to the alveolus, and the alveolar epithelium. In many instances, the red blood cells are just barely able
to fit through the small capillaries, so the blood cell wall is often in very close proximity to this
membrane complex with the alveolus.
Figure 9.5 illustrates how the remarkable design discussed above facilitates gas exchange. Carbon
dioxide and oxygen readily cross this membrane complex in a process of simple diffusion. Many
inhaled airborne industrial chemicals will also readily cross this membrane and will enter the
bloodstream. These potential toxins thus enter the blood circulatory system in a manner analogous to
someone receiving an intravenous infusion of a drug. A unique view of the alveoli is provided in Figure
9.6. The small holes, called
pores of Kohn,
provide for some ventilation between adjacent alveoli.
Toxicologic insult to the lung as well as various disease states can result in a functional derangement
of this membrane system. Exposure to some chemicals may result in an increase in fluid in the
interstitial space. If sufficient fluid accumulates, a condition known as pulmonary edema, gas exchange
can be hindered sufficiently to result in severe difficulty in breathing and even in death. Damage to the
membrane itself can result in scarring, which may increase the thickness of the membrane or decrease
the elasticity of the lung tissue, or both. As with pulmonary edema, an increase in the thickness of the
membrane can deleteriously affect pulmonary gas exchange. Alterations in elasticity make the work
of breathing harder, which can decrease the volume of respiration as the individual tires from the
increased effort required. Of course, whenever gas exchange or the volume of respiration is sufficiently
decreased, the amount of oxygen pressure in the circulatory system will also decline. If this decline
proceeds to a sufficient extent, affected individuals can become seriously compromised in their health
status.
Figure 9.4 Photomicrograph of lung tissue, showing the relationship of a terminal bronchiole (TB) and its
accompanying blood vessel, the pulmonary artery (PA), to the alveoli. (Reproduced with permission from J. F.
Murray, The Normal Lung. The Basis for Diagnosis and Treatment of Pulmonary Disease, Saunders, Philadelphia,
1976.)
9.1 LUNG ANATOMY AND PHYSIOLOGY
173
Physiologic Differences between Inhalation and Ingestion
Following inhalation, the chemical goes directly into the bloodstream without being first processed by the

temperature inversions. The toxicity of inhaled particulates has been known for a long time, especially
in relation to occupational exposure. The early (1493–1541) famous toxicologist Paracelsus described
the relationship between mining occupations and pulmonary toxicity in the sixteenth century.
Particle Size
In the case of particulates, size is the primary critical determinant of how much of and where the agents
will be deposited. The range in particle size for various aerosols is generally as follows: dusts, up to
100 µm; fumes, from 10 Å to 0.1 µm; smokes, less than 0.5 µm. The pattern of airflow in the respiratory
system and anatomic features of the exposed individual are also important.
Most inhaled particles are not spherical, but highly irregular in shape. In order to categorize the
highly heterogeneous nature of inhaled particles, the aerodynamic diameter is calculated for the
population of particulates of interest. This value is based on the settling velocity of the population of
particles and roughly approximates what the particles’ diameter would be if it were compared to a
Figure 9.6 Scanning electron micrograph showing interior of an alveolus and its pores of Kohn. (Reproduced
with permission from D. V. Bates et al., Respiratory Function in Disease. An Introduction to the Integrated Study
of the Lung. Saunders, Philadelphia, 1971.)
9.1 LUNG ANATOMY AND PHYSIOLOGY
175
spherical particle in the time it takes the particles to settle in the air. This calculation is also referred
to as the
mass median aerodynamic diameter.
If the number of particles is of primary interest (and not
necessarily particle shape), the count median diameter is determined. Of course, the size of particles
may change during the course of traversing the respiratory tract. Since the respiratory tract is highly
humidified, particles that absorb water could be expected to undergo chemical reactions and increase
in size as they descend.
Lung Deposition Mechanisms
Particles tend to deposit in the lung according to size, air velocity, and regional characteristics of the
respiratory system. In the nares, nose hairs tend to block out the very large particles that enter the nose.
Once inside the nares, the abrupt turn in the nasopharyngeal system of humans (from going up to going
down) results in the impact of many of the larger particles on the walls of this region of the respiratory

