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OCR for page 415
Biochemical and Cellular
Interrelationships in the
Development of Ozone
Tnclucec! Pulmonary Fibrosis
JEROLD A. LAST
University of California, Davis
Difficulties in Relating Ozone Exposure to Lung Disease / 416
Response to Fibrogenic Insult / 417
Acute Lung Injury and Edema / 417 Transition from Pulmonary
Edema to Cellular Inflammation / 418 The Middle
Phase / 420 Lung Fibrosis / 425
Models of Exposure / 427
Experimental Approaches / 427 Chronic Exposure / 429
Progression of Lung Injury After Cessation
of Exposure / 430 Synergistic Interactions / 430
Determination of Human Risk / 431
Identification of Susceptible Populations / 432
Summary 1 433
Summary of Research Recommendations / 434
Air Pollution, the Automobile' and Public Health. @) 1988 by the Health Effects
Institute. National Academy Press, Washington, D.C.
415
OCR for page 416
416
Biochemical and Cellular Interrelationships
Difficulties in Relating Ozone
Exposure to Lung Disease
The focus of this chapter is on the evidence
that exposure to ozone (O3) can cause pul-
monary fibrosis, generally considered to be
a chronic lung disease. A description of the
underlying mechanisms of lung injury that
might be predictive of other adverse health
effects provides a background to the discus-
sion of O3-induced pulmonary fibrosis.
True chronic experiments have rarely
been performed with O3. Examinations of
the development of lung fibrosis, a disease
widely perceived as chronic, have been
made primarily on the basis of data from
experiments performed in an acute time
frame, usually days or weeks. Long-term
data are generally products of earlier exper-
iments not performed under present-day
standards of care and precision. Animal
hygiene in those experiments was not al-
ways adequate to prevent outbreaks of
pneumonia and other confounding disease.
It may be that the human disease analo-
gous to that caused by O3 is not chronic
lung fibrosis but acute forms of pulmonary
fibrosis such as the adult respiratory dis-
tress syndrome. The many similarities be-
tween the acute disease in humans and the
responses of animals to O3 are also dis-
cussed in this chapter.
The misconception that O3 can cause
emphysema deserves special note. Early
work of Stokinger and coworkers (1957)
suggested this to be the case. However, in
those studies O3 was routinely generated
from air rather than oxygen, and there was
concurrent exposure to nitrogen dioxide
(arising from oxidation of atmospheric ni-
trogen), a known emphysema-provoking
agent. Intercurrent animal illness might
also have influenced those results.
Perhaps the frequency of occurrence of
chronic obstructive pulmonary disease
(COPD) vis-a-vis fibrosis tempted investi-
gators to find a solution to this much more
prevalent disease in humans. Although data
are lacking that suggest that exposure to
O3, by itself, can cause emphysema, with-
out large-scale, careful, lifetime exposures
of animals to O3, the possibility that expo-
sure to O3 may contribute to the develop
ment of COPD in humans cannot be to-
tally ruled out.
Another difficulty lies in trying to distin-
guish between an "effect" and an "adverse
health effect." Acute exposure to O3, espe-
cially at higher-than-ambient concentra-
tions, causes changes in a wide variety of
measurable biological parameters. Some of
these changes are probably relevant to the
mechanisms of action of O3. However,
some are not they are more likely to be
epiphenomena (for example, changes in
barbiturate-induced sleeping time) or arti-
facts (for example, sleeplessness and lack of
appetite in rats for 1-2 days after the start of
exposures to levels of O3 that presumably
are irritating enough to cause "sore
throats". Interpretation of whether mea-
sured changes, even statistically significant
changes, are "adverse health effects" may
be a particularly difficult problem when
human pulmonary function tests are per-
formed on volunteers and one or two inter-
dependent parameters out of 20 are shown
to change slightly.
Such "experiments of nature" as epide-
miologic studies of the risk of breathing
polluted atmospheres in, for example, the
Los Angeles air basin, have essentially
nothing to tell us thus far. This is true for
several reasons. Most important, from the
perspective of this chapter, the clinical rec-
ognition of pulmonary fibrosis is difficult,
except for the most severe manifestations
of this disease. Routine spirometry (simple
pulmonary function testing), such as may
be performed on large populations, is a
very insensitive technique for scoring lung
fibrosis. There have been very few studies
(and no large-scale ones) on autopsies of
people routinely exposed to O3 and dying
of random causes. Costs of such a large-
scale study might be prohibitive.
Confounding effects of cigarette smok-
ing further complicate large-scale epidemi-
ologic studies, as do definitions of appro-
priate control groups. The relationship
between outcome in an individual and ac-
tual dose of O3 to that individual is never
known; personal monitoring of individuals
in a large-scale study probably has not been
performed for O3. Finally, epidemiologic
studies are most powerful when the disease
OCR for page 417
Jerold A. Last
417
at risk is a rare one, and fibrosis is not rare.
All of these limitations also pertain to de-
termination of whether O3 exposure in-
creases the risk of cancer.
Finally, the characteristics of particularly
susceptible populations at risk for pulmo-
nary fibrosis are unknown. Intuitively-
and limited animal data support this intu-
ition it is hypothesized that neonates and
youngsters whose lungs are still developing
might be one such sensitive population.
Populations receiving higher-than-ordi
nary doses of 03, such as joggers and other ~ '
outdoor exercisers and athletes, and obli
gate mouth breathers, might constitute an
other. Definition of such potentially sus
ceptible populations has profound impact
on the proper design and interpretation of
animal exposure studies, but this is an area
where little has been done experimentally.
Some may argue that mathematical
modeling of calculated dose delivered to
tissue sites can replace such actual experi-
mentation, but this is not yet possible. Not
enough is known about lung structure,
target cells and molecules, thickness and
reactivity of protective layers at different
sites in the lung, or the role of secondary
reactions in amplification of injury, to in-
terpret such modeling exercises, either bi-
ologically or for regulatory purposes. The
requisite data to refine such models to the
point where they may replace animal ex-
periments will most probably not be avail-
able in the near future. In the meantime,
measurement of equivalence of biological
responses may be the best indicator for
interpretation of exposure/dose relation
of fibroblasts to these sites (Reiser and Last
1979~. These fibroblasts would then syn-
thesize the collagen that constitutes the fi-
brotic "scar." Macrophages may release
substances that stimulate fibroblasts to pro-
duce collagen, or to produce more collagen
than their baseline synthesis levels (Reiser
and Last 1979~.
