Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 91
c
Markers of Inflammatory and Immune Response
Inflammation in the respiratory tract
can be caused by injury, immune response,
or infection. All three routes of induc-
tion of inflammation are of interest in
studying the health effects of respirable
pollutants. Inflammation due to injury
from inhaled toxicants is a measure of the
cytotoxicity of the pollutants; markers
of the progress of the inflammation could
potentially be used in a predictive fashion
to detect the early stages of irreversible
structural changes in the lung, such as
fibrosis and emphysema. Many inhaled
toxicants-such as isocyanates, cotton
dust, and beryllium-induce an immune
response that constitutes the major ad-
verse health effect of exposure. The
inflammatory response to infectious
agents is not the topic of this report, but
is of interest in relation to the potential
for some inhaled pollutants to reduce the
ability of the body to resist infection.
In this chapter, we will discuss markers
to detect and require the inflammation
induced by all three types of agents. First
is a discussion of markers of injury in-
duced in the respiratory tract by inhaled
toxic materials. That is followed by a
discussion of the effect of pollutants
on the infectivity of pathogens in the
respiratory tract. Finally, a major
portion of the chapter is devoted to a
91
discussion of the immune response of the
lung, markers of this type of response
and the use of memory cells of the immune
system as markers of both exposure and
adverse health effects in the respiratory
tract.
INFLAMMATORY RESPONSE TO
INHALED TOXINS
Epithelial cells and resident macro-
phages in the respiratory tract are the
points of first contact of the body with
inhaled toxicants. The ensuing injury
or death of those cells induces an in-
flammatory response characterized by the
release of cytoplasmic enzymes from the
damaged or lysed cells and the recruitment
of neutrophils to the site of injury. An
increase in the permeability of the alveo-
lar-capillary barrier is accompanied by
the transudation of serum protein. Macro
. .
~
phages may increase in number; it the
toxic material is a particle, there will
be a release of hydrolytic enzymes from
the macrophages, either during phagocyto-
sis, after lysis of the macrophage, or as
an active secretory process.
The inflammatory process provides many
markers that can be used to detect and
measure the response and to follow its
progress toward resolution or chronic
OCR for page 92
92
inflammation.
. . .
Lactate dehydrogenase,
a cytopiasm~c enzyme released from dam-
aged or lysed cells, can be used as a marker
of cytotoxicity. An increase in this en-
zyme can be used to distinguish between
toxic events and physiologic responses.
The presence of neutrophils or increased
serum protein in the epithelial lining
fluid can be used as a marker of the inflam-
mation induced by the injury. The activity
of hydrolytic and proteolytic enzymes
released by the phagocytic cells has been
shown to correlate with the toxicity of
particles (Beck et al., 1982; Henderson,
1988a,b).
The advent of fiberoptic bronchoscopy
has allowed sampling of fluids lining
the respiratory tract for analyses of the
above markers. The use of the technique
is described fully in Chapter 6. The analy-
sis of epithelial lining fluid (ELF) for
markers of inflammation has some advan-
tages over older methods of detecting in-
flammation. First, a pulmonary inflamma-
tory response can be detected with analysis
of the bronchoalveolar fluids before it
can be detected with radiography (Fahey
et al., 1982~. In addition the use of the
markers in the lining fluids allows meas-
urement of the degree of inflammation.
which is useful not only for determining
the progress of an inflammatory response,
but also for ranking inhaled compounds
for toxicity in animal studies. In£1am-
matory responses in the nose and in the
upper respiratory tract can be detected
by site-specific sampling of the lining
fluids. The biochemical and cellular
content of the ELF can also provide infor-
mation on the type of inflammatory response
and the stage of the disease process.
Several examples of the use of ELF analysis
for the detection of toxicant-induced
respiratory tract inflammation are given
in Chapter 6. The major uses of the markers
has been in animal toxicity studies to rank
a series of compounds for toxicity and to
study the mechanisms of toxicant-induced
lung disease. Studies in this field have
been reviewed (Henderson, 1988a,b).
MARKERS OF PULMONARY TOXICOLOGY
INFLAMMATORY RESPONSE TO
MICROBIAL INFECTIONS
The respiratory tract constitutes the
primary mammalian portal of entry for many
pathogens. For some microbial infec-
tions, the bronchopulmonary mucosa serves
as a benign substrate for initial replica-
tion events that lead to eventual systemic
spread without producing any clinical
disease locally. For other infectious
agents, however, the respiratory tract
is the principal target for the disease-
producing potential. It is estimated that
respiratory viruses are responsible for
5-6% of all deaths and about 60% of deaths
related to respiratory disease (Ogre et
al., 1984~.
Ample evidence supports the conclusion
that normal AMs can ingest and kill many
types of microorganisms that gain en-
trance to the respiratory tract (Jakab,
1984~. Some microorganisms, particularly
the virulent intracellular parasites,
can survive in normal macrophages; it is
apparent that this class of parasite can
be controlled only when the forces of ac-
quired immunity orchestrate the macro-
phage system into antimicrobial action
that is more potent, both qualitatively
and quantitatively.
Various chemicals in the environment
and workplace affect the immune response,
as determined by one or more of the many
tests available to measure various com-
ponents of the immune system (Faith et
al., 1980; Sharma, 1981; Luster et al.,
1988~. Extensive evidence from animal
lung infectivity models points to the det-
rimental effects of air pollution on vari-
ous defense mechanisms of the lung (Neiman
et al., 1977; Fauci et al., 1984). Results
of epidemiologic studies indicate that
living in urban areas increases the in-
cidence of airway infections (Hong, 1976;
Penn, 1978), although direct correlations
of infection with specific pollutants have
not been found. In vivo and in vitro stud-
ies, using mainly animal models, have shown
that ozone impairs AM phagocytic function
(U.S. Environmental Protection Agency,
1984~. The success of AMs in digesting
organic particles and organisms depends
on lysosomal hydrolytic enzymes, includ
OCR for page 93
INFLAMAL4TORYAND IMMUNE RESPONSE
.
sing acid phosphatase, cathepsins, lyso-
zyme, beta- glucuronidase, beta- galac -
tosidase, arylsulfatase, and beta-gluco-
saminidase. Exposure to ozone in viva and
in vitro depressed the intracellular
activity of lysozyme, beta-glucuronidase,
and acid phosphatase from rabbit AMs (Hurst
etal., 1970~.