from the body is achieved through the gastrointestinal system, the lymphatics, and the pulmonary
blood.
In the nasopharyngeal and tracheobronchial regions, there is a
mucociliary escalator
mechanism.
In the respiratory wall, there are pseudostratified columnar epithelial cells together with specialized
goblet cells, which produce a layer of mucous along the wall of epithelial cells. Hundreds of cilia,
which resemble small hairs, protrude from the epithelial cells (Figure 9.7). The mucous itself is in two
layers: the lower layer, known as
sol,
contains the cilia and is thin and watery so that cilia movement
is not impeded; the upper layer, the
gel,
is thick and viscous. The cilia beat in unison and move the gel
layer along like a continuous sheet (Figure 9.8). Inhaled particles and other toxins become trapped on
the gel layer. In the tracheobronchial region, the cilia beat upward, and the entrapped particles in the
gel are propelled up toward the mouth. Typically, an individual will solubilize the material in saliva,
which is then eliminated via the gastrointestinal tract. Occasionally the material may be coughed out
of the body. In the nasopharyngeal region, the cilia beat downward toward the mouth and rely on the
same mechanisms of removal. Typically, mucociliary clearance will occur within hours of the
deposition of most particles, and in healthy individuals, the process is usually completed within 48 h.
176
PULMONOTOXICITY: TOXIC EFFECTS IN THE LUNG
In the alveolar region,
macrophages
provide a mobile and effective defense against particles,
bacteria, and other offensive agents that reach the lower respiratory tree. Chemotactic factors are
released when these inhaled agents deposit in the lung, and these factors alert the phagocytic cells to
the location of the agents. The macrophages then engulf them and attempt to ingest them with
proteolytic enzymes. An example of a macrophage moving from one alveolus to another through a

alveocapillary membrane
complex
and enter the pulmonary blood. This complex consists primarily of the capillary and alveolar
membranes, separated by an interstitial space (sometimes with fluid in it). The lining of the alveolar
membrane also has a lining of surfactant (dipalmitoyl lecithin), which serves to equalize the inflation
pressures of the heterogeneously sized alveolar sacs.
The passage of the inhaled gases and vapors across the alveocapillary membrane complex, or the
diffusion efficiency, is influenced by several factors. The solubility of the inhaled compound is
important, as highly water-soluble compounds are often more likely to deposit in the upper respiratory
Figure 9.9 Scanning electron micrograph of the interior of an alveolus showing pores of Kohn (P) and a
macrophage (arrow). [Reproduced with permission from Murray (1976) (see Figure 9.4 source note).]
178
PULMONOTOXICITY: TOXIC EFFECTS IN THE LUNG
system, before reaching the alveolar regions of the lung. The condition of the alveocapillary membrane
is also important. Poor health conditions in a patient might lead to the engorgement of the interstitial
space with fluid, which would impair the diffusion of toxic chemicals across the alveocapillary
membrane. While this protects the affected individual from the toxic effects of the inhaled
chemical, it also prevents the free exchange of oxygen and carbon dioxide, which can have obvious
life-threatening outcomes.
The degree of uptake of inhaled gases and vapors can be quite significant in workers in many
occupations. Following the initiation of inhalation, rapid uptake of perchloroethylene, a commonly
used dry cleaning solvent for which there are thousands of potential exposures, can be observed in
many different tissues (Figure 9.10). In this case, the uptake of perchloroethylene in circulating blood
and seven tissues was remarkably rapid, and for many industrial chemicals, it is often within minutes
of exposure. It is often interesting to note that the levels of the inhaled solvent remained fairly constant
throughout the inhalation exposure period. This can have important ramifications in occupational
exposures, as workers who enter an environment with a potentially toxic gas can experience systemic
toxic effects almost immediately, and these effects can persist for long periods of time (while the
inhalation exposure period continues). For instance, many industrial solvents cause neurobehavioral
depression following inhalation exposure, and workers have been known to be injured as a result of

and lung cancer in many thousands of Americans each year.
Interference with Pulmonary Defense
Tobacco smoke inhalation results in the derangement of the
pulmonary defense mechanisms necessary to protect against the inhalation of industrial toxins. It has
been shown that, following chronic cigarette smoking, the cilia in the mucociliary escalator become
increasingly paralyzed. The decrease in ciliary activity slows or prevents the removal of deposited
toxins from the nasopharyngeal and tracheobronchial regions, as the gel layer becomes more sedentary.
Many of the more than 2000 components of tobacco smoke are known to be respiratory irritants, and
these irritating properties lead to an increased production of mucous in the respiratory system.
Therefore, there is a decreased movement (and removal) of mucous simultaneously with an increase
in mucous production. Eventually, some of the airways can become impeded and even blocked,
severely limiting the respiratory volume of the affected individual. Sometimes the overworked mucous
glands will increase in size sufficiently to block the airways themselves, further impeding airflow and
increasing resistance.
It has been shown that the cellular defense mechanisms of the lung, particularly the alveolar
macrophages and the alveolar polymorphonuclear leukocytes, are significantly impacted by tobacco
smoke inhalation. In many cases, these cells may be killed, causing the release of proteolytic enzymes,
which come in contact with the respiratory membrane surfaces. Pulmonary emphysema can result, if
this process is extensive, from the severe rupturing of the septa walls. Even short of cell death, these
cells become less efficient in the removal of particulates and other toxins. Therefore, the inhalation of
toxic agents in industrial environments has the potential to exert greater toxicity in smokers than in
equally exposed nonsmokers. This has been shown repeatedly for many exposures to toxic chemicals
in occupational studies, such as with asbestos. For this reason, occupational epidemiologists and
physicians will often look for correlations between toxicity in an industrial worker population and
tobacco use.
Lung Cancer and Tobacco Smoke
Bronchogenic carcinoma data from the 1980s estimated that
approximately 90 percent of the more than 100,000 lung cancer cases each year in the United States
are due to tobacco smoke inhalation. A very distressing aspect of this unpleasant data is that the
incidence of lung cancer, previously occurring more often in men, is growing rapidly in the female