Other cells, including lymphocytes,
granulocytes, and eosinophils, also seem
able to release substances chemotactic for
one another (Sober and Gallin 1979~. Some
or these same inflammatory cell types also
seem to be able to release factors that stim-
ulate fibroblast mitogenesis (Nathan et al.
1980), which in turn increases the capacity
for collagen synthesis at such sites. The
interplay of chemotactic, mitogenic, and
collagen synthesis-stimulating factors in
the etiology of organ fibrosis and the reg-
ulation of cell infiltration and turnover in
the damaged lung is poorly understood but
presently under very active investigation.
It will be discussed more thoroughly be-
low.
Acute Lung Injury and Edema
The earliest quantifiable response of the
lung to many types of injury, including
exposure to 03, is pulmonary edema. At
high levels of injury, this response is easy to
quantify; the animal is sacrificed and the
lung weighed. An increase in lung wet
weight over weight of lungs from matched
control animals exposed to filtered air can
be equated with the increase of fluid that
r-~ r- ~constitutes edema. To control for differ
ships in humans vis-a-vis animal experi
ments.
Response to Fibrogenic Insult
When the lung is exposed to O3 or other
fibrogenic agents administered systemically
or by inhalation, the critical response seems
to be an inflammatory influx of macro-
phages to sites of injury, with accompany-
ing edema. This influx may be responsible
for the release of chemotactic factors that,
in turn, are responsible for the recruitment
ences in animal size, data are expressed as
the ratio of lung weight to body weight.
However, the best way to quantify edema
in most animal models that is, the ratio of
wet weight to dry weight of the lung
is not appropriate with O3 (Cross et al.
1981~.
The edema fluid accumulating in lungs of
rats exposed to less than 1 part per million
(ppm) of O3 has a wet weight to dry
weight ratio similar to lung and to blood
serum; therefore, pulmonary edema in-
duced by O3 has little or no effect on the
lung wet weight: dry weight ratio, even in
lungs whose wet weight has doubled. Mea
OCR for page 418
418
Biochemical and Cellular Interrelationships
surement of wet weight (either directly or
normalized to body weight) alone, how-
ever, can be used to quantify O3-induced
edema. Such measurements show concen-
tration/response behavior between about
0.5 and 1.0 ppm O3, but these measure-
ments are insensitive compared to quanti-
fication by other methods.
Two sensitive methods deserve special
mention: accumulation of serum proteins
in lung ravage fluid (Hu et al. 1982; Guth et
al. 1986) and tracer transport from blood to
ravage fluid (and vice versa) (Alpert et al.
1971; Guth et al. 1986~. Both techniques
have been used to examine effects of expo-
sure of laboratory animals to O3 at low
concentrations. Under the assay conditions
used in those studies, the increased protein
content of ravage fluid from lungs of rats
exposed to O3 was due almost completely
to movement of serum albumin from
blood to broncho-alveolar and pulmonary
interstitial fluid, as determined by electro-
phoretic analysis of proteins in the ravage
fluid. More recent studies (Warren et al.
1986; Warren and Last 1987) document a
positive response by this assay at 0.12 ppm
O3, the current peak hourly National Am-
bient Air Quality Standard.
Increased permeability of the lung to
serum proteins can be equated with pulmo-
nary edema, a recognized adverse health
effect. Increased permeability has been ob-
served in animal experiments at O3 concen-
trations routinely encountered, or even
routinely exceeded, as peak hourly values
in southern California smog episodes. At a
given concentration of O3, rats as obligate
nose breathers probably receive a lower
total dose to the peripheral lung than do
humans. Further work is necessary to as-
certain the reversibility of these changes
suggestive of pulmonary edema and the
long-term significance of a succession of
short-term episodes of pulmonary edema.
Current experiments in humans, using la-
beled aerosols of diethylenetriaminepenta-
acetic acid (DTPA) to probe for permeabil-
ity changes of the lung after exposure to O3
in humans (see, for example, Gellert et al.
1985) and rats (for example, Minty and
Royston 1985) should be correlated with
similar animal work.
Recommendation 1. Using experimen-
tal animals, anatomic regions in the lung
associated with tracer transport from the
blood to ravage fluid and vice versa after O3
exposure should be localized and correlated
with the bulk movement of albumin into
the airspaces.
~ Recommendation 2. To facilitate inter-
pretation of human studies, sensitive lav-
age-based assays of protein content need to
be correlated with DTPA aerosol transport
. .
assays in anlma s.
~ Recommendation 3. Human studies us-
ing DTPA aerosols or other tracers in
conjunction with concentration/response
assays for edema should be undertaken.
There are two important barriers to the
accumulation of fluid in the lung in pulmo-
nary edema. The first, the endothelial cell
permeability barrier, is thought to be in-
trinsically leaky. Fluid is constantly cross-
ing this barrier and constantly being
pumped from the interstitial region back to
the vasculature to maintain homeostasis.
O3 does not appear to damage endothelial
cells directly since it is too chemically reac-
tive to traverse the distance from the air-
ways to the vasculature and thereby to
reach these cells. However, products of O3
reaction with epithelial cells or mucous
constituents might well have long enough
lifetimes to affect cells deeper in the lung or
elsewhere in the body.
The other barrier the epithelial cell
layer with its tight junctions between
cells is thought to prevent fluid move-
ment between the vasculature (and intersti-
tial space) and the airways. It is thus easy to
visualize the relationship between damage
to epithelial cells in the small airways and
centriacinar regions of the lung and pulmo-
nary edema.
Transition from Pulmonary Edema to
Cellular Inflammation
The first lung cells to encounter inhaled or
intratracheally instilled fibrogenic agents
are the epithelial cells lining the respiratory
tract. Oxidant gases such as nitrogen diox
OCR for page 419
.lerold A. Last
419
ide (NO2) and O3 have long been known to
damage epithelial cells directly; much has
been published `,n the mechanisms bv
which such oxidant gases may exert their
cytotoxic effects (see, for example, Mustafa
and Tierney 1978~. Systemically adminis-
tered fibrogenic agents such as paraquat,
radiation, and possibly bleomycin may also
exert a direct cytotoxic effect on lung pa-
renchymal cells through generation of free
radicals in vitro, and thus these agents may
share common pathways of injury and fi-
brogenesis with the oxidant gases.
