the corticosterone concentration versus time curve in B6C3F1 mice
subjected to exogenous corticosterone or to restraint
stress. Toxicol Sci 49:272–280
3. US Food and Drug Administration (2002) Guidance for
Industry: Immunotoxicology evaluation of investigational
new drugs. FDA, Rockville
4. Whitnall MH (1993) Regulation of the hypothalamic
corticotropin-releasing hormone neurosecretory system.
Prog Neurobiol 40:573–629
S
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Stressor
5. Yang EV, Glaser R (2002) Stress-induced immunomodulation and the implications for health. Int Pharmacol
2:315–324
Structure Activity Relationships
Chemical Structure and the Generation of an Allergic Reaction
3
Stressor
A stimuli that elicits a stress reaction.
Stress and the Immune System
STZ-SHR Rat
3
Diabetes and Diabetes Combined with Hypertension, Experimental Models for
3
Stroma
3
Stromal Cell-Derived Factor-1 (SDF-1)
Stromal cell-derived factor-1 (SDF-1) is a CC chemokine also known as CCL12 which is important in the
migration of lymphocytes, hemopoietic stem cells and
other types of cells. It binds to CXCR4, an HIV coreceptor.
Chemokines
3
Stromal Cells
Stromal cells are epithelial cells present in immune
tissues (thymus, lymph node, spleen, bone marrow)
that express numerous growth factors, ligands, and
receptors, and which facilitate lymphocyte development, maturation, apoptosis, and immune responses.
Steroid Hormones and their Effect on the Immune
System
3
Structural Alert
Sunlight
Sunlight includes various parts—the ultraviolet (UV)
part, the visible part and the infrared (IR) part. Most
important for the induction and elicitation of reactions
due to a combination of radiation and a light absorbing
chemical is the UV-A part (315–400 nm) of the solar
spectrum. The UV-A part of sunlight in general does
not induce sunburn reactions. Sunburn reactions are
mainly induced by the UV-B part (280–315 nm).
Photoreactions
3
Matrix of adherent multilayered cellular complex that
provides the microenvironment for hematopoiesis.
Composed of endothelium, fibroblasts, reticular cells,
fat cells, and macrophages. Furnishes lodging and issues short-range growth factors.
Colony-Forming Unit Assay: Methods and Implications
Superantigens
Thomas Herrmann
Institute for Virology and Immunobiology
University of Würzburg
Versbacher Strasse 7
D-97078 Würzburg
Germany
Synonyms
sAg, superantigens.
Definition
Proteins that bind and activate most or all T cells that
express a particular set of T-cell antigen receptors
(TCR) β chain variable genes (Vβ). In the broader
sense, superantigens are lymphocyte-activating molecules binding specifically to V gene-encoded parts of
antigen receptors.
Characteristics
The term superantigen (SAg) has been coined by Kappler and Marrack (1) as an operational definition of
various T-cell activating substances with specificity for
T cell antigen receptors comprising certain superantigen-specific Vβ. This means that many or all T cells
3
A chemical substructure commonly associated with
reactivity and thus with the ability of a substance to
behave as an allergen.
Chemical Structure and the Generation of an Allergic Reaction
3
Superantigens
expressing these Vβ respond to a superantigen, independent of their original antigen-specificity and
MHC restriction: e.g. the bacterial superantigen Staphylococcus enterotoxin B (SEB) activates Vβ7- and
Vβ8-positive mouse T cells while SEA, for example,
activates Vβ 3-, 10-, 11- and Vβ12-positive mouse
T cells. The Vβ specificity, the limited number of
Vβ genes in mouse (up to 25) and man (about 60)
together with the reactivity of several Vβ for one
superantigen results in a high frequency of superantigen-specific T cells. This frequency can reach nearly
half of all primary T cells compared with less than 1 in
10 000 responding to a typical peptide antigen. Table 1
gives a comparison of the key features of antigens and
superantigens, and Figure 1 shows a highly schematic
view of the binding of superantigens to TCR and
MHC molecules.
Various methods are used to determine the Vβ specificity of a T cell response. In vitro, cell cultures of
unprimed T cells are performed with or without superantigen and subsequently Vβ−specific activation is
determined. This can be done by parallel measurement
of early activation markers such as CD69 and Vβ expression or by testing changes in the Vβ frequency
after several days of cell culture. An alternative method is the analysis of activation of T cell clones or T cell
hybridomas expressing known Vβ. In vivo, superantigens can be administered by various routes and cause
a transient increase in the frequency of cells expressing superantigen-specific Vβ, which often is followed by apoptosis or anergy of the superantigen-activated cells.
Superantigens cannot be defined by structural criteria,
therefore it is extremely important to ensure origin and
purity of a T-cell activating agent before claiming its
superantigenicity. Particularly, this holds true for products of Staphylococcus aureus and Streptococcus
pyogenes. Both bacteria produce a wide range of
615
superantigens (Table 2) and (cross)-contamination
has led to erroneous claims of superantigenic features.