as pulmonary edema. Although most of these individuals will recover completely, many people have
died from irritation of the airways following industrial chemical inhalation, and every incident must
be treated as a serious episode. It is highly recommended that workers have a baseline pulmonary
function test on file with which to compare after an irritant exposure.
Fibrosis and Pneumoconiosis
A variety of lung diseases resulting from the inhalation of dusts has been encountered in occupational
environments. The disease mechanism, known as
fibrosis,
results when the lung gradually loses
elasticity as a result of the pulmonary response to long-term dust inhalation. The disease condition is
referred to as
pneumoconiosis,
derived from the Latin and Greek root words
pneumo,
which means
breath or spirit, and
coniosis,
which means dust.
TABLE 9.1 Chlorine Dose–Response Relationships
<4 ppm Can be tolerated up to 30 min
15 ppm Severe respiratory symptoms begin
30 ppm Coughing, choking, chest pain
>40 ppm Pulmonary edema
>1000 ppm Immediate death
9.2 MECHANISMS OF INDUSTRIALLY RELATED PULMONARY DISEASES
181
Silicosis
Following long-term inhalation of silica-containing dusts, many workers have developed irreversible
lung damage known as silicosis. One-half to two-thirds of the rocks in the crust of the planet contain
silica, so it is to be expected that many industrial processes result in the production of silica-containing

neoplasms in the respiratory tract. One form of cancer, mesothelioma, is so rare in situations outside
of asbestos exposure that many physicians consider it a “ marker” disease for asbestosis. A higher
incidence (up to an 80-fold increase) of bronchogenic carcinoma is distinctly correlated with tobacco
smoke inhalation and asbestos exposure. These asbestos related cancer deaths generally occur from
25–40 years after the asbestos inhalation.
Excess Lung Collagen
Most types of pulmonary fibrosis involve distinct changes in the proportion of the types of lung collagen
that is produced in the affected lung. Such information is used by pathologists today in determining
the degree of pulmonary fibrosis that has occurred. In most normal lungs, the two most common
collagen types, type I and type III, are observed at a ratio of approximately 2:1. When pulmonary
fibrosis occurs, there is generally an increase in type I collagen in relation to type III collagen.
Mechanistically, the presence of the fibers causes macrophages to release lymphokines and various
growth factors, which leads to an increase in the production of certain collagen types. Since type III
182
PULMONOTOXICITY: TOXIC EFFECTS IN THE LUNG
is considered to be more compliant than type I, this might be the cause of the “stiffening” of the lung
tissue, but this is not known for certain.
Emphysema
Whenever inhaled toxins result in the progressive destruction of the alveolar walls of the lung tissue,
there is an enlargement of the lung air spaces accompanied by a decrease in the surface area of the
lung available for gas exchange. This is commonly referred to as
emphysema,
and it is a relatively
common pulmonary disease condition in the United States. Although emphysema is due primarily to
tobacco smoke inhalation, a number of inhaled industrial toxins may also be responsible for the
development of emphysematic conditions. For instance, the inhalation of coal dust by miners over
extended periods has been shown to result in both pulmonary fibrosis and emphysema.
Recent research has indicated that a genetically related deficiency in α-1-antiprotease, of a
biochemical inhibitor of elastase, is clinically related to the relatively early onset of emphysema. It is
believed that the breakdown of the alveolar walls is modulated by elastases, which are released by