There appears to be a wide range of re-
sponse of the type I and II pneumonocytes
to such agents. In general, type I pneumo-
nocytes appear to be more sensitive to
damage than type II pneumonocytes, al-
though the much greater surface area of
type I cells (about 95 percent of normal
alveolar epithelium) may merely present a
correspondingly larger target for injury.
Exposure of an animal to a pneumotoxic
agent often results in a characteristic pattern
of injury and repair in which type II cells
rapidly begin proliferating to repair the
epithelial lesions resulting from injury and
death of type I cells. Despite their greater
cytoplasmic differentiation, type II cells
apparently serve as progenitor cells of type
I pneumonocytes (Haschek and ~Vitschi
1979).
Cellular damage may also result in the
local release of soluble mediators thought
to be chemotactic for phagocytotic cells.
Such mediators could include products
from fibrin, fibronectin, albumin, prosta-
glandins, leukotrienes, and a vast array of
poorly characterized chemoattractants and
other factors.
Present concepts of the critical sequence
of events in lung injury suggest that the
response of the lung to cellular damage
and/or to pulmonary edema is the move-
ment of alveolar macrophages and (per-
haps) the recruitment of leukocytes to sites
of damage within or near the lung epithelial
surface. This process may also contribute
to increased airway reactivity. For most
models of lung damage, the neutrophil is a
ubiquitous participant in this inflammatory
response.
O3 iS unusual as a pneumotoxicant; the
inflammatory response of the lung to O3
seems to be predominantly macrophagic,
and there is as yet no evidence for a role of
the neutrophil in this process, especially in
the critical early stages. It is tempting to
speculate that the lack of a neutrophilic
response to O3 may have mechanistic sig-
nificance, especially with regard to lung
fibrosis and, perhaps, to the failure to ob-
serve an emphysematous response to O3.
This would be consistent with the pre-
sumed role of neutrophil elastase in the
pathogenesis of emphysema, as discussed
by Wright (this volume).
Accumulation of alveolar macrophages
at sites of lung damage can be beneficial or
detrimental. If the response is limited,
dogma suggests that the lung repairs itself.
However, the long-term consequences to
the lung of multiple episodes of this type
and whether the lung maintains its putative
ability for self-repair in the face of repeated
limited insults are unknown. If the macro-
phagic response is more exuberant, then
the alveolar macrophages elaborate factors
that stimulate the proliferation of lung fi-
broblasts. Such fibroblast proliferation
gives rise to increased numbers of intersti-
tial lung cells producing collagen, the hall-
mark of pulmonary fibrosis. This process is
not well understood. Specifically, the ex-
tent of macrophage accumulation at sites of
lung damage cannot be used to determine
whether lung repair or progression to fi-
brosis occurs. However, macrophage accu-
mulation elicited by "inert" particles (for
example, fly ash) does not result in lung
fibrosis.
The concept of cellular damage is often
invoked in discussions of toxicity but sel-
dom defined in biochemical terms. There
seems to be a general consensus as to the
mechanism of action of 03, which is as-
sumed to initiate lipid peroxidation in lungs
by reacting with unsaturated fatty acids of
cell membranes. This putative mechanism
of action, although widely accepted, has
not been proven. Neither is the chemical
basis for generation of free radicals by these
reactions completely straightforward. It is
likely that if free radicals and active oxygen
species do play a role in cellular damage
elicited by 03, macrophages and leuko
OCR for page 420
420
Biochemical and Cellular Interrelationships
cytes participating in the inflammatory re-
sponse might well be the source of such
active oxygen species.
The initiation of lipid peroxidation in the
lung is a possible mechanism for oxidant
damage by gases such as O3 and NO2
(Fridovich 1976; Waling 1963~. In recent
years a considerable body of evidence has
been amassed in support of the hypothesis
that lung damage may be mediated in large
part by reactions involving free radicals.
Such radicals may be directly generated by
reaction of toxicants with target molecules
(for example, O3 or NO2 reaction with
polyunsaturated fatty acids), or indirectly
as a result of phagocytotic activity by alve-
olar macrophages or neutrophils. These
cells release free radicals such as superoxide
and hydroxyl during increased metabolism
which is often termed "oxidative burst."
Evidence for the role of free radicals in
lung damage includes a wide variety of
observations. Uncontrolled clinical trials
have shown increased survival of patients
suffering from adult respiratory distress
syndrome after they are treated with vita-
min E (Wolf and Seeger 1982~. Treatment
with various hydroxyl radical scavengers
protected rats from pulmonary edema in-
duced by high doses of thiourea or from
otherwise lethal levels of gamma irradia-
tion (Fox et al. 1983~. Recent studies have
shown protection against hyperopia by su-
peroxide dismutase or catalase stabilized by
encapsulation in liposomes (Turrens et al.
1984~.
Production of oxygen radicals in rat
lungs during hyperoxia has been directly
demonstrated by Freeman and Crapo
(1981), and in vitro studies by Martin
(1984) support this observation. Numerous
studies, among them Crapo et al. (1978)
and Mustafa and Tierney (1978), have re-
ported increases in the activity of free rad-
ical-scavenging enzymes in lungs of ani-
mals surviving insult with 03, NO2, or
other toxicants, thereby indirectly support-
ing this hypothesis. In vitro experiments
also support the concept that O3 can di-
rectly injure cells by a mechanism involv-
ing free radicals (see, for example, Morgan
and Wenzel 1985~.
This is a field of intense investigation at
present, with new observations reported in
practically every issue of the relevant jour-
nals. The linkages by which early damage
are connected to "late" pulmonary fibrosis
have not been defined and remain an area
for future studies.
Recommendation 4. Animal studies
should be done to determine the role of free
radicals and active oxygen species in lung
. .
1nJury.
The Middle Phase
The middle phase, or the events that occur
between injury to cells and deposition of
excess collagen, is the component of the
lung's response to injury that we under-
stand the least. The recruitment of alveolar
macrophages to sites of injury and the
subsequent proliferation of fibroblasts are
key cellular events in the pathogenesis of
pulmonary fibrosis. In addition, there are
published studies (reviewed by Haschek
and Wits chi 1979) suggesting an important
role for the alveolar type II cells in the
determination of the balance between cell
repair and fibrogenesis in the damaged
lung.