An example of the practical importance is the originally reported activation of mouse Vβ 3 T cells by SEB,
which is due to the minute contamination from SEA
found in some commercial preparations. Another instructive example is the Vβ-specific stimulation of
human T cells by Epstein-Barr (EB) virus-infected
cells, which is not caused by an EBV product, but
by a protein of the human endogenous retrovirus K
(HERV-K), which becomes transactivated after EBV
infection.
It also should be pointed out that changes in the Vβ
distribution in a T cell pool found after antigenic challenge or in medical conditions do not necessarily result
from action of superantigens. Indeed, a nonrandom
Vβ usage of TCR comprising certain Vβ has been
documented also for the immune response to various
peptide antigens.
The superantigens produced by the gram-positive bacteria S. aureus and S. pyogenes are characterized in
some detail. They show little sequence similarity to
each other but share a carboxy domain, which is structurally similar to the immunoglobulin-binding motifs
of streptococcal proteins G and L and an amino-terminal domain with an oligosaccharide oligonucleotide
(OB)-binding fold, which can be found in the B subunits of cholera and pertussis toxin. Based on sequence similarity, they can be further divided into
five groups as listed in Table 2. Table 2 also lists
superantigens of gram-negative bacteria and viruses
which are less well characterized with respect to structure, MHC and TCR-binding (2–5).
The most intensively studied viral superantigens are
those encoded by germline-integrated proviruses
(mtv) of the retrovirus mouse mammary tumor virus
(MMTV). They are found in all laboratory mouse
strains and also in wild mice. Thymic expression of
mtv superantigen induces a Vβ specific deletion of
3
S
Superantigens. Table 1 Comparison of superantigens (SAgs) and peptide antigens
Superantigens
Peptide Antigens
Genes encoding TCR contact sites
BV
AV, AJ, BV, BJ, DJ, BJ
TCR structures interacting with (S)
Ags
CDR1, 2, HV4 of the β-chain and framework
region
CDR1, 2, 3
Frequency of specific primary T cells 1/2–1/100
1/10000–1/100000
Presenting molecule
MHC class I and class
II
MHC class II
Processing required for presentation No (with exception of mtv-SAgs)
Yes
Size of presented molecule
1–3 kDa
15–30 kDa
HV4, hypervariable loop 4; MHC, major histocompatability complex; TCR; T cell receptor.
616
Superantigens
Superantigens. Figure 1 Schematic diagram of the
complex of T cell receptor, superantigen (Sag) and
MHC class II. Numbered boxes in the T cell receptor
show CDR1-3 and hypervariable loop 4 (HV4). Note
that some SAg contact also the antigenic peptide and/
or bind both chains of the MHC class II molecule.
mtv-SAg-specific thymocytes. The mtv superantigen
expressed by peripheral B cells can lead to a massive
stimulation in mixed leukocyte cultures which was
originally used to define the so-called “minor lymphocyte stimulatory locus” (Mls).
Like antigens, superantigens need to be presented. Efficient binding has been only reported for MHC
class II molecules and, in contrast to peptide antigens,
bacterial superantigens are presented as intact molecules. MHC class II binding varies between superantigens (Table 2) and can involve the α chain, the
β chain, or both chains of the MHC class II molecule.
Exact binding sites of the TCR vary for different types
of superantigen but, consistent with the Vβ specificity,
contacts are mostly found in the BV-encoded domain,
especially the complementarity determining regions 1
and 2, the framework regions, and the fourth hypervariable loop (Figure 1; Table 1).
Due to the unique topology of the TCR-Sag-MHC II
complex, the original MHC restriction of the T cells
plays only a minor (or no) role in superantigen recognition. Consequently—although to a variable extent—
CD4 as well as CD8 cells respond to the superantigens
presented by MHC class II molecules. Furthermore,
presentation across the species barrier occurs. It can
even improve the T cell response if the presenting
xenogenic MHC class II molecule binds superantigen
better than the autologous restriction element.
MHC class II binding of superantigens varies not only
between species but also between MHC class II isotypes (in human leukocyte antigens(HLA)-DR, - DP,
- DQ or in mouse H2A, H2E) and their alleles. So far,
most superantigens have been isolated from human
pathogens. Often they bind much better to human
than to mouse or rat MHC class II molecules, which
may be because of an adaptation of the superantigen
producing pathogen to the host, and this to some extent explains species-specific differences in the dose
response.
The biological function of most superantigens is unclear. Bacterial superantigens induce a massive antigen-independent activation of the immune system.
This activation can cause a flooding of the organism
with cytokines and, subsequently, disease symptoms.
It can also interfere with an antigen-specific immune
response and thus may be advantageous for the invading microorganism. In the case of MMTV, superantigen-induced lymphocyte activation is needed for virus
replication and the replication cycle is disturbed if
superantigen-specific T cells are missing. As mentioned above, this can happen as a consequence of
intrathymic mtv-SAg-induced deletion, meaning that
expression of a superantigen by an endogenous retrovirus may protect from infection by an exogenous retrovirus.