hazard for exposed workers. Usually, the edema fluid is not readily detected by the exposed individual
or by clinical examination for at least several hours after the termination of exposure.
In a typical occupational exposure, the worker may experience short-term symptoms involving
irritation of the airway, which influences them to seek immediate medical assistance. Since the
short-term symptoms usually have no immediate cytotoxic sequelae, the medical examination will
result in no revelation of significant morbidity, and the patient will be released. Then, 4–24 h later, the
pulmonary edema rapidly develops, usually while the patient is asleep. Often, when patients awake
9.2 MECHANISMS OF INDUSTRIALLY RELATED PULMONARY DISEASES
183
with difficulty breathing, they are already in an advanced stage of pulmonary decline, and the condition
is difficult to treat. It is critical that individuals who have been exposed (or potentially exposed) to
agents known to cause pulmonary edema, be kept overnight (or at least 24 h following the exposure)
at a medical facility where they can be closely monitored. A series of chest X rays during the “ critical
period,” when pulmonary edema could be initiated, should be taken and examined for the appearance
of fluid in the lung.
Respiratory Allergic Responses
Among the potential allergic reactions of the respiratory system in industrial exposures, there are many
well-characterized conditions, as well as somewhat mysterious and hard-to-define personnel histories.
Many of the characterized diseases have historically involved certain occupations and are often named
after the occupations in which they were first observed. The allergic reactions involve antibody
formation against certain inhaled toxins or to dusts and organic particles. Subsequent exposure to the
same agent then often results in a more severe reaction, which is understandably a real problem in the
workplace where individuals often work in the same environment and receive repeated exposures. In
the less characterized occurrences, it often appears that exposure to one agent might result in a
nonspecific reaction to a multitude of other compounds inhaled at some later time.
Occupation-Related Inhaled Allergic Disorders
A very old disease, known as “farmers’ lung,” involves the allergic reaction to the Actinomycetes
spores found in hay. Hay that is collected in the field is often damp, and the high temperatures that can
arise inside damp hay over time may give rise to large numbers of the thermophilic Actinomycetes
spores. When the farmers inhale these spores, IgG antibodies are produced (against the spores), and

of the disease, however, has been shown to result in permanent injury. In addition, the symptoms
associated with byssinosis are usually more severe in smokers than in nonsmokers.
184
PULMONOTOXICITY: TOXIC EFFECTS IN THE LUNG
Industrially Related or Occupational Asthma
Many individuals develop asthma following workplace exposure, and some asthmatics suffer addi-
tional provocation following the inhalation of certain industrial toxins. The inhalation of wood dusts,
for instance, has been implicated in both situations. Some grocery workers have developed an asthmatic
condition following the wrapping of meats with plastic film. Apparently, heating the plastic to seal it
releases toluene diisocyanate, which is then inhaled. Subsequent exposure to even very low levels of
the plastic, or its component, may result in a severe reoccurrence of symptoms.
It has been shown that the bronchiolar muscles of asthmatics will undergo constriction at a lower
concentration of inhaled industrial chemicals than will those of nonasthmatics. Not surprisingly, these
individuals often find themselves reacting in situations in which their co-workers do not respond. A
further complication for these workers is that exercise tends to exacerbate the asthma symptoms.
Physical exertion, obviously required in many industrial situations, along with the simultaneously
chemical exposure can lead to severe complications for the affected worker.
Lung Cancer
Until the twentieth century, lung cancer was relatively rare. The rapid promotion of lung cancer to the
number one cancer killer is directly related to the inhalation of tobacco smoke (probably 80–90 percent
of all lung cancers) and industrial/atmospheric chemicals. The relationship between tobacco smoke
inhalation and lung cancer was discussed previously. Many industrial chemicals have also been linked
to lung cancer in workers and laboratory animal studies.
The dusts and fumes of many metals have been demonstrated to be carcinogenic in lung tissue.
Epidemiologic studies conducted on worker populations in smelting operations have long shown
definitive relationships between metal inhalation and lung cancer. Industrial metal carcinogens include
nickel, arsenic, cadmium, chromium, and beryllium. Workers in mining operations, including metal
recovery from ores, are at risk for developing lung cancers because of exposure to certain metals such
as chromium and uranium. The inhalation of benzo(
a

cantly. Industrial chemicals that are inhaled as gases and vapors are often taken up very rapidly, and
the effects in workers can be substantial, both in the lung and at distant sites.
Inhaled industrial toxins exert toxicity by several distinct physiological mechanisms, which have
historically led to many deleterious disease states in workers. Specific mechanisms of respiratory-
related toxicity include
•
Irritation of respiratory airways
•
Fibrosis and pneumoconiosis
•
Pulmonary edema
•
Respiratory allergic responses
•
Lung cancer
Some inhaled agents exert toxic effects by more than one mechanism, and many workers may suffer
from more than one lung-related disease condition. Potential interactions between different inhaled
toxins, especially tobacco smoke and various industrial chemicals, pose an additional threat. There is
a tremendous potential for inhalation exposure to toxic chemicals in the workplace; therefore, workers
must be monitored thoroughly by vigorous programs in industrial hygiene, environmental monitoring,
occupational physicals, and toxicology.
REFERENCES AND SUGGESTED READING
Church, D. F., and W. A. Pryor, “The oxidative stress placed on the lung by cigarette smoke,” in
The Lung,
Vol II,
R. G. Crystal, J. B. West, P. J. Barres, et al., eds., Raven Press, New York, 1991, pp. 1975–1979.
Dosman, J. A., and D. J. Cotton, eds.,
Occupational Pulmonary Disease. Focus on Grain Dust and Health,
A c a d e m i c
Press, New York, 1980.