Kimura and Goldstein ~ 1981 ~ demonstrat-
ed a decrease in the bactericidal enzyme
lysozyme in AMs from rabbits exposed to
ozone at 0.25 ppm. That finding paralleled
the increased susceptibility of those
animals to various infectious agents
(Coffin et al., 1968, Ehrlich et al., 1977;
Miller et al., 1978~. Other products of
the AMs that are important in phagocytosis
and have antibacterial activity are su-
peroxide anions (O2-), hydroxyl radical
(OH.), and H2O2. In viva exposure of rats
to ozone at 0.9-3.2 ppm resulted in a dose-
dependent decrease in O2- production by
AMs from the exposed animals (Amoruso et
al., 1981; Witz et al., 1983~. Thus, the
studies in animal models have shown that
ozone depresses many components of AM
function that are important in phagocy-
tosis and in protecting the lung from
microorganisms or particles present in
the environment and that can be measured
and therefore can serve as markers of
exposure.
In vitro studies can also be used to im-
prove understanding of in viva phenomena.
Phagocytosis is a well-organized process,
made up of several integrated steps, many
of which are adversely affected by ozone.
McAllen et al. (1981) have noted that AMs
obtained from rats after in viva exposure
to ozone at 1 ppm exhibit decreased mobili-
ty. AMs in ravage fluid from the lungs
of rodents that had been briefly exposed
to ozone at 0.5-1.0 ppm display a lower
ability to engulf bacteria than AMs from
control animals. However, if the animals
are exposed to ozone at 0.8 ppm for a longer
period before lung ravage, the ability
to incorporate carbon-coated latex micro-
spheres of isolated AMs is increased.
The data suggest that phagocytic activity
is impaired soon after exposure to ozone,
but that AM function recovers if the insult
persists. Whether those changes are due
to the influx of unexposed AMs into the
93
lung or to adaptation by resident AMs is
not clear.
Several investigators have recently
started to examine the effect of inhaled
pollutants on human subjects' ability to
resist infections. Frampton et al.
(1987) exposed normal human subjects to
NO2 in controlled chamber conditions.
AMs were obtained from the subjects by
BAL 3.5 hours after exposure and exposed
in vitro to influenza virus. It was ob-
served that the AMs obtained from subjects
exposed to NO2 at 0.6 ppm continuously
(3.5 hours) were able to inactivate the
virus significantly less than those ob-
tained from subjects exposed to clean air.
No major changes in the cell numbers in
the BAL fluid from the exposed subjects
were observed, but several biochemical
changes indicated that NO2 did induce in-
flammation in the exposed subjects. In
another study, Kulle et al. (1987), exposed
normal human subjects to NO2 at different
concentrations, and then a live attenuated
cold-adapted influenza A virus was ad-
ministered intranasally to all subjects.
Infection was determined by virus recov-
ery and a 4-fold or greater increase in
antibody titer. The results suggest that
subjects that were exposed to NO2 at 1 or
2 ppm for 2 hours/day for 3 days and inhaled
the virus on day 2 had small, reproducible
signs of infectivity. The approaches taken
by the different investigators were dif-
ferent-Frampton et al. exposed subjects
to the pollutant in viva and studied infec-
tivity in vitro, and Kulle et al. exposed
subjects to both the pollutant and the
infectious agent-but the results collec-
tively suggest a decrease in host defense
against the viral infection. In both
studies, spirometrically measured pul-
monary functions were unchanged by ex-
posure to the pollutants.
IMMUNE RESPONSE
The human respiratory tract contains
a complex array of host defenses-anatom-
ic barriers, mucociliary clearance, phag-
ocytic cells, and various components of
cellular and humoral immunity-that col-
lectively cleanse inhaled air and inac-
tivate infectious and other injurious
OCR for page 94
94
agents that are inhaled (Reynolds, 1979;
Reynolds and Merrill, 1981~. In particu-
lar, the mucosal lining of the small air-
ways and alveolar airspaces contains many
components of the immune system that are
important in providing protection of the
normal lung. However, some of the compo-
nents also play an important role in im-
munologic lung disease. This section re-
views the general features of the immune
system and how it operates in the lung.
Antigen-antibody complexes are the
basis of immune response. The afferent
phase of the immune response usually be-
gins with antigen processing by phago-
cytes, such as macrophages. That includes
degradation of foreign substances and
exposure of lymphocytes to antigens,
which stimulates the production of anti-
body, sensitized cells, or both (Figure 5-
1~. Interactions of macrophages stimu-
lated by antigens with cells in lymphoid
tissue result predominantly in a cellular
or humoral immune response. Cellular im-
mune responses (delayed hypersensitivity)
are mediated by thymus-derived lympho-
cytes-T cells. Antigen interaction with
T cells usually leads to their prolifera-
tion. It is now recognized that T-cell
proliferation and the generation of effec-
tor cells occur separately. Antigens
interact with macrophages, and inter-
leukin- 1 (I1- 1 ) stimulates resting T
cells. The T cells then can respond to a
growth factor called interleukin-2 (I1-
2~. Among the progeny of antigen-stim-
ulated T cells are memory cells, which
M'4R=RS OF PULMONARY TOXICOLOGY
respond quickly to later challenge with
the original antigen; killer cells (or
natural killer cells, NK cells), which
destroy alien cells; and effecter T cells,
which produce molecules called lympho-
kines. 11-2 signals T cells to produce
more T cells and effecter T cells. Lympho-
kines can play an important role in the
generation of an inflammatory response,
particularly one involving cell-mediated
immunity. For example, initiation and
development of granulomas are thought
to arise from the secretion of lymphokines
that influence macrophage motility, ac-
tivation, and function.