Preclinical Relevance
The Vβ specificity of superantigen-mediated T cell
activation, together with the possibility to track superantigen reactive cells with Vβ-specific monoclonal
antibodies, has made superantigen a widely used tool
in T cell immunology. Moreover, superantigen-induced T cell activation interferes with antigen-induced
immune responses, so that both exacerbation and suppression have been reported for animal models of autoimmunity (especially experimental autoimmune encephalomyelitis) as well as for animal models of allergy and asthma.
The contribution of some of the staphylococcal enterotoxins (SE) and toxic shock syndrome toxin 1 (TSST1) to the etiology of the toxic shock syndrome is undisputed, but not faithfully reflected by the commonly
used mouse models. In these models, SEB in conjunction with hepatotoxic agents such as d-galactosamine
induces a toxic shock syndrome-like disease, but notably this disease shows a tumor necrosis factor
(TNF)α−induced lethal hepatocellular apoptosis,
which has not been reported for patients with clinical
toxic shock syndrome.
Relevance to Humans
Many superantigens are potent toxins—but some aspects of their toxicity like the food poising caused by
SE—are independent of the T cell activating properties. Nevertheless, there is a clear correlation between
the massive superantigen-induced cytokine release and
various forms of toxic shock syndrome, scarlet fever
and illness caused by systemic Yersinia pseudotuberculosis infection. In other medical conditions (such as
atopic dermatitis, psoriasis, Kawasaki syndrome-like
SEH5,4
SEG
SEE
SED
SEA5
SPE-A5
SSA5
SEG
SEC3
2
SEC22
SEC12
SEB2
TSST-15
SPE-I
SPE-H
SMEZ-2
SMEZ-1
SPE-J
SPE-G
Streptococcus pyogenes SPE-C5
Staphylococcusaureus
Streptococcuspyogenes
Staphylococcus aureus
Organism and Superantigen
Toxic shock syndrome
?
?
1, 3, 15
2, 12, 14, 15
Class II α + β
?
?
18.1, 9.1, 22a
β
2.1, 4.1, 7.3, 8.1
?
?
2.1
2.1, 7.3, 9.1
?
?
2.1, 6.9, 12.3
4.1, 8.1
?
β
1, 2, 5.1, 10
?
?
?
?
?
Toxic shock syndrome,
scarlet fever
?
Food poisoning (preferentially)3, toxic shock
syndrome
Toxic shock syndrome,
scarlet fever
?
?
3, 12, 13a, 14
5.1, 6.3, 6.4, 6.9, 8.1, 18 Class II α + β
5, 12
1.1, 5.3, 6.3, 6.4, 7.3, 7.4, Class II α + β
9.1, 18
?
3, 12, 13a, 14
5, 12, 13.2
12, 13.2, 14, 15, 17, 20
12, 13.2
Toxic shock syndrome
(preferentially), food poisoning3
Class II α
2
3, 12, 13.2, 14, 15, 17,
205
Associated Disease (s)
Binding to MHC
Chain
BV (Vβ) Specificity in
Humans
Superantigens. Table 2 Vβ specificity of superantigens (SAgs) produced by different organisms (grouped according to reference (4).
IV
III
II
I
Group
Superantigens
617
S
Various types stimulate
different sets of Vβ
8
Specific for 8.3 mouse
T cells
Mouse mammary tumor virus (MMTV)
Rabies virus nucleocapsid8
Urtica dioica agglutinin UDA
Class I and II
?
?
?
?
Rabies3
SAg required for replication cycle of virus
Juvenile diabetes type I?
Systemic disease after
Yersinia infection
Arthritis in rodents; unclear association in
humans3
Class I α + β
?
Scalded skin syndrome3
?
?
Associated Disease (s)
?
?
?
?
Binding to MHC
Chain
V
Group
EBV, Epstein-Barr virus; ETA, exfoliate exotoxin A; MAS/MAM, Mycoplasma arthritidis SAg/mitogen; MHC, major histocompatability complex; SEB, Staphylococcus enterotoxin B;
SMEZ, Streptococcus pyogenes mitogenic toxin; SPE-A Streptococcus pyrogenic exotoxin A; SSA Streptococcus SAg; TSST, toxic shock syndrome toxin; YPM, Yersinia
pseudotuberculosis mitogen.
1
Vβ specificity of staphylococcal and streptococcal products according to reference (5).
2
3-D crystal structure for substance or of substances complexed with TCR and/or MHC available.
3
No (or unclear) contribution of superantigenic activity to disease symptoms.
4
Appears to be an “untypical” SAg, which binds to MHC II β chains and activates only human Vα 10 T cells
5
Also designated as 19.
6
Transactivated during EBV infection.
7
Exclusively found in mus spp.
8
So far tested only by one laboratory.