Epithelial Cells. Alveolar type II cell pro-
liferation may represent a critical period in
terms of repair (Haschek and Witschi
1979~. Disruption of the normal sequence
of events during this period may favor the
development of pulmonary fibrosis. For
example, systemic administration of buty-
lated hydroxytoluene to mice results in
widespread necrosis of type I cells within
24 hr. On days 2 and 3, most dividing cells
are type II pneumonocytes. If mice are
exposed to high (70 percent) concentrations
of oxygen (02) during this period, type II
cells are severely affected, whereas dividing
interstitial cells are not. Inhibition of the
epithelial cell proliferation allows the inter-
stitial cells to proliferate relatively un-
checked, resulting in increased collagen pro-
duction and pulmonary fibrosis. Animals
exposed to x rays instead of O2 showed
similar results (Witschi et al. 1980~.
These experiments also showed that the
timing of the second insult was critical. If it
OCR for page 421
Jerold A. Last
421
did not coincide with the period of reepi-
thelialization, then the enhanced fibrosis
was not observed. Other investigators have
found a similar enhancement of fibrosis in
rats exposed to bleomycin followed by
high levels of O2 (Rinaldo et al. 1982;
Tryka et al. 1982~. Interestingly, paraquat,
which by itself can induce irreversible fi-
brosis, has been observed to destroy type I
as well as type II cells (Vejeyaratnam and
Corrin 1971; Smith and Heath 1973; Skill-
rud and Martin 1984~. These data suggest
that epithelial cell control of fibroblast pro-
liferation may be an important early mech-
anism in pulmonary fibrosis of various
etiologies (Bowden 1984~.
· Recommendation 5. Studies of the link-
ages between epithelial cell damage and
repair and changes in populations of pul-
monary macrophages and interstitial cells
should be undertaken in whole animals.
Pulmonary Alveolar Macrophages. Many
investigators have allocated a central role to
the pulmonary alveolar macrophage in the
initiation of fibrosis. The role of the mac-
rophage in inflammatory and immunologic
processes is enormously complex and the
topic is reviewed periodically (Hocking and
Golde 1979; Nathan et al. 1980~.
A related topic is the effects of fibrogenic
agents on pulmonary alveolar macrophages
in terms of their recruitment and functional
response. The time course of macrophage
accumulation in relation to a fibrogenic
stimulus appears to vary with the insult
and, to some extent, with the experimental
design. In many models of fibrosis, macro-
phages begin accumulating extremely rap-
idly after exposure to the fibrogenic agent.
Brody and coworkers (1985) hypothe-
sized that complement may play a key role
in particle-induced macrophage migration.
They found that rats depleted of comple-
ment by cobra venom and mice genetically
deficient in complement had far less mac-
rophage accumulation following asbestos
exposure. In addition, the molecular
weight of the chemotactic factor from lav-
age fluid was consistent with its being C5a.
These researchers did not speculate on the
source of the complement component, ex
cept to point out that edema fluid (serum
transudate) is an important potential source
of lower-molecular-weight components of
complement in the damaged lung.
Other investigators have shown that
macrophages themselves may participate in
macrophage recruitment. Dauber and Da-
niele (1980) found that guinea pig lung
macrophages secreted a chemoattractant
for macrophages as well as for neutrophils
and lymphocytes. Kagan and coworkers
(1983b) found that alveolar macrophages
from rats exposed to asbestos appear to
secrete a chemoattractant for macrophages.
That study did not, however, examine the
effects of this chemoattractant on other
effecter cells. Partial characterization sug-
gested it to be a protein and heterogeneous
with regard to molecular weight. Circulat-
ing immune complexes, whose etiology is
unknown, are also believed to stimulate
macrophage accumulation (Hunninghake
et al. 1981~.
The direct effects of fibrogenic agents on
macrophages also seem to vary consider-
ably, depending on the specific agent and
the experimental design. The role of the
macrophage in fibrogenesis has often been
envisioned as passive; that is, the macro-
phage may release various soluble media-
tors that play a role in fibrogenesis. More
recent studies suggest that macrophages
may respond to a fibrogenic agent in vari-
ous ways.
Silica-exposed rat lungs showed numer-
ous silica-containing macrophages in lav-
age fluid as well as in situ immediately after
exposure, with high percentages of silica
. . . . .
contaln1ng macrop aages remalmng SIX
weeks later (Brody et al. 1982; Warheit et
al. 1984). Ultrastructural examination re-
vealed that the silica-containing macro-
phages appeared normal, and functional
studies (measurement of oxygen consump-
tion and phagocytosis) of ravaged macro-
phages revealed no abnormalities.
A decrease in phagocytic capacity is not
necessarily synonymous with functional
impairment. For example, Tryka and co-
workers (1984a) found that alveolar macro-
phages ravaged from lungs of hamsters
exposed to bleomycin and O2 at time
points ranging up to 120 days increased in
OCR for page 422
422
Biochemical and Cellular Interrelationships
number but had a decreased amount of cell
surface antigen, indicating that they were
relatively immature. Those researchers at-
tribute the markedly decreased phagocytic
capability at least partly to the decrease in
surface antigen. Other data from that study
suggest that fibrosis may be associated with
increased macrophage turnover.
Once macrophages have been recruited,
they are capable of releasing many media-
tors that modulate the behavior of other
effecter cells in the lung. Kazmierowski et
al. (1977) observed that macrophages ob-
tained from normal primate lungs secreted
at least two chemotactic factors. One factor
had properties consistent with its identity
as the complement component Csa, and
was chemotactic for neutrophils, mononu-
clear cells, and eosinophils. The other fac-
tor had an apparent molecular weight of
about 5000, did not appear to be a comple-
ment component, and was a specific che-
moattractant for neutrophils.
Similarly, Merrill and coworkers (1980)
found that human alveolar macrophages
secreted two chemoattractants for neutro-
phils, and Dauber and Daniele (1980) found
that macrophages from guinea pig lung
secreted chemoattractants for macro-
phages, neutrophils, and lymphocytes. A
phagocytic stimulus increased the release of
the neutrophil chemoattractant.
Recommendation 6. Basic research in
cell culture systems should be performed to
examine the biochemical basis of cell-cell
communication and the molecular nature
of various mediators, released by leuko-
cytes and macrophages from damaged
lungs, that enhance or prolong the cellular
inflammatory response.
Macrophages may also modulate effecter
cells in another way. Stimulated macro-
phages are capable of releasing arachidonic
acid from cell phospholipid pools. Further
metabolism through the cyclooxygenase
pathway results in the production of the
prostaglandins PGE~ and PGI~. among
other products. In vitro data show that
PGE2 and PGI2 suppress a variety ot neu-
trophil, macrophage, and lymphocyte
functional responses. They may also partic
~ ~O
mate in the induction of suppressor T cells.