Humoral responses are the end result
of antigen interaction with marrow-de-
rived or bursar-cell-equivalent lympho-
cytes (B cells). B-cell function is regu-
lated by at least two subpopulations of
T cells: helper T cells (Th cells) are re-
quired for optimal production of anti-
body to most antigens, and suppressor T
cells (Ts cells) are required for inhibi-
tion or modulation of the humoral re-
sponse once it is initiated.
T-cell subsets in humans have been shown
to express distinct differentiation anti-
gens, which can be identified with mono-
clonal antibodies to T cells.
At each step in the sequence of immune
stimulation, immunocompetent cells are
activated and liberate soluble mediators.
For example, when activated by immune
stimulation, macrophages can produce
various potentially injurious agents,
including arachidonic acid metabolites,
~> ~ Ceil
Antigen 111/' \\ \~J immunity
~ ~ J ( Helper ~(Suppresso)
~\ ~ ~
Macrophage \ ~\
\~ ( ~Ant~body
Plasma
cell
FIGURE ~1 Cellular interactions involved in generation of immune response. Antigen presentation leads to stimulation
of T-cell or B-cell systems. Factors involved in T-cell system include interleukin-1, which stimulates T cells to acquire
receptor for T-cell growth factor called interleukin-2 (I1-2~; same subpopulation of T cells can also secrete 11-2.
OCR for page 95
INFLAMMATORY AND IMMUNE RESPONSE
free oxygen radicals, and growth factors
capable of initiating abnormal growth and
metabolism of fibroblasts. Similarly,
activated lymphocytes can produce soluble
mediators that can act on immunocompetent
cells and other cells, including inter-
feron-gamma capable of activating macro-
phages and chemotactic factors capable
of activating other phagocytic cells,
particularly neutrophils. Thus, although
immune stimulation is initiated by a speci-
fic antigen, secondary inflammatory medi-
ators can appear as the cascade of cells
and inflammatory signals progresses. Many
of the cells, particularly macrophages,
can be stimulated by nonspecific agents,
and the appearance of soluble mediators
does not necessarily indicate that an im-
mune response has been initiated. The
potential use of some of the inflammatory
mediators as specific biologic markers
of injury must be viewed in light of the
fact that they can be initiated by both
specific and nonspecific stimuli.
The pulmonary immune cells are hetero-
geneous, and external stimuli, such as
pollutants, can modulate their behavior
both qualitatively and quantitatively.
The changes can be used to assess effects
of exposure. Characteristics that can
be monitored, such as cell numbers and
cell-surface markers, can be considered
biologic markers. Some of the changes can
be transient (reversible); others can
be chronic or irreversible. Examples of
modulation in the immune cells in the lung
include changes in the proportions of sub-
populations of immune cells (e.g., the
TH/TS ratio), in the extent of expression
(density) of cell-surface markers, in
the cytolytic capacity of T cells or nat-
ural killer cells, and in the ability of
phagocytes to ingest particles.
Various components of lung fluid that
are associated with the inflammatory re-
sponse can serve as markers. The fiberop-
tic bronchoscope has made access to the
trachea and major airways routine, and
this versatile instrument has contributed
enormously to the care and diagnosis of
patients with lung diseases (Sackner et
al., 1972~. Since the late 1960s, BAL has
been incorporated into fiberoptic bron-
choscopy and has proved to be safe and
95
reliable for sampling airway and alveolar
fluid and cellular components in normal
lungs (Reynolds and Newball, 1974) and
diseased lungs (Reynolds et al., 1977;
Crystal et al., 1981~. BAL has made pos-
sible extensive study of the pathogenic
roles played by immune and inflammatory
reactions in the respiratory tract and
important advances in understanding the
pathogenesis of various forms of obstruc-
tive, inflammatory, and interstitial lung
disease. Much of the information reviewed
in this chapter has been obtained with
BAL (Reynolds, 1987~.
In ravage of the lungs of a normal non-
smoker, approximately 10-15 million res-
piratory cells typically are recovered.
The number of cells obtained in BAL can
vary widely. For instance, the number
of cells is increased by a factor of ap-
proximately 4-5 in ravage fluid from a
cigarette-smoking patient and is cor-
related roughly with the intensity of smok-
ing. Approximately 90% of the cells in
ravage fluid are alveolar macrophages
(AMs). Most of the remaining cells are
lymphocytes, and usually only a few poly-
morphonuclear leukocytes (PMNs) are
found. Eosinophils and basophils are rare-
ly detected. In smokers, the cell yield
far exceeds that of nonsmokers. Therefore,
although the percentage of lymphocytes
is diminished, the absolute number is ac-
tually increased. Analysis of the differ-
ential counts and more recently the use
of monoclonal antibodies and flow cytomet-
ry of the cells in BAL specimens have been
found to provide useful markers of a vari-
ety of pulmonary disorders, particularly
the granulomatous and nongranulomatous
interstitial lung diseases, and of effects
of exposure to pollutants.
AMs are the principal phagocytic cell
in the airways and seem to play a pivotal
role in initiating and modulating the pul-
monary immune response (Merrill et al.,
1982~. Phagocytosis of microorganisms
and other foreign particles in the alveoli
by macrophages is an important defense
mechanism of the lung against Ins Eaton
and other forms of external assault by
inhalation. AMs interact with other cells
and foreign material in the lung via mem-
brane receptors. Surface receptors for
OCR for page 96
96
the Fc portion of IgG and for complement
fragments Cab and Cad have been identified
(Reynolds et al., 1975~. The activity
of the phagocytic system generally de-
pends on the specific immunoglobulins
IgA and IgG-IgA probably functions main-
ly as an antitoxin and in the neutraliza-
tion of viral infectivity, whereas IgG
is also important in promoting
phagocytosis.