7, 13
Human endogenous retrovirus K HERK6
2
ETB
3, 9, 13
2
ETA
Yersinia pseudotuberculosis YPM
5.1, 5.2, 6.7
SEK
8, 175
1, 5, 6b, 9, 23
SEI
Staphylococcus aureus
Mycoplasma arthritidis MAS/MAM
BV (Vβ) Specificity in
Humans
Organism and Superantigen
Superantigens. Table 2 Vβ specificity of superantigens (SAgs) produced by different organisms (grouped according to reference (4). (Continued)
618
Superantigens
Suppressor Cells
illness) and various autoimmune diseases the role of
superantigens remains to be clarified.
The analysis of the human T cell response to superantigens can be complicated by neutralizing antibodies
found in many human sera. Also, differential binding
of Vβ−specific monoclonal antibodies to Vβ alleles
can lead to misinterpretation in the analysis of a superantigen response.
619
cept of suppressor cells fell into disfavor for several
years, following an inability to clone the cells or to
precisely characterize the mechanisms of their activity,
new evidence for a population of natural suppressor
T cells, now referred to as T regulatory cells, has reinvigorated the field (1). At the same time, significant
advances have been made in the characterization of
non-lymphoid natural suppressor cells derived from
the myeloid lineage (2).
Regulatory environment
No specific regulation for superantigens as such.
References
1. White J, Herman A, Pulle AM, Kubo R, Kappler JW,
Marrack P (1989) The Vβ specific superantigen staphylococcal enterotoxin B: Stimulation of mature T cells and
clonal deletion in neonatal mice. Cell 56:27–35
2. Acha-Orbea H, MacDonald HR (1995) Superantigens of
mouse mammary tumor virus. Ann Rev Immunol
13:459–486
3. Sundberg EJ, Li Y, Mariuzza RA (2002) So many ways
of getting in the way: Diversity in the molecular
architecture of superantigen-dependent T cell signaling
complexes. Curr Opin Immunol 14:36–44
4. McCormik JK, Yarwood JM, Schlievert PM (2001) Toxic
shock syndrome and bacterial superantigens: An update.
Ann Rev Microbiol 55:77–104
5. Alouf JE, Müller-Alouf H (2003) Staphylococcal and
streptococcal superantigens: Molecular, biological and
clinical aspects. Int J Med Microbiol 292:429–440
Suppressor Cells
Nancy I Kerkvliet
Oregon State University
Dept. Environmental and Molecular Toxiology
Corvallis, OR 97331
USA
Synonyms
CD4; CD25 regulatory T cells, TR1 cells, myeloid
suppressor cells, MSC
Definition
Cells that function to actively suppress the activity of
other cells within the context of an immune response
are called suppressor cells. Non-specific as well as
antigen-specific suppressor cells have been described.
Together, they comprise a basic homeostatic mechanism within the immune system that has been recognized for more than 25 years. Suppressor cell activity
is appreciated for its role in the maintenance of tolerance to self-antigens, and is maligned for its ability to
prevent effective immunological responsiveness to tumors and certain other pathogens. Although the con-
Characteristics
T Regulatory Cells
CD4+ T regulatory cells have been characterized phenotypically by their natural expression of CD25 (the
alpha chain of the interleukin-2 receptor). In mice,
these cells also express high levels of CD62L and
low levels of CD45RB, distinguishing them from antigen-activated T cells that transiently express CD25
but downregulate the expression of CD62L and upregulate expression of CD45RB. In some models, regulatory T cells also express CD152 (CTLA-4), a signaling molecule that inhibits T cell activation. In rats,
T regulatory cells express the CD4+CD45RC− phenotype. In humans, CD4+CD25+ T cells with regulatory
cell functions have been isolated from peripheral
blood.
CD4+ T regulatory cells have a low proliferative capacity in vitro, which is dependent on IL-2. When
repetitively stimulated in the presence of IL-10 a subpopulation of CD4+ T cells develop (designated
TR1 cells) that secrete high amounts of IL-10 as well
as transforming growth factor-β (TGF-β), and suppress the immune response of other T cells in vitro
and in vivo. In other models, IL-4 production is also
associated with suppressive activity.
Activation of CD4+ T regulatory cells is T cell receptor
(TCR)-dependent. However, once activated, these
cells inhibit proliferation of other T cells in an antigen-non-specific manner. The inhibitory effect of
T regulatory cells induces the target cells to undergo
cell-cycle arrest at the G0/G1 stage.
CD4+ T regulatory cells have been shown to inhibit
autoimmune-related pathology in several experimental
disease models including experimental allergic encephalitis (EAE), diabetes, inflammatory bowel disease, as well as the immunopathology associated
with neonatal thymectomy (for primary references
see reference (1)). Depletion of CD4+CD25+ T cells
can also enhance immune responses to foreign antigens associated with microbial pathogens and to tumor
antigens. Furthermore, expansion of CD4+CD25+
T cells or augmentation of their activity can suppress
allograft rejection. Thus, the activity of natural
CD4+CD25+ T regulatory cells not only influences
the occurrence of autoimmune disease but can also
suppress the response of T cells to exogenous antigens.