In marked contrast, metabolic products of
arachidonic acid resulting from the li-
pooxygenase pathway appear to enhance
the inflammatory response (Hunninghake
et al. 1984~.
Recommendation 7. Mediators possi-
bly released by damaged lung epithelial
cells or derived from damaged matrix com-
ponents, which maintain and amplify lung
injury after acute cellular or organ damage,
should be characterized.
Macrophage secretion of effecter cell
chemoattractants appears to be increased in
fibrosis. Schoenberger and coworkers
(1982) observed that asbestos stimulates
alveolar macrophages to increase neutro-
phil migration to the lung. Wesselius and
coworkers (1984) found that macrophages
ravaged from exposed rat lungs between 5
and 20 days after bleomycin instillation
secreted increased amounts of neutrophil
chemoattractant as compared with macro-
phages from controls. Since bleomycin did
not directly stimulate macrophages, the
mechanism for the increased macrophage
secretion is unclear. The authors postulate
that the stimulus may result from cell in-
jury caused by bleomycin. In addition, data
from a study of macrophage-derived che-
moattractants from hamsters instilled with
bleomycin suggest that macrophages may
be regulating the sequence of effecter cell mi-
gration following injury (Kaelin et al. 1983~.
The interaction between macrophages
and fibroblasts is particularly complex since
macrophages appear to have the capacity to
stimulate fibroblasts as well as to suppress
them. Stimulation of fibroblast prolifera-
tion alone is also complex. Stiles and co-
workers (1979) proposed dividing growth
factors into competence factors (which pro-
vide a signal early in the G. phase of the cell
cycle) and progression factors (which pro-
vide a signal later in Gil, stimulating the
cell to replicate.
Bitterman and coworkers (1982) showed
that alveolar macrophages secrete a growth
factor (alveolar macrophage-derived growth
factor, mol. wt. = 18,000) for fibroblasts that
is distinct from other described growth fac
OCR for page 423
lerold A. Last
423
tars. The alveolar macrophage-derived
growth factor is believed to function as a
progression factor and to stimulate fibro
blasts to produce their own progression
factor.
Macrophages secrete fibronectin, a large
glycoprotein known to mediate cell/matrix
interactions through a variety of functions
including its chemotactic properties (Hun
ninghake et al. 1984~. Fibronectin is be
lieved to act as a competence factor for
fibroblast growth (Bitterman et al. 1983~.
In addition, fibronectin is chemotactic for
fibroblasts. Indeed, macrophage-derived fi
bronectin is 1,000-fold more potent as a
chemoattractant than is plasma fibronectin
(Rennard et al. 1982~.
Macrophages also appear to be capable of
suppressing fibroblast growth. Elias and
coworkers (1985) found that supernatants
from normal human alveolar macrophages
inhibit growth of log-phase fibroblasts.
Their study showed that the inhibitory
capacity of the supernatant was directly
related to its capacity to stimulate fibroblast
prostaglandin production. They separated
macrophage subpopulations by density
gradients and found that the factor(s) an
peared to be preferentially elaborated by
smaller and denser macrophages. In some
respects this factor resembles those de- r -a
scribed by Korn et al. (1980) and Clark et
al. (1982~.
Investigation of the effects of specific
fibrogenic agents on macrophage regula
tion of fibroblast growth illustrates the
complexities involved. For example, Lu
gano and coworkers (1984) found that 2
and 14 days after silica exposure, ravaged
macrophages depressed fibroblast prolifer
ation, whereas at 42 days macrophages
stimulated fibroblast proliferation. Clark
and others (1982) discovered that macro
phages from hamsters instilled with
bleomycin had a greater suppressive ettect
on fibroblast proliferation and collagen
synthesis compared with control macro
phages. They found that suppression was
associated with increased PGE2 and intra
cellular cAMP, and that fibroblast suppres
sive activity decreased in the first days after
bleomycin instillation, and then increased
after 8 days. This suppressive activity may
~ . .
be a mechanism for modulating fibroblast
proliferation and fibrosis following fibro-
gen~c exposure.
Recently published data of Schmidt et al.
(1984) suggest that interleukin-1 may play a
role in fibroblast proliferation. Although
other researchers have not found that inter-
leukin-1 has fibroblast-stimulating proper-
ties, Schmidt and coworkers point out dif-
ferences in experimental design that might
account for this discrepancy. Peripheral
blood monocytes were used in that partic-
ular study, and it should be noted that
alveolar macrophages are also capable of
secreting interleukin-1. No detailed studies
have been reported with macrophages or
effecter cells from animals exposed to O3.
Such work should be done to test whether
(and which of) these pathways might be
operative in the lungs of animals exposed to
o3.
Broncho-alveolar ravage was used to ob-
tain macrophages in many of the studies
discussed above. Given the heterogeneity
of macrophages, it is unclear if the popu-
lations present in ravage fluid accurately
reflect the populations actually present in
lung tissue, particularly in disease states.
The problem is compounded by the fact
that there is no consensus as to the ap-
oronriate functional and/or structural def-
~n~t~ons of such macrophage subpopu-
lations. Brain and coworkers (1977),
Mason (1977), and Lum and coworkers
(1983) have reviewed some of the potential
problems in studying ravaged macro-
phages.
In detailed morphometric analyses of
centriacinar macrophages in situ and pul-
monary macrophages ravaged from control
and O3-exposed rats, Lum and coworkers
(1983) observed significant differences in
most parameters studied between the la-
vaged and in situ macrophages in the con-
trol as well as the O3-exposed rats. These
data suggest that, at least in this model of
fibrosis, ravaged macrophages are not rep-
resentative of macrophages present at the
sites of greatest lung damage. Alterna-
tively, it is questionable whether interstitial
macrophages, and those resident in airways
and therefore accessible by ravage, are a
homogeneous population in equilibrium or
OCR for page 424
424
Biochemical and Cellular Interrelationships
are somehow "different." Clearly, similar
problems may exist in studying any of the
effecter cells obtained by ravage.
Fibroblasts. Presumably the cells respon-
sible for synthesizing the "abnormal" col-
lagen of pulmonary fibrosis whether it be
abnormal in amount, location, or type are
the fibroblasts. Although they often are
perceived as passive target cells for the
effecter cells and their mediators, fibro-
blasts may on occasion play a more active
role in directing the course of fibrosis.