Although complement concentrations
are low in the airways, complement can
be an important participant in infections
when the inflammatory response promotes
transduction of complement, as well as
of serum immunoglobulins. Once phago-
cytosis is completed, killing of an in-
gested organism is mediated by phagosome-
lysosome fusion, degranulation, and the
elaboration of digestive enzymes and toxic
oxygen species. Present evidence suggests
that AMs use metabolically generated H2O2
in conjunction with some type of oxidase
to kill microorganisms. The hydrolases
in the lysosomes probably play a major role
in digesting a phagocytosed microbial
carcass. It is also likely that mobilized
and activated lymphocytes and macrophages
can produce an exterior milieu that is
adverse for at least some microorganisms.
The various specific immunologic mechan-
isms probably act in concert with nonspeci-
fic factors in mucus, such as lysozyme and
other nonantibody antimicrobial agents;
some events of inflammation, as well as
the mucociliary approaches, also are im-
portant contributions to the overall de-
fense of the lung.
AMs participate further in the regula-
tion of the inflammatory and immune proc-
ess in the lungs by secreting a variety
of soluble mediators, including products
of the arachidonic acid pathway, which
seem to play an important role in inflamma-
tion (Hunninghake et al., 1980a; Slauson,
1982~.
AMs secrete chemotactic factors for
neutrophils that cause influx of these
cells into the lung parenchyma and alveo-
lar space, where they can participate ac-
tively in the inflammatory response.
AMs have a wide range of other secretory
capabilities and have been shown to se-
crete such products as colony-stimulating
factor, superoxide anions, and various
M,4R=RS OF PULMONARY TOXICOLOGY
enzymes, including collagenase, neutral
protease, and elastase. Among the most
potent inflammatory substances produced
by AMs are products of arachidonic acid
(Slauson, 1982~. Some of the better-known
mediators of the inflammatory response
include prostaglandin F2 (PGF2 alpha) and
the chemotactic factor LTB4. Those arachi-
donic mediators and their occurrence are
described below. AMs also play a unique
role in the development of an immune re-
sponse to a novel antigen by presenting
bound or ingested antigen to T lympho-
cytes. About 8-10% of the cells in the
BAL fluid recovered from normal human
lungs are lymphocytes. As in blood, three
subpopulations of lung lymphocytes (T.
B. and NK cells) can be discerned on the
basis of their differing surface mark-
ers. The proportions of T and B lympho-
cytes in ravage fluid from the airway lumi-
na have been found to approximate closely
those in peripheral blood (Hunninghake
and Crystal, 1981a). Whereas T lymphocytes
make up about 60-70% of the lymphocytes
found in normal BAL fluid, only about 10-
15% of the lymphocytes have surface im-
munoglobulin and can be identified as B
cells. A portion of the lymphocyte popula-
tion that cannot be classified by classi-
cal T- and B-cell markers has been iden-
tified as NK cells.
PMNs are not usually found in large num-
bers in BAL fluid from normal lungs
(Reynolds, 1987~. In ravage fluid from
the lungs of smokers (Young and Reynolds,
1984) or subjects that were exposed to
ozone (Seltzer et al., 1986; Koren et al.,
1989), an increased percentage and greatly
increased absolute numbers of neutrophils
are found. As participants in the inflam-
matory response in lung tissue, PMNs mi-
grate into the lung from the blood under
the influence of one or several chemotactic
factors, including complement component
C5a and soluble factors secreted by AMs.
They might be considered as a secondary
line of phagocytic defense of the lungs,
which can be recruited into the airspaces
in response to exposure to microbial agents
or other inhaled materials. Some of the
observed changes have been shown to have
a predictive value; others represent more
progressive biologic changes.
OCR for page 97
INFL4MMATORYAND IMMUNE RESPONSE
Acquired Local Immune Response
In defining the role of the lungs' im-
mune system in the generation of specific
biologic markers of injury or disease,
it is important to consider the central
feature of an immunologic response-that
it is a specific reaction to an antigenic
stimulus and is capable of distinguishing
proteins that differ by only a few amino
acids. A substance that is inhaled and
can act as a unique and foreign antigen
elicits an immune response that is mani-
fested predominantly in the form of anti-
bodies or sensitized cells. (This mani-
festation is the cellular immune re-
sponse.) As discussed below, local immune
responses in the lung operate in concert
with other mechanisms (such as mucocil-
iary clearance) in recognizing, trans-
porting, and eliminating inhaled foreign
agents. Obviously, local immune responses
are very important; their failure can
result in injury and tissue damage in the
lung.
With respect to local immune system as
a generator of markers, two underlying
possibilities or conditions need to be
considered: the presence of antigen-spe-
cific antibodies or cells indicates that
the host has been exposed to an antigen
at some time, even if no longer harboring
it; and specific immune responses might
indicate the presence and persistence of
an antigen that produces a chronic inflam-
matory response that leads to tissue in-
jury. Thus, the products of the immune
system can be used as markers of a host's
exposure to an antigen that might or might
not be responsible for tissue injury and
disease.
In an operational model, the lung's im-
mune system can be viewed as having three
distinct compartments, each containing
immunocompetent cells (lymphocytes and
macrophages): the bronchoalveolar air-
spaces, the submucosal or secretory an-
tibody system lying beneath the lamina
propria of the tracheobronchial tree,
and a network of lymphatic vessels and
lymph nodes lining the tracheobronchial
tree (Daniele, 1980~. In each of these
compartments, the potential exists for
lymphocyte-macrophage interaction and
the generation of immune responses.