S
620
Suppressor Cells
Myeloid Suppressor Cells
Myeloid suppressor cells (MSC) morphologically resemble granulocyte-monocyte progenitor cells, expressing the granulocyte-monocyte markers CD11b
(Mac-1) and Gr-1 (Ly-6G), and appear to be similar
to the "natural suppressor (NS) cells" described more
than two decades ago. When cultured, MSC lost their
expression of Gr-1 but retained high expression of
CD11b. They also stained positive for F4/80 and
CD31. The cells did not express class II, B220,
CD3, or CD16, whereas a subpopulation of the cells
expressed a low level of CD11c, DEC 205, and/or
CD86. Depending on the cytokines present during differentiation in vitro, suppressive (IL-4) or activating
(IL-4 + GM-CSF) functions could be induced, suggesting a common myeloid progenitor.
As summarized in Mazzoni et al. (2), numerous investigators have shown that MSC accumulate in the
spleens and lymph nodes of tumor-bearing mice,
where they inhibit antibody production, generation
of cytotoxic T lymphocytes (CTL), and lymphocyte
proliferative responses. MSC activity correlated with
the release of GM-CSF by the tumor cells and was
simulated by administration of GM-CSF to normal
mice. In addition to tumor-bearing states, MSC also
accumulate in the spleens of mice undergoing intense
immune stimulation—such as that associated with
graft-versus-host disease or treatment with superantigens—and may function to restrain otherwise overwhelming immune responses. Several soluble mediators, including inhibitory cytokines (TGF-β and IL10), prostaglandins, reactive oxygen intermediates, hydrogen peroxide, and nitric oxide (NO) have been
implicated in the suppressive mechanism of MSC, of
which NO appears to be particularly important. Production of peroxynitrite (ONOO−), a potent oxidant
that inhibits T cell activation and induces T cell apoptosis, has been shown to mediate the suppressive effects of MSC in some studies.
Preclinical Relevance
Suppressor cells can profoundly influence the outcome
of an immune response. If suppressor cell activity is
selectively augmented by exposure to an exogenous
chemical, the subsequent immune response will be
suppressed. Depending on the immune context in
which suppressor cell activity is induced, the suppression may be beneficial or deleterious. In the 1980s,
suppressor cell activity was invoked to explain numerous situations of immune suppression following chemical exposure—however, definitive characterization of
the suppressor cells remained elusive. The possibility
that 2,3,7,8-tetrachlorodibenzo-p-dioxin and other immunotoxic AhR ligands act through induction of suppressor cell activity was debated for many years and
remains unresolved. The enhanced susceptibility of
mice to tumor growth following exposure to ultraviolet (UV) light was also attributed to the induction of
suppressor cell activity. Similarly, induction of suppressor cells by UV light was associated with suppressed systemic immune responsiveness to contact
sensitizers.
Loss of suppressor cell activity induced by exposure to
an immunotoxicant could also lead to uncontrolled
immune responses, resulting in development of immune-mediated pathologies and exacerbation of autoimmune disease. For example, treatment of mice with
cyclophosphamide (CY) induced the onset of diabetes
in male and female non-obese diabetic mice at an age
when spontaneous diabetes rarely occurs. Since spleen
cells from CY-treated diabetic mice were capable of
transferring the disease to irradiated non-diabetic recipients, the loss of suppressor cells by CY was concluded. CY was also reported to act as a selective
toxicant toward suppressor cells, resulting in a potentiated allergic contact dermatitis response. Loss of suppressor cells has also been investigated for a role in
mercury-induced and d-penicillamine-induced autoimmunity. Recently, mercury-induced autoimmunity
was shown to be exacerbated by treatment of the
mice with a reagent that blocked CTLA-4; similar
treatment induced disease in mice genetically resistant
to the disorder. These results suggest that the activity
of T regulatory cells may be important in this model.
Relevance to Humans
Numerous disease states may benefit from therapeutic
manipulation of suppressor cell function, ranging from
induction of effective antitumor immunity to prevention and treatment of autoimmune diseases and transplant rejection. On the other hand, inadvertent changes
in suppressor cell function by exposure to immunotoxicants could induce or exacerbate the disease process.
As our understanding of suppressor cell immunology
progresses, and as therapeutic reagents that target suppressor cells are developed, monitoring suppressor cell
activity may become a routine measurement for assessing immunologic status, and regulatory requirements
for their assessment may logically ensue.
Regulatory Environment
There are no regulations that require the analysis of
suppressor cells in immunotoxicity assessments.
References
1. Maloy KJ, Powrie F (2001) Regulatory T cells in the
control of immune pathology. Nature Immunol 2:816–
821
2. Bronte V, Serafini P, Mazzoni A, Segal DM, Zanovello P
(2003) l-arginine metabolism in myeloid cells controls
T lymphocyte functions. Trends Immunol 24:302–306
Systemic Autoimmunity
3. Mazzoni A, Bronte V, Visintin A et al. (2002) Myeloid
suppressor lines inhibit T cell responses by an NOdependent mechanism. J Immunol 168:689–695
621
stranded DNA and upon excitation emits light. There
is no need to design a probe for the gene target being
analyzed. The dye cannot distinguish between specific
and non-specific DNA products.