Press, San Diego, 1993, pp. 259–303.
Mauderly, J. L., “Effects of Inhaled Toxicants on Pulmonary Function,” in
Concepts in Inhalation Toxicology,
R.
O. McClellan, and R. F. Henderson, eds., Hemisphere, New York, 1989, pp. 347–402.
McClellan, R. O., and R. F. Henderson, eds.,
Concepts in Inhalation Toxicology,
Hemisphere, New York, 1989.
Menzel, D. B., and M. O. Amdur, “ Toxic responses of the respiratory system,” in
Doull’s Toxicology: The Basic
Science of Poisons,
3rd ed., Macmillan, New York, 1986.
Morgan, W. K. C., and A. Seaton, eds.,
Occupational Lung Diseases.
Saunders, Philadelphia, 1975.
Morrow, P. E., “Dust overloading in the lungs: Update and appraisal,”
Toxicol. Appl. Pharmacol.

113
: 1–12 (1992).
186
PULMONOTOXICITY: TOXIC EFFECTS IN THE LUNG
Muir, D., ed., Clinical Aspects of Inhaled Particles, Davis, Philadelphia, 1972.
Parent, R. A., Treatise on Pulmonary Toxicology, Vol. I, Comparative Biology of the Normal Lung. CRC Press,
Boca Raton, FL, 1991.
Parkes, W. R., Occupational Lung Disorders, 2nd ed., Butterworths, Woburn, MA, 1982.
Samet, J. M., “ Epidemiology of lung cancer,” in Lung Biology in Health and Disease, C. Lenfant, ed., Marcel
Dekker, New York, 1994.
Shami, S. G., and M. J. Evans, “Kinetics of pulmonary cells,” in Comparative Biology of the Normal Lung, Vol.
1. Treatise on Pulmonary Toxicology, R. A. Parent, ed., CRC Press, Boca Raton, FL, 1991, pp. 145–155.

of immune origin, including acute and chronic respiratory distress, dermal reactions, and manifesta-
tions of autoimmune disease. The types of substances associated with immune system effects is
extraordinarily diverse, and include chemicals found in occupational and environmental settings,
infectious materials, certain foods and dietary supplements, and therapeutic agents. As discussed in
this chapter, dysregulation of the immune system by toxicants can lead directly to adverse health effects,
as well as rendering the body more susceptible to infectious disease and cancer.
The immune system is highly complex, with many facets poorly understood. Because of this,
assessment of potential immunotoxic effects of drugs, chemicals, and other agents is not a simple task.
Often, measurement of a variety of components of the immune system and/or their functionality is
required to gain an appreciation of the likelihood of immune dysfunction from drug or chemical
exposure. Increasingly, there is realization that the immune system may be among the most sensitive
target organs for toxicity for many chemicals and, as a result, merits special attention.
10.2 BIOLOGY OF THE IMMUNE RESPONSE
The immune system has evolved primarily to defend the body against the invasion of microorganisms,
although normal immune function is important in regulating and sustaining the internal environment
as well, such as recognition and removal of malignant cells. There are two types of immunity: natural
immunity (also termed
innate immunity
) and acquired immunity (also termed
specific immunity
).
Natural immunity
is nonspecific in that it is directed to a wide variety of foreign substances, and is
rarely enhanced by prior exposure to these substances. Natural immunity arises from several mecha-
nisms, including complement, natural-killer (NK) cells, mucosal barriers, and the unique activity of
189
Principles of Toxicology: Environmental and Industrial Applications, Second Edition
, Edited by Phillip L. Williams,
Robert C. James, and Stephen M. Roberts.
ISBN 0-471-29321-0 © 2000 John Wiley & Sons, Inc.

specific rejection of allografts. Recognition of “ self” is known to be guided, in part, by genetic
variations in proteins of the class I and II
major histocompatibility complex
(MHC). Initially, the ability
of the immune system to differentiate “self” from “ nonself” is an educational process. During
maturation, the system must ignore an infinite variety of self-molecules and yet be primed and ready
to respond to an array of exogenous antigens. Immunomodulatory control mechanisms lead to immune
tolerance of self and carefully orchestrate the immune response to targets and removal of foreign
macromolecules and cells. These control mechanisms arise from interactions among the several
different cell types with roles in proper immune function.
Lymphocytes are considered to be the major cells involved in a specific immune response in
humans. They are derived from pluripotent stem cells and undergo an orderly differentiation and
maturation process to become T cells or B cells (see Figure 4.1 in the chapter on hematotoxicity), with
critical functional roles in the host defense. T-cell development occurs primarily in the thymus, where
cell surface protein markers are acquired during the selection and differentiation process. These protein
markers are called CD antigens (for
cluster of differentiation
), and at least 78 different CD antigens
have been identified in humans. The presence of certain CD antigens, detectable by immunofluores-
cence, has been used to positively identify immunocytes. In general, mature T cells are characterized
by the presence of CD3
+
and CD4
+
or CD8
+
surface markers and are devoid of surface or cytoplasmic
immunoglobulin. There are various subtypes of T cells, such as T-helper (T
H
) cells, T-suppressor (T