97
In the last 5 years, most of our knowledge
about cell-mediated immune responses in
the lung has come from studies involving
cells recovered from bronchoalveolar
airspaces with ravage in humans or in ex-
perimental animals (Daniele et al., 1985~.
Until the advent of the flexible fiber-
optic bronchoscope, little was known ab-
out the cells and secretions in the bron-
choalveolar airspaces of the human lung.
It has since been observed that, in non-
smoking adults, cell yields equal 10- 15
x 106 cells/100 ml of ravage fluid (Dauber
et al., 1979~; AMs are the predominant
cell type (80-90%), lymphocytes con-
stitute about 10% of the cells (Reynolds
et al., 1977; Dauber et al., 1979), and
neutrophils, eosinophils, and basophils
constitute less than 1 % of the cells. In
smokers, the cell yield is some 4 times
as great; macrophages usually account
for 90% or more of recovered cells, and
lymphocytes for 1-5% (Daniele et al.,
1977b; Reynolds et al., 1977; Dauber et
al., 1979~; there can also be a slightly
higher proportion of neutrophils ~ 1 -4%~.
The distribution of lymphocyte subpopu-
lations in the ravage fluid is similar
to that in blood, with T cells accounting
for 60-70% of the lymphocytes and B cells
5-10%. The ratio of Th cells to Ts cells
is 1.6:1 (Daniele et al., 1975; Dauber et
al., 1979; Hunninghake et al., 1979b;
Hunninghake and Crystal, 1981 a).
The major soluble constituents in la-
vage fluid are IgG and IgA; their concen-
trations reflect rates of active trans-
port across the bronchial epithelium
(Reynolds et al., 1977; Low et al., 1978;
Hunninghake et al., 1979b). Little or no
IgM is present. Components of both the
classical and alternative pathways have
been identified in ravage fluid, but CS
appears to be absent (Robins et al., 1982~.
Other inflammatory derivatives have been
detected in ravage fluid, including al-
phal-antitrypsin.
Much needs to be learned about the role
of lymphocytes in the normal human lung,
particularly with respect to their initi-
ation and development of immune responses.
The evidence is more substantial in experi-
mental animals (Daniele, 1980~. Lympho-
cytes recovered with lung ravage from
guinea pigs and rabbits respond to
OCR for page 98
98
antigens introduced into the respiratory
tract by producing antibody and lympho-
kines (such as macrophage-migration in-
hibition factor, MIF). Furthermore, lung
lymphocytes can demonstrate an anamnestic
response to airborne antigens and, de-
pending on the type and dose of antigen,
exhibit a capacity to respond that is inde-
pendent of systemic lymphoid tissue.
Thus, results of animal experiments indi-
cate that localized immune responses can
occur in the bronchoalveolar airspaces.
Alternatively, it has been proposed
that the cells and secretions in the bron-
choalveolar airspaces are deployed so
as to prevent entry of antigenic particles
beyond the mucosal barrier and to deter
antigen interaction with organized lung
lymphoid tissue. According to the notion
of "immune exclusion," the primary func-
tion of AMs is to ingest particles and re-
move them from the lung, rather than to
transport them to submucosal and tracheo-
bronchial lymph nodes, where lymphocytes
and tissue macrophages might interact.
Which of the two hypotheses is correct
remains to be settled.
The two hypotheses might not be mutually
exclusive. The nonspecific activities
of AMs and the mucociliary blanket might
be entirely adequate for expelling some
inhaled inert substances. Nonspecific
clearance mechanisms would not suffice
for other antigens, such as microorgan-
isms with capsular membranes that resist
phagocytosis, and the aid of specific anti-
body and cells in bronchial secretions
would be required for effective phagocyto-
sis, killing, and clearance. The genera-
tion of a specific immune response consist-
ing of either antibody or cells in bronchi-
al secretions requires, however, that the
inciting antigen in some way penetrate
the mucosal barrier and stimulate submuco-
sal lymphoid cells. That condition is also
required for any inhaled particles (e.g.,
allergens and organic particles) that
result in local immoral and cellular immune
responses. It should also be emphasized
that initially only a relatively small
fraction of the inhaled antigenic load
might be required for stimulating submuco-
sal lymphoid tissue. Once initiated, the
secretion of antibody or the appearance
AL4RKERS OF PULMONARY TOXICOLOGY
of sensitized cells in the airspaces would
greatly increase the exclusion of the same
or similar inhaled antigens on later
challenges.
The degree to which nonspecific de-
fenses interact with specific immune re-
sponses in the lung remains ill defined.
It probably depends on the size of the par-
ticle, the antigenic load, and the physico-
chemical characteristics of the particle,
which are related to its antigenicity,
toxicity, and, perhaps most important,
biologic properties (e.g., type of virus
and capsulated bacteria).
Those are some of the variables that
determine whether inhaled particles and
microorganisms are contained or eliminat-
ed or result in lung injury and disease.
Perhaps equally or more important are
the unique genetic properties of the host,
especially the immune responses that are
linked and controlled by the immune-re-
sponse genes. The latter consideration
is particularly relevant for two immuno-
logic diseases, hypersensitivity pneumo-
nitis and chronic berylliosis, that are
discussed below. In both, only a minority
of persons equally exposed to the airborne
agents develop disease.
Examination of cells and secretions
in BAL fluid from patients with immunolo-
gic lung diseases has provided important
insights into pathogenesis.