Polymerase Chain Reaction (PCR)
3
Surface Plasmon Resonance (SPR)
Syngeneic
The genetic relationship between individuals of a species in an inbred population is designated as syngeneic. In humans syngeneic refers to the fact that within
an individual chromosomes are identical.
Idiotype Network
Graft-Versus-Host Reaction
3
Surface-acting agents that form a monomolecular layer
over pulmonary alveolar surfaces, to stabilize alveolar
volume by reducing surface tension and protecting
against risk of collapse (atelectasis).
Respiratory Infections
Primate Immune System (Nonhuman) and Environmental Contaminants
3
3
Surfactant
Symian
3
A biophysical method for detecting binding of ligands,
such as antibodies, to immobilized antigens on a sensor chip surface in real-time. In SPR-based assays the
signals (resonance units) are generated when the binding of a molecule to the biosensor surface causes a
change in refractive index (i.e. light reflection from a
conducting film at the interface between two media).
Immunoassays
3
Systemic Anaphylaxis
A statistically-validated health outcome measure
which is not the true object of screening or diagnosis
but which is intended to substitute for the final health
outcome of interest. Surrogates are usually chosen because they can be measured before the primary health
outcome.
Polymerase Chain Reaction (PCR)
An allergic reaction to systemically administered antigen which in serious cases (life-threatening) is known
as anaphylatic shock. It is characterized by circulatory
collapse and suffocation due to tracheal swelling. It is
mediated by antigen-specific IgE antibodies binding to
mast cells of connective tissue throughout the body.
Hypersensitivity Reactions
3
Surrogate
3
Systemic Autoimmunity
Sweetbread (When Used as Food)
Thymus
3
Swine
S
K Michael Pollard
Department of Molecular and Experimental Medicine
The Scripps Research Institute
10550 North Torrey Pines Road
La Jolla, CA 92037
USA
Synonyms
3
SYBR Green
In real-time PCR detection, this dye binds to double-
Autoimmunity, multisystem autoimmunity, systemic
autoimmunity, connective tissue diseases
Definition
An exact definition of autoimmunity is difficult because the etiology of the majority of autoimmune diseases is unknown. There is also confusion as to what
3
Porcine Immune System
Systemic Autoimmunity
3
3
3
3
3
Systemic autoimmune diseases are disorders of unknown etiology. They are thought to be influenced
by genetics, gender, and the environment. Systemic
autoimmune diseases are classified clinically by their
respective spectrum of signs and symptoms (1). There
is no individual diagnostic test which identifies any of
these disorders and their complexity makes diagnosis
particularly difficult. For disorders such as SLE and
3
Characteristics
3
Systemic autoimmunity comprises a group of disorders best characterized by systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren syndrome (SS), and scleroderma. Systemic autoimmune
diseases are complex clinical entities that may involve
all of the organ systems of the body so that two patients without overlapping clinical findings may still
be classified as having the same disease. Individual
systemic autoimmune diseases are identified according
to clinical features (see below).
scleroderma patients are classified according to a list
of criteria that have been drawn up for each disorder
after clinical evaluation of the signs and symptoms of
selected patients. Certain clinical features may be more
common in one type of disease than another. For example RA is most commonly associated with an inflammatory polyarthritis of the small joints, and the
presence of rheumatoid factor. In contrast the arthritis
of SLE is non-deforming and non-erosive. SLE patients commonly have photosensitivity, serositis, lymphopenia, renal disease, and antibodies against doublestranded (ds) DNA. Scleroderma includes disorders
ranging from the generalized form of diffuse cutaneous systemic sclerosis to the far less severe limited
cutaneous form, also known as CREST (calcinosis,
Raynaud’s phenomenon, esophageal involvement,
sclerodactyly, and telangiectasia). Skin-thickening is
a common feature of the forms of scleroderma, as is
fibrosis and vasculopathy.
A characteristic feature of autoimmune diseases is the
presence of autoantibodies. In organ-specific autoimmunity autoantibodies react with the particular
organ or tissue associated with the disease (e.g. antiacetylcholine receptor autoantibodies in myasthenia
gravis). In systemic autoimmunity autoantibodies
react with common cellular components that appear
to bear little resemblance to the underlying clinical
picture (e.g. anti-dsDNA autoantibodies in SLE). In
both types of autoimmune disease autoantibody specificities can serve to aid diagnosis. Autoantibody specificities may occur at very different frequencies in a
variety of diseases, and the resulting profile of specificity versus frequency can be indicative of a particular
clinical syndrome.
With regard to the relationship between systemic autoimmunity and immunotoxicology it is important to
note that numerous studies have shown that interactions between certain xenobiotics (chemicals and therapeutics) and the immune system can result in autoimmunity. The most common cases resemble SLE, or
scleroderma. Drug-induced SLE is most commonly
associated with drug treatment as the induced clinical
signs and symptoms are temporally related to starting
a drug, and generally abate with discontinuation.