In some cases fibroblasts may directly
interact with the fibrogenic agent. Several
workers have examined the effects of
bleomycin on fibroblasts. Sterling and co-
workers (1982) found that collagen synthe-
sis increased in human fetal lung fibroblasts
exposed to bleomycin for 48 hr. but deg-
radation also increased. They also found
that polysomes from bleomycin-treated fi-
broblasts synthesized twice as much colla-
gen as control polysomes, but noncollagen
protein synthesis was not affected. Similar
results have been reported by Clark et al.
(1980) and Phan et al. (1985~.
In an examination of the effects of
bleomycin, hyperopia, and the presence of
lung macrophages on collagen synthesis by
human WI-38 fibroblasts, Robin and Juhos
(1983) found that bleomycin alone directly
stimulated collagen synthesis, as measured
by hydroxyproline in the culture dishes.
The addition of hyperoxia and/or the pres-
ence of lung macrophages did not further
increase collagen synthesis, and hyperopia
alone significantly decreased collagen syn-
thesis. However, hyperopia in the presence
of lung macrophages increased collagen
synthesis about the same extent as did
bleomycin.
There is also evidence that bleomycin
affects fibroblast proliferation. In a study of
the effects of in vitro and in viva exposure
to bleomycin on growth characteristics of
fibroblasts, Absher and coworkers (1984)
observed that both types of bleomycin ex-
posure appeared to decrease growth of fi-
broblasts in comparison with controls. In a
similar system of in vitro exposure, Phan
and coworkers (1985) found that bleomy-
cin exposure had no effect on growth.
However, since that study examined iso-
lated fibroblasts 14 days after bleomycin
instillation, its authors suggest that the
difference in timing may account for the
apparently discrepant findings. That is,
during the first week after instillation,
bleomycin toxicity may impair fibroblast
growth, whereas during the second week,
recovery may be occurring. Furthermore,
this recovery may involve recruitment or
selection of a population of fibroblasts with
different growth characteristics. Whether
these provocative findings in the bleomycin
system accurately model events in lungs of
animals exposed to O3 remains to be tested.
Fibroblasts are believed to play a role in
epithelial cell growth and function. Fibro-
blast pneumonocyte factor has been iso-
lated from glucocorticoid-treated fibro-
blasts and has been shown to stimulate
disaturated phosphatidylcholine synthesis
in whole lung in vitro (Smith 1979) and in
isolated type II pneumonocytes (Smith
1978~. A small somatomedin-like growth
factor specific for pneumonocytes is be-
lieved to be secreted by fibroblasts after
pneumonectomy (Smith et al. 1980~. Pul-
monary fibroblasts exposed to hyperoxia in
vitro secrete an epithelial cell growth factor
as well as a lipid-synthesis-stimulating fac-
tor (Tanswell 1983~. These particular fac-
tors appear to differ from any of the others
previously described.
Fibroblasts may also affect effecter cells.
Although their predominant function is
apparently production of collagen, other
matrix components, and mucopolysaccha-
rides, they also secrete biologically active
products such as Cal and interferon (Al-
Adnani and McGee 1976~. Cultured fibro-
blasts produce a factor chemotactic for mo-
nocytes as well as for neutrophils (Sober
and Gallin 1979~. In addition, fibroblast
culture fluid is capable of generating
chemotactic activity from human serum,
probably by cleaving C5a from C5. Fibro-
blasts are capable of producing macrophage
migration inhibition factor (Tubergen et al.
1972~. Whether such "fibrokines" play a
role in pulmonary fibrosis has yet to be
demonstrated.
Collagen itself is another fibroblast prod-
uct with chemotactic properties. Type I
OCR for page 425
.lerold A. Last
collagen and its isolated chains are chemo-
tactic for monocytes but not neutrophils
(Stecher 1975; Postlethwaite and Kang
1976~. In contrast, rat collagen is chemo-
tactic for rat neutrophils in viva (Chang
and Houck 1970~.
~ Recommendation 8. Examination of
factors released by inflammatory cells that
modulate collagen synthesis or fibroblast
proliferation, especially in response to 03,
might help to define the mechanisms un-
derlying the transition from the damaged,
inflamed lung to the fibrotic lung.
Lung Fibrosis
Lung fibrosis, as defined clinically, refers to
interstitial fibrosis as is seen in the later
stages of idiopathic pulmonary fibrosis
(also called cryptogenic fibrosing alveolitis
in the literature of the United Kingdom). In
this disease the hallmark of pulmonary
fibrosis as seen by the pathologist is in-
creased focal staining of collagen fibers in
the alveolar interstitium. Despite earlier
misconceptions, it seems clear that fibrotic
lungs from humans with either acute or
chronic pulmonary fibrosis contain in-
creased amounts of collagen as evaluated
biochemically, in agreement with the his-
tological findings.
In the normal lung, interstitial collagen
fibers are thought to provide structural
matrix or scaffolding upon which the lung
cells are assembled. These fibers are also
thought to be responsible for the limits to
which the alveoli can be stretched during
inhalation or to which they can relax dur-
ing expiration. The deposition of additional
collagen in the fibrotic lung is presumably
responsible for the increased stiffness of
these lungs, whereby the volume to which
they can expand at a given distending pres-
sure is decreased as compared with normal
values.
Unfortunately, pure interstitial fibrosis
does not generally occur in lungs damaged
by toxicants. In many ways such toxicants
cause disease that more closely resembles
adult or infant respiratory distress syn-
drome than chronic fibrosis. Excess lung
collagen is usually observed not only in the
425
alveolar ~nterst~t~um, but also throughout
the centriacinar region, including the alve-
olar ducts and respiratory bronchioles. The
relationship between increased collagen
deposition around small airways and lung
mechanics is not understood, either theo-
retically or empirically.
Recommendation 9. The reversibility
of excess collagen deposition in the fibrotic
lung, as reflected by increased hydroxy-
proline content of the lung, should be
determined.
There are at least 13 genetically distinct
collagen types known to occur in all mam-
mals, of which.at least seven have been
found in normal lungs or synthesized by
isolated lung cells. Two types predominate
in the lung, representing about 90 percent
or more of the total lung collagen. Type I
and III collagens are the major interstitial
components and are found in the normal
lungs of all mammals in an approximate
ratio of 2:1.