First, the lung can be the site of a com-
partmentalized inflammatory response
(Daniele, 1980), as in hypersensitivity
pneumonitis, in which the disease is re-
stricted to the lung. In other systemic
disorders, the inflammatory response that
evolves in the lung might not be reflected
in the peripheral blood (Daniele et al.,
1980~. The reason for the difference is
unclear; one hypothesis is that the lung,
when it is involved, acts as a selective
target for acute (neutrophils) or chronic
(lymphocytes and monocytes) inflammatory
cells, which are increased in the pulmon-
ary parenchyma as well as in the ravage
fluid (Crystal et al., 1981~.
Second, pulmonary ravage has establish-
ed the existence of two predominant types
of chronic inflammatory response in the
lung, one involving neutrophils and mac-
rophages (idiopathic pulmonary fibrosis)
OCR for page 99
INFL4MMATORYAND IMMUNE RESPONSE
and the other involving lymphocytes and
macrophages (hypersensitivity pneumoni-
tis and berylliosis).
Finally, several laboratories have
found in studies of pneumonitis a height-
ened state of activation of these inflam-
matory cells (Daniele et al., 1980; Crys-
tal et al., 1981~. Lymphocyte activation
probably reflects immune stimulation in
cases of hypersensitivity pneumonitis
and berylliosis.
In summary, the ability to detect sen-
sitized cells or antibodies that are spe-
cifically reactive to large complex or-
ganic antigens (as in hypersensitivity
pneumonitis) or simple elements that be-
have as haptens (as in berylliosis) can
serve as a useful paradigm for investigat-
ing other inhalational diseases in which
an immunologic response is predominant
in pathogenesis. The presence of specific
responses in the lung indicates that the
subject has been exposed to a foreign anti-
gen; it does not necessarily mean that
the antigen is causing disease. for ex-
ample, in both hypersensitivity pneumoni-
tis and chronic berylliosis, it is still
unclear whether the presence of sensitiz-
ed cells or antibodies in BAL fluid indi-
cates that a patient has or will have dis-
ease related to the foreign substances
found.
Acquired Antigen-Specific Immune
Response
In Vivo Challenge
Testing for immune response has often
included testing of whole animals or hu-
mans. With such testing, the interaction
of several components of the immune system
can be tested at once, and actual body re-
sponse can be measured, so that one need
99
not rely on extrapolation from results
obtained in vitro. However, in viva chal-
lenge has several difficulties: the risk
of a serious adverse reaction, including
anaphylaxis; the difficulty of separating
· an- ~ · ~
a nonspec~t arc ~ rom a specie ic response;
the difficulty of interpreting whether
a response in one area reflects a response
in another area; and the difficulty of
purifying an antigen to be specific enough
for testing and suitable for administra-
tion without causing nonspecific damage.
The immune response has been divided
into four groups summarized in Table 5-
1 (Bellanti, 1985~. Skin testing usually
elicits Type I reactions, although Type
IV reactions can be detected in skin.
Cell-mediated immunity is tested by ex-
amining the skin site 24-48 hours after
-
injection; this can be done to determine
whether a subject has been infected with
tuberculosis-as with the PPD skin test
(Snider, 1 982-or to determine whether
a patient is allergic (not reacting to any
of the common antigens, such as those of
tetanus or mumps). Testing for granuloma
formation can use the Kveim antigen (sar-
coid tissue antigen) (Chase, 1961~. Skin
testing for delayed reactions has not been
routinely used for detecting sensitivity
to pulmonary toxicants.
With further sophistication, a chal-
lenge might be graded not only by the am-
ount of visible inflammation present,
but by other factors, such as the influx
of inflammatory cells and the presence
of inflammatory mediators, including
histamine, immunoglobulins, and immune
complexes.
Skin Testing
A standard method for testing for reac-
tion to a possible pollutant is skin test
TABLE 5-1 Immunologic Mechanisms of Tissue Injury
-
Type Manifestations Mediators
II
III
IV
Immediate hypersensitivity reactions
Antibody-directed reactions
Formation of antigen-antibody complexes Mainly IgG
IgE and other immunoglobulins
IgG and IgM
Delayed hypersensitivity (cell-mediated) Sensitized T lymphocytes
reactions
OCR for page 100
100
ing. Allergists have used skin testing
extensively to identify substances to
which a patient is allergic (Norman,
1980~. Its potential use in environmental
studies in toxicology is based mostly on
its simplicity of application and inter-
pretability. The procedure is relatively
safe, although some subjects are allergic
to antigens and anaphylaxis has been re-
ported after skin testing in a few sub-
jects. Usual precautions in skin testing
include testing with the lowest possible
dose and observing subjects for some time
after testing.
Methods of skin testing include prick
testing, scratch testing, and intradermal
injection, in order of increasing dose.
The skin prick test is the safest, in that
a very small amount of antigen is injected.
Reactions to a prick test are read at
10 minutes; a reaction indicates an im-
mediate type of sensitivity. Patients
with dermatographism will have a false-
positive wheel; otherwise, the test is
readily assessed. Pepys's group has used
the prick test for many years to evaluate
exposure of platinum workers (Cleare et
al., 1976~. Platinum salts can be highly
reactive, and systemic reactions can oc-
cur even to scratch tests, which therefore
were considered too risky for general
surveillance testing. Nevertheless, the
authors also found that the prick test was
better for differentiating between con-
trols and reactive subjects.
A major difficulty with skin testing
has involved the preparation of a suffi-
ciently reactive antigen. Most sub-
stances studied are haptens and become
antigenic on combination with high-molec-
ular-weight carriers. That can happen
at the injection site. For example,
phthalyl acid anhydride is an essential
reagent in the manufacture of epoxy resins
and some paints. It is highly reactive,
and skin testing can be performed direct-
ly. Positive skin tests have been seen
in documented cases of asthma induced by
phthalyl anhydride (Maccia et al., 1976~.
Most substances do not induce responses
by themselves and have to be conjugated
to human serum albumin before testing.
Some have been found to be reliable skin-
test reagents for particular environmen-
tal pollutants (Zeiss et al., 1983~.