However, disease induction can require months to
years of exposure, and symptoms such as autoantibodies may persist for many months after cessation of
exposure. Approximately 40 medications are associated with the appearance of systemic autoimmune
features. In some cases drug-induced SLE appears to
be mediated by reactive drug metabolites generated
from the ingested medication by neutrophil-mediated
oxidative transformation. The two most frequently described are procainamide and hydralazine. When
injected into the thymus of mice, the reactive metabolite of procainamide—procainamide hydroxyla3
constitutes an autoimmune disease. In general terms
the immune system (of healthy individuals) is tolerant
of the tissue constituents of its host, but intolerant of
non-host (or “non-self”) matter such as invading
viruses, bacteria or other pathogens. In autoimmunity
the immune system mounts responses against tissue
constituents of the host organism. This response is
most commonly referred to as recognition of “self”,
as opposed to “non-self” (e.g. viruses, bacteria). The
“self/non-self” paradigm is argued to be the basis for
understanding how the immune system determines
whether or not to respond to a suspected challenge.
Considerable debate exists as to the definition of “self”
and “non-self” with both “infection” and “danger”
being suggested as possible discriminators. Irrespective of the final outcome of these ongoing discussions
it is clear that autoimmunity constitutes a significant
disruption in the mechanisms that regulate the immune
systems’ ability to discriminate at the molecular level.
An autoimmune disease may be described in one of
two ways. The presence of autoantibodies is deemed
by some to be sufficient indication that the immune
system has failed to regulate “self/non-self” discrimination. Others argue that it is important to determine if
the presence of autoimmunity is causally related to
disease, and not the byproduct of disease or simply
an innocent bystander. Clearly the most rigorous definition is that autoimmunity (e.g. autoantibodies) is the
cause of disease, however this can be difficult to establish, particularly in human patients.
Autoimmune diseases consist of two types:
* organ-specific, in which the autoimmune response
targets a particular organ system
* systemic autoimmunity, which targets multiple
organ systems of the body.
3
622
Systemic Autoimmunity
USA in 1989 the ingestion of impure l-tryptophan
was associated with scleroderma-like hardening of
the skin and subcutaneous tissues. The syndrome
was characterized by myalgia and eosinophilia, followed by fatigue, muscle cramps, and sclerodermalike skin changes. This illness was subsequently
named eosinophilia-myalgia syndrome (EMS). EMS
shares features with TOS and eosinophilia fasciitis.
These and other scleroderma-like disorders share a
number of clinical findings among which are activation of eosinophils and disordered regulation of fibroblast collagen synthesis, apoptosis, and proliferation.
Several animal models have been described for scleroderma, including a substrain of white leghorn chicken called L200 from the University of California at
Davis. Several murine strains have also been described
with a phenotype of tight skin ( TSK) which is associated with thickened skin and fibrosis due to mutations in the fibrillin gene. Scleroderma in American
Choctaw Indians has been localized to the fibrillin
gene, suggesting that biosynthesis of this extracellular
matrix component by fibroblasts may underlie the cutaneous and visceral fibrosis seen in scleroderma.
Although overproduction of collagen by fibroblasts
may be a common feature of scleroderma, defects in
the fibrillin gene are not. Animal models of TOS and
EMS continue to be examined using exposure to the
presumptive causative agents. However these studies
have been largely unsuccessful, suggesting that either
the true causative agent has not been identified or that
the metabolism of these compounds differs between
human and animal species.
The association of fibrillin with scleroderma should
not be confused with the similar sounding fibrillarin.
Fibrillarin is a nucleolar protein and the target of autoantibodies in a portion of patients with diffuse scleroderma. An intriguing aspect of fibrillarin immunotoxicology is that a major histocompatibility complex
(MHC) restricted anti-fibrillarin autoantibody response
can be induced in mice following mercury exposure.
Although the human scleroderma and mercury-induced murine anti-fibrillarin responses share many similarities, the systemic autoimmunity elicited in mice
by mercury is more similar to SLE than to scleroderma
(3). These animal model studies suggest that MHCrestricted autoantibody responses can occur irrespective of the underlying diseases symptoms. As anti-fibrillarin is not a feature of human SLE these findings
suggest that xenobiotics may not only exacerbate idiopathic disease but may elicit xenobiotic-specific responses.
3
3
mine—produces an autoantibody response typical of
drug-induced SLE. The mechanism responsible for
procainamide-induced autoimmunity in the mouse
has been shown to involve disruption by procainamide
hydroxylamine of positive selection of T cells in the
thymus (2). The end result is a failure to establish
unresponsiveness to low-affinity selecting self antigens, which leads to the seeding of peripheral lymphoid organs with T cells that can be activated by
interaction with antigen-presenting cells carrying self
antigen. Although this mechanism has yet to be shown
to operate in human drug-induced lupus, it is the only
model to unambiguously elicit the autoantibody response described in human procainamide-induced
SLE.