This ratio is altered in fibrotic lungs. It is
not known whether shifts in collagen
types, as compared with absolute increases
in collagen content, account for the in-
creased stiffness of fibrotic lungs. Type III
collagen is much more compliant than is
type I; thus, an increasing proportion of
type I relative to type III collagen might
result in a stiffer lung as is observed in
pulmonary fibrosis. Changes in collagen
cross-linking in fibrotic lungs may also
contribute to the increased stiffness ob-
served. In addition, because type I collagen
is the material that stains histologically as
"collagen," whereas type III collagen does
not, an increased proportion of type I rel-
ative to type III collagen would be appre-
ciated histologically as an "increased
amount of lung collagen." Therefore, it is
unclear whether the observed increase in
stainable collagen is due solely to the in-
crease in collagen content of the lungs
observed biochemically, or whether altered
collagen types or cross-linking might also
contribute to the histological changes seen.
Some types of pulmonary fibrosis, in-
cluding that induced by 03, involve abnor-
malities in the type of collagen being made.
OCR for page 430
430
Biochemical and Cellular Interrelationships
posed and control rats reached the same
endpoint collagen content after six months,
with the exposed animals reaching this
value more rapidly (at three months). It
seems likely that the changes in hydroxy-
proline content of the lungs from exposed
rats observed over the first three months of
the study were related to excessive deposi-
tion of collagen (fibrosis) in their lungs (as
observed morphologically), but a role for
accelerated aging (or growth) of the lungs
in O3-exposed rats cannot be ruled out as
an alternative explanation of these observa-
tions. One disadvantage of rats as an ex~oer-
imental animal for chronic studies Is that
rats continue to grow throughout their en-
tire lifetime, and lung growth is not "com-
plete" at maturity as in humans.
Recommendation 12. The response of
the developing lung versus the mature lung
in animals exposed to O3 should be studied
to ascertain whether the developing lung is
more susceptible to damage, fibrosis, or
change.
Progression of Lung Injury After
Cessation of Exposure
What are the consequences of allowing
animals to "recover" during a postexpo-
sure period? In another set of experiments,
intermittent exposure to high levels of O3
(0.64 or 0.96 ppm) for 8 hr/day elicited the
same increases in lung collagen content of
adult rats as did a continuous exposure
protocol of 23.5 hr/day (Last et al. 1984b).
This finding emphasizes the importance of
not assuming that effects of O3 exposure
may be estimated by a simple concentration
x time relationship. A six-week postexpo-
sure "recovery period" breathing filtered
air exacerbated the increase in lung collagen
content appreciated immediately after ces-
sation of exposure. This result suggests that
not only are these effects irreversible, at
least in this time frame, but that they are
also progressive.
The mechanism for exacerbating dam-
age, or repairing such damage, to the lung
by a postexposure period breathing filtered
air is not obvious. Before any meaningful
O ~
hypothesis can be presented, further exper-
iments examining cellular changes within
the lung during such a postexposure period
are necessary. Examination of the time
course and cellular components of reepithe-
lialization of the alveolar ducts and walls
during the postexposure period would be
especially important in this regard.
Haschek and Witschi (1979) stressed the
importance of this component of lung re-
pair in potentiation by O2 of pulmonary
fibrosis after lung damage with butylated
hydroxytoluene. Further, to avoid misin-
terpretation of data because of altered
growth rates in exposed and control ani-
mals, use of appropriate (pair-fed) controls
in experiments with growing animals ex-
posed to high levels of O3 iS obligatory.
This specific area of research on postexpo-
sure effects of O3 iS an important one for
further study.
Recommendation 13. Lung structure
and biochemistry over long postexposure
periods should be studied in detail.
Synergistic Interactions
The design of experiments to assess fibro-
genicity of a given agent becomes much
more complex when the possibility of syn-
ergistic (or antagonistic) actions between
agents is considered. For example, Haschek
and coworkers found that exposure of mice
to 70 percent O2 enhances pulmonary fi-
brosis previously induced by intraperito-
neal injection of butylated hydroxytoluene
(Haschek and Wits chi 1979; Has chek et al.
1983~. They concluded that the severe fi-
brosis seen after the combination of buty-
lated hydroxytoluene and O2 is the result of
synergism, and proposed the following
mechanism: when butylated hydroxytol-
uene causes lung damage, there is an initial
phase of epithelial proliferation. If mice are
exposed to 70 percent O2 during this pe-
riod, the epithelial type II cells are either
inhibited from dividing or killed. Damage
to the epithelial cells may then lead to unin-
hibited interstitial cell growth. This specific
interaction between butylated hydroxytol-
uene and O2 seems to occur only in mice.
OCR for page 431
Jerold A. Last
431
A similar synergism apparently occurs
with bleomycin and 70 percent O2 in rats,
hamsters, and other laboratory rodents
(Tryka et al. 1984b) and is not specific to
the mouse. Other such two-agent models
also seem to work in multiple species (Has-
chek et al. 1983~; such combinations in-
clude drugs and 02, radiation and 02, and
cytotoxic agents and bleomycin.
In another study of synergism between
particulates and gases, McTilton and
Charles (1976) examined the effects on
guinea pigs of exposure to sodium chloride
aerosols and SO2. When the mixture was at
a high humidity, those researchers found
decreases in airway flow resistance. Nor-
mally, SO2 does not penetrate deep into the
lung, and McTilton and Charles proposed
that the highly soluble SO2 dissolved in
droplets and thus was able to "piggyback"
into the lower respiratory tract. Ellison and
Wailer (1978) have reviewed this topic.
In a study of oxidant gases plus SO2,
Gardner and coworkers (1977) found that a
protocol of O3 and sulfuric acid (H2SO4)
aerosol presented sequentially was more
toxic than either agent alone, based on
mortality rates of mice exposed to Strepto-
coccus pyogenes. Juhos and coworkers (1978)
found evidence for synergism between O3
and H2SO4, based on very limited his-
tologic evaluations of rat lungs. Last and
Cross (1978), using several biochemical cri-
teria and studying the effects upon tracheal
mucous-producing cells, reported a syner-
gism between O3 and H2SO4 aerosols at
relatively low concentrations of each. Ha-
zuka and Bates (1975) observed synergism
between O3 and sulfate aerosols and sug-
gested that it was responsible for decreased
maximal flow rates observed during human
exposure to O3 and SO2 or H2SO4 at
near-ambient levels.