MARKERS OF PULMONARY TOXICOLOGY
Another difficulty with skin testing
has involved the need to relate the find-
ings with the pulmonary symptoms of the
subjects. A positive skin prick test in
platinum refiners is a more specific and
sensitive index of disease than are some
clinical symptoms (Dally et al., 1980~.
For example, skin prick tests of mouse
and rat urine extracts in laboratory-ani-
mal workers have yielded a sensitive meas-
ure of asthma, but not of rhinitis or urti-
caria (Newman Taylor et al., 1981~.
Work with skin testing has extended be-
yond the routine measurement of size and
character of skin reaction. As mentioned
above, skin biopsies are routinely used
to examine for the presence of granulomas
after a Kveim test; studies are underway
to characterize the earlier stages of the
inflammation (Mishra et al., 1986~. The
studies have included examination of in-
flammatory cell population and mediators
in the biopsies of skin lesions during
various phases of immediate skin reaction
and have led to a better understanding
of early pathologic response. During the
late-phase reaction, skin biopsies can
show neutrophil and lymphocyte influx
(Felarca and Lowell, 1971~.
A novel method is the injection of anti-
gen into bullae in the skin. This particu-
lar challenge allows one to measure the
influx of mediators, including histamine,
into the site of a skin reaction (Warner
etal., 1986~.
In summary, skin testing has several
advantages, including low cost, wide ap-
plicability, ready acceptance by pa-
tients, and relative safety. Its major
drawbacks include difficulty in assessing
observations regarding skin reactions
and in correlating reactions with symp-
toms in other organs and identifying prop-
er antigens for testing.
Nasal Challenge
The upper airways, especially the nose,
are a major target of toxic damage. Rhini-
tis is a common complaint after exposure
to toxicants; but research into rhinitis
has been limited, because it is not asso-
ciated with substantial morbidity and
its relationship with lower respiratory
symptoms is not clear. Nasal challenges
OCR for page 101
INFLAMMATORYAND IMMUNE RESPONSE
do provide information that might not be
obtainable with any other method and thus
should always be considered when examin-
ing new ways of studying toxicants are
being examined. Nasal challenges date
at least to 1873, when Blakley placed grass
pollen in the noses of allergic patients
and induced the signs and symptoms of al-
lergic rhinitis (Naclerio et al., 1983~.
The method of intranasal challenge var-
ies. Again, identification of the correct
antigen is difficult. One method is to
study patients with known intradermal
reactions. The specific known antigens
are then delivered by nebulizer (Naclerio
et al., 1983) or by direct application
of an extract (Naclerio et al., 1985~.
Methods of assessing inflammatory reac-
tions after nasal challenge also vary.
They include measurement of airway resis-
tance in the challenged nostril (McLean
et al., 1976), measurement of mucus pro-
duction (Maim et al., 1981), subjective
assessment (Cornell, 1979; Naclerio et
al., 1985), and objective assessment of
hyperemia and stenosis (Naclerio et al.,
1983~.
Nasal challenge is fairly safe. The
usual symptom is rhinitis, and an oc-
casional patient develops wheezing. Un-
like skin tests, it has not been used in
large populations. But there is little
to suggest that it could not be performed
in a similar manner, with the patient ob-
served for some period after challenge.
The cost would depend on the extent of as-
sessment. For example, if measurement
of mediators in nasal washes were the goal,
the assays could become expensive and thus
impractical for screening large popula-
tions. Observation for the presence of
edema would be simple, although difficult
to measure. Nasal airway resistance can
be measured by anterior rhinomanometry,
which is relatively simple and inexpen-
sive (McLean et al., 1976; Naclerio et
al., 1983~.
In summary, nasal challenge has dis-
tinct advantages over skin testing, be-
cause it uses a mucosal surface. Direct
observation can be used to assess inflam-
matory response, so it might be appropri-
ate for screening large populations. In
addition, when more objective data are
101
required, nasal airway conductance is
easily measured.
The best method for assessing airway
response to an antigen would be direct
observation. The antigen is chosen on
the basis of intradermal response. The
antigen dose, described in protein nitro-
gen units (1 unit is the amount that causes
a 4 x 4-mm wheel after intradermal
injection), is determined. Intrabron-
chial challenge is then begun at one-hun-
dredth of that unit. Intrabronchial chal-
lenge is usually complemented by BAL in
the contralateral lung and in the edema-
tous bronchus after challenge.
Bronchoscopy is performed in the usual
manner. Subjects are premedicated with
atropine, metaproterenol (a beta
agonist), and topical Xylocaine. The
bronchoscope is advanced to a subsegmen-
tal bronchus, the initial dose of antigen
is injected through the bronchoscope,
and the bronchus is observed for 3 minutes.
If there is no change in the bronchus, the
dose is increased. A recordable response
consists of blanching, edema, or narrow-
ing of the airways.
The major advantage of Intrabronchial
challenge is its specificity for identi-
fying an inflammatory response in the
bronchus. Visualization lasts for only
3-5 minutes, so it would detect only an
immediate response. However, repeat
bronchoscopy has been done 2-3 days after
bronchial challenge to assess persistent
changes, and persistent abnormalities
in the cell population have also been ob-
served in BAL fluid (Metzger et al., 1987~.
Patients challenged to date have been
challenged only with antigen to which they
have a good skin response. Patients were
usually far more sensitive to intrabron-
chial than to intradermal exposure. Of
11 patients studied by Metzger et al.
(1987), nine responded to less than one-
twentieth of the intradermal dose.