also perform this function.
190
IMMUNOTOXICITY: TOXIC EFFECTS ON THE IMMUNE SYSTEM
In the specific immune response, antigen may be taken up by APCs and presented to T or B cells.
In order to present the antigen to T cells, the antigen must be processed, or partially digested by the
APC and then presented on its cell surface bound to an MHC class II molecule. Presentation of antigen
to B cells does not require this processing, and in fact B cells are capable of recognizing antigens
directly, without APC presentation. Antigens, either presented by APCs or encountered independently,
interact with immunoglobulins on the cell surface of B-cell clones. Different B-cell clones vary in the
immunoglobulins expressed on their cell surface, and these immunoglobulins can be quite specific in
terms of the antigens with which they will interact. Thus, a particular antigen may interact with only
one or a few B cell clones, a critical aspect in creating a specific immune response. When the antigen
binds to an immunoglobulin receptor on the B cell surface, the antigen–receptor complex migrates to
one pole of the cell and is internalized within the cell. The B cell becomes activated, and the antigen
is processed leading to display of antigenic peptides on the cell surface in conjunction with an MHC
class II protein.
T-cell activation is postulated to require at least two signals. The first signal is thought to be an
interaction between the CD4
+
T-cell receptor of T-helper (T
H
) lymphocytes and antigenic peptides and
MHC class II proteins presented by APCs or B cells. The second signal may be under the influence of
other receptor–ligand pairs on the T cell and cognate interactions through adhesion molecules of APCs,
MHC complex, and the various cytokines produced by T-cell subsets and accessory cells, such as
macrophages. When activated, T
H
cells proliferate, creating more cells for interaction with APC and
B cells.
An effective immune response requires the activation of specific subsets of T

expansion leads to increased production of antibody specific to that B cell, and this antibody, in
turn, has reactivity directed rather specifically to the antigen initiating the response. Through this
mechanism, the immune system is able to produce the necessary quantities of antibodies targeting
specific molecules (antigens) regarded as foreign. The synthesis of the antibody is tightly
regulated, however, and the proliferation of plasma cells and antibody synthesis are controlled by
cytokines and interactions with T cells. T-amplifier cells (T
A
) and T-suppressor cells (T
S
), as their
names imply, function to enhance or suppress the immune response, respectively. Control of the
immune response is achieved by balancing the stimulatory and inhibitory effects of T cells and
various cytokines.
After an encounter with an antigen, the immune system appears to retain “ memory” of that
antigen and is able to mount a more rapid and greater antibody response on subsequent contact,
even if the period between exposures to the antigen span several years. The basis for this memory
is still not well understood. Initial (
primary
) immune responses to T-dependent antigens require
a proliferative response by naive T and B cells. As these cells mature, they differentiate and
become
effector cells
. The elimination of effector T cells and the factors controlling the survival
of memory cells is still controversial. Because immune responses to viruses or immunization
encountered in childhood generally result in lifelong immunity, it has been presumed that memory
10.2 BIOLOGY OF THE IMMUNE RESPONSE
191
is afforded by long-lived cells that become activated only following repeat exposure to the antigen or
immunization. While it has been assumed that “ memory cells” last indefinitely following a single
antigen contact, recent evidence suggests the life-span of memory cells may be related to repeat contact

β
)
Several cell types, including
neutrophils and
macrophages
Variety of effects, including neutrophil and macrophage
activation, T- and B-cell chemotaxis, and increased IL2
and IL6 production
IL2 T cells Stimulates replication of T cells, NK cells, and B cells
IL3 T cells Involved in regulation of progenitor cells for several
different cell types, including granulocytes, macrophages,
T cells, and B cells
IL4 Activated T cells Activates T and B cells; suppresses synthesis of IL1 and
TNF
IL5 T cells and activated B cells Increases secretion of immune globulins by B cells
IL6 Several cell types, including T
and B cells
Important in inflammatory reactions and in differentiation
of B cells into Ig-secreting cells
IL7 Bone marrow stromal cells Important in regulating lymphocyte growth and
differentiation
IL8 Activated monocytes and
macrophages
Activates neutrophils; important for chemotaxis of
neutrophils and lymphocytes
IL9 T
H
cells Stimulates growth of T
H
cells