The role that drug/chemical exposure plays in eliciting
idiopathic systemic autoimmunity in humans remains
to be elucidated. However studies with mice predisposed to develop SLE have shown that exposure to
many of these compounds (e.g. UV radiation, lipopolysaccharide, halothane, mercury) leads to exacerbation of disease. Such studies clearly show that environmental exposures can influence disease expression and suggests that environmental exposures of disease susceptible human hosts may lead to earlier or
more severe presentation of symptoms of systemic autoimmunity.
Xenobiotic-induced scleroderma, or pseudoscleroderma, can be drug-related but has also been associated
with occupational exposures. Scleroderma related to
environmental exposures may share features in common with idiopathic scleroderma, particularly cutaneous features, but may also present unique clinical features unrelated to the idiopathic form. An important
feature of pseudoscleroderma is the similarity between
diseases induced by seemingly disparate exposures,
which suggests a common disease mechanism. Some
of the agents implicated in scleroderma are silica,
vinyl chloride, amines, bleomycin and organic solvents.
Two significant outbreaks of pseudoscleroderma due
to environmental exposure have occurred in recent
years (1). Toxic oil syndrome (TOS) occurred in
Spain in 1981 following ingestion of adulterated rapeseed oil. The causative toxic agent was identified as
illicit oil—originally destined for industrial use—
which had been refined to remove the aniline denaturant and then sold as olive oil in unlabeled 5-liter containers. Over 20 000 people were affected, with more
than 1200 deaths. The most distinctive lesion of TOS
is a non-necrotizing vasculitis involving different
types and sizes of vessels in every organ. The most
obvious scleroderma lesion consisted of skin changes
and was seen in all three phases of the clinical presentation. However pulmonary hypertension was also a
severe feature of the later stage of the disease. In the
623
Preclinical Relevance
A significant proportion of xenobiotic-induced systemic autoimmunity is due to therapeutic drug exposure. Such adverse drug reactions may be detected
S
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624
Systemic Lupus Erythematosus (SLE)
during clinical trials and other regulatory testing of
compounds destined for human use. Systemic autoimmunity due to human exposure to unregulated or illicit
chemicals (such as toxic oil) may be difficult to diagnose without careful epidemiological study.
Biological Systems, Volume 34: Mercury and its Effects
on Environment and Biology. Marcel Dekker, Basel,
pp 421–440
4. US Department of Health and Human Services (2002)
Autoimmune Diseases Coordinating Committee (ADCC)
Research Plan (Report) Publication No 03-5140. National
Institutes of Health, Bethesda
Relevance to Humans
Systemic Lupus Erythematosus (SLE)
Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease of unknown etiology,
characterized by involvement of multiple target organs
(skin, heart, lungs, kidneys, joints and nervous system)
and typified by the production of autoantibodies, esp.
antinuclear antibodies (ANA). It is mediated by autoantibodies against DNA, RNA, and proteins associated
with nucleic acids. These form immune complexes
which damage small blood vessels and/or glomeruli
in the kidney by complement activation. SLE disproportionately affects females, with women comprising
nearly 90% of all cases.
Synonymous with disseminated lupus erythematosus.
Graft-Versus-Host Reaction
Autoimmune Disease, Animal Models
Antinuclear Antibodies
Systemic Autoimmunity
Autoimmune Disease, Animal Models
Hypersensitivity Reactions
Steroid Hormones and their Effect on the Immune
System
Chemotaxis of Neutrophils
Complement Deficiencies
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Some form of autoimmunity, including both systemic
and organ-specific forms, is argued to occur in 14–
22 million individuals or 5%–8% of the US population
(4). The list of autoimmune diseases comprises about
80 individual disorders with varying degrees of severity, prevalence and economic cost to healthcare services and the national economy. The most common
systemic autoimmune disease is rheumatoid arthritis
with 2.1 million suffers in the USA. Other systemic
autoimmune diseases are less common; SLE is found
in about 240 000 individuals, while diseases like scleroderma are found less often. The number of individuals diagnosed with xenobiotic-induced systemic autoimmunity constitutes a relatively small proportion of
patients with any specific disorder. However the true
significance of environmental exposure in the etiology
of idiopathic systemic autoimmunity remains to be
determined. It is important to note that when considered together autoimmune diseases place an equal or
even greater burden on society than cancer or heart
disease, conditions more commonly thought responsible for the health burden on modern-day society (4).
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The major regulatory body involved in identifying potential adverse reactions to drugs and other therapeutics in the United States is the Food and Drug Administration (FDA).
3
Regulatory Environment
Systemic Vascular Resistance (SVR)
References
The resistance to blood flow through the systemic vascular bed.
Septic Shock
3
1. Rose NR, Mackay IR (eds) (1996) The Autoimmune
Diseases, 3rd ed. Academic Press, San Diego
2. Kretz-Rommel A, Rubin RL (2000) Disruption of
positive selection of thymocytes causes autoimmunity.
Nature Med 6:298–305
3. Pollard, KM, Hultman P (1997) Effects of mercury on the
immune system. In: Sigel H, Sigel A (eds) Metal Ions in