Intrabronchial challenge presents many
problems, mostly because it is relatively
new. Although the bronchial changes are
visually dramatic, there is little objec-
tive measure of response. Because of prob-
lems with parallax from a flexible fiber
OCR for page 102
102
optic bronchoscope, it is difficult to
determine size in the bronchus without
a reference object at the same plane as
the area one wishes to measure. That is
commonly provided by touching the area
with an open biopsy forceps or attempting
to pass a bronchoscope or a bronchoscopy
brush through a narrowed bronchus (Zavala,
1978~. Obviously, touching the walls of
the bronchus that one wishes to evaluate
for edema can lead to local trauma and in-
correct interpretation of edema. In addi-
tion, accurate estimation of airway nar-
rowing might not be possible when a bron-
chus has responded to an antigen.
Intrabronchial challenge poses a sub-
stantial risk in some persons. The risk
associated with bronchoscopy in asthmatic
subjects is dealt with in the section on
BAL; the risk associated with intrabron-
chial challenge conceivably is even high-
er. In the studies reported so far, pa-
tients have been carefully selected, many
precautions have been observed, and es-
tablished guidelines have been followed
(NHLBI, 1985~. Patients have been observ-
ed closely for evidence of bronchospasm.
In one study (Metzger et al., 1987), three
of 11 asthmatic subjects developed wheez-
ing; two were treated with local epine-
phrine, and the other with aerosol ther-
apy. Pulmonary function of all asthmatics
returned to normal within 15 minutes of
the procedure.
A final problem in intrabronchial chal-
lenge is cost. With the current system,
including close observation, studies are
expensive and require highly trained med-
ical and technical assistants.
In conclusion, the utility of intra-
bronchial challenge as a screening tool
for identifying patients sensitive to
pulmonary toxicants seems limited. In
studies to date, only patients who were
highly responsive to skin tests responded
to intrabronchial challenge. In most of
the reported studies, patients were chal-
lenged with an antigen clearly associated
with pulmonary symptoms. Although the
research data obtained after intrabron-
chial challenge are considerable, their
application to a large group of subjects
remains questionable.
AL9RKERS OF PULMONrARY TOXICOLOGY
In Vitro Challenge
Proliferation of lymphocytes exposed
to antigen in vitro is an indication of
sensitization. In general, lymphocyte
proliferation requires the participation
of accessory cells and products of Type
I or Type II histocompatibility antigens
expressed on accessory cell surfaces.
Accessory cells are usually macrophages,
but dendritic cells, B cells, and perhaps
other cells (such as fibroblasts) can act
as accessory cells. The exact relation-
ships between lymphocytes and accessory
cells in the lung remain to be defined.
The pulmonarylymphocytes obtained with
BAL are functionally competent-they
can proliferate and produce lymphokines
when exposed to antigens to which they
are sensitized (Schuyler et al., 1978;
Moore et al., 1980; Pinkston et al., 1983~.
The exact population of lymphocytes re-
sulting from proliferation and the level
of lymphokine secretion are not known.
Proliferation of antigen-induced and
mitogen-induced BAL lymphocytes is lower
than proliferation of peripheral blood
lymphocytes.
Increases in the percentage and number
of BAL lymphocytes are characteristic
of granulomatous lung diseases, such as
hypersensitivity pneumonitis, sarcoidos-
is, berylliosis, and tuberculosis
(Reynolds et al., 1977; Rossman et al.,
1978; Godard et al., 1981, Epstein et al.,
1982~.
Recent reports indicate that pulmonary
lymphocytes from patients with sarcoido-
sis spontaneously secrete interleukin-
2 (Pinkston et al., 1983), which provides
a signal for responsive lymphocytes to
proliferate. There is evidence that I1-
2 secretion by pulmonary lymphocytes from
patients with sarcoidosis is secondary
to an altered milieu in the lung, rather
than being a reflection of changes of the
constitutive properties of T lymphocytes
(Muller-Quernheim et al., 1986~.
Pulmonary lymphocytes from patients
with hypersensitivity pneumonitis are
sensitized: they proliferate and produce
lymphokines on exposure to the appropri-
ate antigen (Schuyler et al., 1978; Moore
et al., 1980~. There is evidence that
OCR for page 103
INFL4MMATORYAND LINE RESPONSE
cells that suppress lymphocyte prolifera-
tion are present in asymptomatic exposed
persons, but not in symptomatic exposed
persons (Amrein et al., 1970~. Therefore,
lack of suppressor cells in some subjects
could be associated with development of
symptoms of hypersensitivity pneumonitis
after the same amount of systemic exposure
that does not cause clinical symptoms in
subjects with suppressor cells.
In general, lymphocytes from patients
with berylliosis, but not from control
populations, proliferate when exposed
to beryllium salts. The results with sub-
jects exposed to beryllium but without
apparent disease are controversial (Hani-
fin et al., 1970; Deodhar et al., 1973;
Epstein et al., 1982; Williams and Wil-
liams, 1982, 1983; Rom et al., 1983;
Bargon et al., 1986~. The relationship
of lymphocyte proliferation and beryl-
liosis is complex. Beryllium salts have
multiple effects on lymphocytes in cul
103
sure: at high concentrations, beryllium
is toxic to lymphocytes and decreases pro-
liferation; at low concentrations, it
increases mitogen- and antigen-induced
proliferation (Williams and Williams,
1982~. Lymphocyte proliferation in peri-
pheral blood has been found to correlate
with beryllium exposure in a beryllium
plant (Rom et al., 1983) and thus might be
a good marker of a population's exposure
to beryllium. Although proliferation of
lymphocytes from peripheral blood has
been studied most extensively, there is
preliminary evidence that bronchoalveo-
lar lymphocytes from a patient with beryl-
liosis also proliferate when exposed to
beryllium (Epstein et al., 1982~.
In summary, lymphocyte proliferation
seems to be an index of exposure to envir-
onmental agents and in some instances a
marker of disease. The relationship of
lymphocyte proliferation and pathogen-
esis in humans is unknown.
OCR for page 104
Representative terms from entire chapter:
inflammatory response