IMMUNOTOXICITY: TOXIC EFFECTS ON THE IMMUNE SYSTEM
very early in an immune response. IgM is much larger than the other Igs, consisting of five sets of
heavy/light-chain pairs bound together at a single point with another peptide (the J chain). Its
molecular weight is about 970,000. IgA may exist as a monomer (one basic unit of two pairs of H and
L chains) or as a dimer—two basic units bound together with a J chain. The monomeric IgA has a
molecular weight of about 160,000 and is the predominant form of IgA found in serum. IgA is the
primary Ig found in secretions (e.g., tears and saliva), mostly in the dimeric form with a molecular
weight of 385,000. IgD has a molecular weight of about 184,000, and is present in very low
concentrations in serum. Its function is unclear, but it may play a role in B-cell differentiation. IgE is
slightly larger than IgG (molecular weight of 188,000), and is normally present in low concentrations
in serum. It can attach itself to leukocytes and mast cells, and is the primary antibody involved in
hypersensitivity reactions.
In cell-mediated immunity, cells carrying the antigen on their surface are attacked directly by
cytotoxic T cells (T
C
) or other cell types such as natural-killer (NK) cells. In the case of T
C
cells,
recognition of cells to be destroyed is through interaction between processed antigen in conjunction
with MHC class I molecules on the target cell surface and an antigen receptor on the T
C
. In order to
be active, the T
C
must also receive stimulation from CD4
+
cells, principally in the form of IL2.
Mechanisms of target cell recognition by NK cells are not well understood.
Figure 10.1 Light and Heavy Chain Structure of IgG. IgG illustrates the basic structure of antibody proteins,
which consists of two long, heavy chains and two shorter, light chains held together by disulfide bonds. Composition

leading to induction of immune hemolytic anemia, thrombocytopenia or granulocytopenia.
Type III
. Soluble immune complexes consisting of a drug or chemical hapten (plus carrier
molecule) and its specific antibody plus complement components are primarily responsible
for immune complex disease. A particular form of immune complex disease arising from
injection of an antigen is called
serum sickness syndrome
. Clinically, a type III reaction may
be characterized by the onset of fever and the occurrence of a rash that may include purpura
and/or urticaria. The immunopathology includes the activation of complement and the
deposition of immune complexes in areas such as blood vessel walls, joints, and renal
glomeruli. Some of the signs and symptoms associated with drug-related lupus may be
included under type III reactions.
Type IV.
These reactions involve cell-mediated and/or delayed-type hypersensitivity responses.
The expression of type IV reactions requires prior exposure to the agent and T-cell sensitiza-
tion. A special subpopulation of T cells (T
D
) appear to be responsible for this reaction. The
T
D
cells react with antigens in tissues and release lymphokines, attracting macrophages to the
site and leading to an inflammatory response. The reaction is termed delayed because the
inflammatory reaction may not peak for 24–48 h, as opposed to responses occurring within
a few minutes to a few hours with other reaction types. These reactions are usually seen as
contact dermatitis occurring after the use of certain drugs or exposure to some chemicals.
Immunosuppression
Impairment of one or more components of the immune system from drug or chemical exposure can
lead to loss of immune function, or
immunosuppression

are not identical to idiopathic SLE,
however. Both are characterized by arthralgia and the appearance of antinuclear antibodies in the blood,
but the pattern of antinuclear antibodies is somewhat different, and renal and CNS complications
dominate idiopathic SLE while these are typically absent in drug-lupus. Symptoms of drug lupus
generally subside after the drug is withdrawn. Demonstration of autoimmune responses from environ-
mental exposure to chemicals (other than drugs) has been difficult, in part because of problems
identifying etiologic agents in retrospective studies of patients developing autoimmune disease. One
concern is that some chemicals may exacerbate underlying autoimmune disease (e.g., SLE), rendering
symptomatic a patient with subclinical disease or increasing the duration or severity of symptoms in
those with active disease. Unfortunately, differentiating the effects of chemical exposure from
progression of the underlying disease is difficult or impossible in practice. Understanding of autoim-
mune consequences of chemical exposure is further hampered by the general lack of satisfactory animal
models—the results obtained in laboratory animals seldom correspond exactly to observations in
humans.
10.4 CLINICAL TESTS FOR DETECTING IMMUNOTOXICITY
In the clinical setting, the use and proper interpretation of immunologic laboratory tests can be
important in establishing a differential diagnosis in a patient who has been exposed to an immunotoxic
agent. Immune system testing for diagnostic purposes can be challenging, however, because of the
complexity of the immune system and difficulty in establishing normal values for many of the tests.
When immune dysfunction from chemical exposure is suspected, it is important to be sure that the
patient is free from infectious disease and not taking medications that can influence immune func-
tion—obvious confounders to interpretation of any immune tests. Also, it is important to recognize
that many immune parameters, such as lymphocyte subpopulation counts, can vary normally by age
and gender, making the use of appropriate controls essential for proper interpretation of results. Finally,
10.4 CLINICAL TESTS FOR DETECTING IMMUNOTOXICITY
195
temporal variations in most tests are common. In order to demonstrate that an abnormality exists, it is
usually advisable to repeat the test one or more times to insure that a consistent result is obtained.
Some of the laboratory tests available provide information relevant to assessing humoral immunity,
others are useful in evaluating cellular immunity, and some can provide insight regarding both.

marker for T
S
cells. As discussed above, these
markers are not specific for T
H
and T
S
cells, however, and interpretation of a decreased CD4
+
to CD8
+
ratio as a loss of T help relative to T suppression is an oversimplification. A significant reduction in
CD4
+
cells is associated with several immunodeficient states (e.g., in patients with AIDS, undergoing
radiotherapy, or chemotherapy), implying that diminished CD4
+
is indicative of impaired antibody
production. This assumption is not infallible, however, because there are also circumstances in which
CD4
+
cells may be reduced without loss of antibody production. Significant changes in absolute or
relative concentrations of lymphocyte subsets may be suggestive of immunotoxic effects from
chemical exposure, but are not, by themselves, reliable indicators of compromised function.
Cutaneous Anergy
Anergy
is a generalized clinical condition of non-responsiveness to ubiquitous
skin test antigens that is frequently observed in patients who are immunosuppressed. Cutaneous anergy
may suggest functional impairment or abnormalities of the cellular immune system. The most
cost-effective method for evaluation of cutaneous anergy is the use of a battery of attenuated,

and B cells, and provides an indication of the capability of these two cells to interact
properly and of B cells to produce immunoglobulins. Lipopolysaccharide (LPS) is a mitogen effective
selectively on B cells, while phytohemaglutinin (PHA) and concanavalin A (con A) are selective T-cell
mitogens. Other stimulants to lymphocyte activation can be used, such as tetanus toxoid, diptheria
toxoid,
Candida
, and PPD, if the subject has been previously exposed to these. The rapid cell division
characteristic of a normal response to these mitogens is typically assessed by measuring incorporation
of
3
H-thymidine into DNA of the cells. Other endpoints of stimulation, such as increased expression
of IL2 receptors on T cells, can also be evaluated. The results of these tests are particularly prone to
variability, and the tests should be repeated on several occasions in order to demonstrate an abnormal
response.
In the mixed-lymphocyte reaction (MLR) test, lymphocytes from the test subject and another
individual are mixed. Normally, contact with the allogenic lymphocytes will cause the test subject’s
lymphocytes to become activated and proliferate. To conduct this assay, the target lymphocytes are
rendered incapable of replication, often by irradiation or by treatment with mitomycin C. Test subject
lymphocytes are then added, and the rate of their replication is evaluated by measuring incorporation
of
3
H-thymidine. The cytotoxic lymphocyte (CTL) assay takes the lymphocyte interactions one step
further to evaluate the ability of cytotoxic T cells (T
C
) to destroy target cells. After incubation of the
test subject and target lymphocytes, the subject T
C
are isolated, washed, and reincubated with target
lymphocytes preloaded with
51

immunotoxic agents.
4. The
nucleolar staining pattern,
which has been restricted to antibodies reactive with nucleolar
RNA. This pattern is associated with a particular form of systemic sclerosis (progressive
systemic sclerosis).
10.5 TESTS FOR DETECTING IMMUNOTOXICITY IN ANIMAL MODELS
For most chemicals, an assessment of their potential to produce immunotoxicity in humans is based
on testing in animals. Many of the tests used in animal studies are the same as, or at least analogous
to, those available for clinical assessment described above. However, studies in animals offer the
10.5 TESTS FOR DETECTING IMMUNOTOXICITY IN ANIMAL MODELS
197
opportunity to evaluate directly toxic endpoints difficult or impossible to assess clinically, such as the
development of immunopathology or loss of resistance to infectious disease.
Currently, a tiered approach is recommended for standardized testing for immunotoxicity in
animals. Tier I consists of a battery of tests intended to evaluate both humoral and cell-mediated
immune system integrity. An assessment of immune system pathology is also included in tier I (see
Table 10.2). If the results of tier I tests are negative, the chemical is considered not to possess significant
immunotoxic potential at the dosages tested. If effects are observed in tier I tests, additional tests are
conducted in tier II to better characterize the immunotoxic properties of the chemical. Tier II does not
consist of a rigid battery of tests, but rather the opportunity to select more specific tests to follow up
on observations made in tier I. Examples of tests that might be used in tier II are included in Table
10.2.
Many of the endpoints examined in tier I are basic. Total and differential white cell counts are
obtained from blood, body and specific organ weights are recorded, and tissues of particular relevance
for immune function (viz., spleen, thymus, and lymph nodes) are examined histologically for evidence
of injury. Humoral immunity is assessed with a plaque-forming cell (PFC) assay. In this assay, the test
animal is injected with sheep red blood cells (SRBCs) as the source of antigen. Four days later the
spleen is removed, and cells isolated from the spleen are cultured with intact SRBCs. B cells producing
IgM directed to SRBC antigens result in lysis of the red cells, producing clear areas in the culture called

hypersensitivity (type IV) response
Host resistance, assessed through challenge with pathogens or tumors
198
IMMUNOTOXICITY: TOXIC EFFECTS ON THE IMMUNE SYSTEM