In a recent study of the effects of ammo-
nium sulfate aerosols in combination with
O3 or NO2 on collagen metabolism in rat
lungs, Last and coworkers (1983a) found
that ammonium sulfate aerosols alone had
no effect on collagen synthesis rates; how-
ever, they significantly potentiated the
effects of the oxidant gases. Guth and
coworkers (1986) found that lavagable pro
tein content increased significantly in lungs
of rats continuously exposed for 1-2 days
to 0.12 ppm O3, and, more important,
Warren and Last (1987) saw elevated colla-
gen synthesis when rats were exposed to
0.12 ppm O3 plus H2SO4 aerosol. The
latter researchers obtained similar results
with rats exposed to 0.2 ppm O3 + 40
,ug/m3 of acid aerosol. We interpret these
results as being indicative of a prefibrotic
response of the lung to injury.
Furthermore, responses in this and other
assays in rats exposed to 0.2 ppm O3 for 8
hr/night and in rats exposed continuously
have been similar. That is, the increase in
lung collagen synthesis rate is not contin-
gent upon continuous exposure of rats to
O3, but may occur under an intermittent
exposure regimen that models human diur-
nal exposures as well. These results must be
confirmed, especially in another species.
Last and coworkers (1984a) suggested a
hypothetical mechanism for this synergy
involving increases in the stability of free
radicals generated in situ. Since the mech-
anisms of injury elicited by individual
pneumotoxins are so poorly understood, it
is hardly surprising that mechanisms un-
derlying synergistic interactions remain
highly speculative.
·Recommendation 14. A rational basis
should be developed for prediction of syn-
ergistic or antagonistic interactions of pol-
lutant mixtures by systematically examin-
ing binary and ternary combinations of
the pollutants. The possibility that agents
known to affect epithelial cell turnover
might interact synergistically with O3 de-
serves special attention.
Determination of Human Risk
What are the implications of these experi-
ments for those charged with setting ambi-
ent air quality standards? Clearly, the tra-
ditional data base used for setting such
standards is heavily skewed in the direction
of detecting acute, short-term effects such
as reflex bronchoconstriction in human
OCR for page 432
432
Biochemical and Cellular Interrelationships
subjects undergoing controlled experimen-
tal exposures. However, increased airway
resistance may occur in response to release
of stored histamine from airway mast
cells in response to signals from irritant
receptors (Dixon and Mountain 1965) and
may have little or no long-term conse-
quences to health. The inflammatory re-
sponse of the lung to O3 may also contrib-
ute to increased airway reactivity after
exposure.
The potential adverse health effects of air
pollution that constitute the major concern,
however, are chronic effects from intermit-
tent, long-term, low-level exposures: can-
cer, emphysema, pulmonary fibrosis, and
chronic obstructive lung disease. Con-
trolled human exposures are of absolutely
no value for assessing these types of risks.
To date, epidemiologic studies have also
been of little value in assessing these risks
(Committee on Medical and Biological Ef-
fects of Environmental Pollutants 1979),
partly because of the overwhelming im-
pacts of smoking and occupational expo-
sures on the incidence of these diseases in
our population, and the uncertainty about
individual doses as compared with expo-
sures of entire populations.
It is not practical to look at each pollutant
and every possible combination of pollut-
ants in long-term dose/response experi-
ments that may require inhalation expo-
sures for six months, or a year, or an entire
lifetime (three years or more in rats or
mice). Thus the need is for short-term
assays that probe for potential structural
changes in the lung, such as those for
collagen and elastin metabolism described
above. Such short-term assays may detect
potential adverse health effects of air pol-
lutants that cannot be ascertained by con-
trolled human exposures or other currently
used methodology for risk assessment.
However, the regulatory climate until now
has tended to ignore animal inhalation tox-
icology experiments in favor of data from
controlled human exposures (Whitten-
berger 1985~.
~ Recommendation 15. Greater use
should be made of animal models of sus-
ceptible populations.
We also seem to be allowing ourselves to
be lulled into a sense of security with the
current ambient air quality standards, on
the basis of the belief that we adapt to
pollutants upon continued exposure. The
concept of adaptation comes from the at-
tenuation of reflex bronchoconstriction in
controlled human exposures to, for exam-
ple, O3 upon continued exposure. There
are no data suggesting that the lung can
adapt to continued exposure to O3 when
the assay for effects is based upon structural
changes in the lung rather than upon tran-
sient responses such as reflex bronchocon-
striction or localized inflammation.
It is only by designing experiments that
correlate data between short-term assays
for structural change (to evaluate dose/re-
sponse characteristics of the lung) with
selected long-term chronic exposures that
include detailed examination of lung struc-
ture that we will be able to evaluate prop-
erly the true risks of exposure to ambient
air pollutants and their mixtures.
Recommendation 16. Long-term (life-
time) exposures of rodents should be per-
formed under realistic protocols with so-
phisticated assays at termination. Such
studies should also examine tumor inci-
dence and, to be meaningful, would require
very large groups of animals.
Identification of Susceptible
Populations
The wording of the 1977 version of the
Clean Air Act raises the question of poten-
tial susceptible populations in relation to
any attempt to evaluate actual or hypothet-
ical human risks of exposure to O3. Are
there identifiable susceptible populations
from the standpoint of potential lung fi-
brosis? First, and most important, are very
young children and infants, whose lungs
are still developing and who would seem
from animal experiments to be at risk of
their lungs developing differently in atmo-
spheres containing O3. "Differently" in this
context relates to the higher collagen con-
tent found in lungs of young rats than in
OCR for page 433
herald A. Last
433
lungs of matched, pair-fed controls (Last et
al. 1984b) and the elongated alveolar ducts
or respiratory bronchioles observed in rats
or monkeys after exposure to O3 (Tyler et
al. 1985~. It is not known if these observa-
tions constitute adverse health effects, hence,
use of the word "differently."
Second, and obvious, is the population
that breathes polluted outdoor air rather
than conditioned indoor air by the nature of
their professions, by choice, or by necessity
during episodes of pollution, especially in
conjunction with strenuous exercise (which
increases the inhaled dose to the lungs the
prototypical "freeway joggers.
Since fibrosis is an insidious disease and
the lung has a substantial reserve capacity
to protect itself from the physiological con-
sequences of fibrosis, a third population at
risk includes people whose lungs are al-
ready severely compromised by chronic
obstructive pulmonary disease, emphy-
sema, congenital disease, or fibrosis.
A fourth group at risk would be those
with diseases of the small airways, the focal
site of O3-induced fibrosis. The largest
group with small airways pathology would
be cigarette smokers. If the cellular mech-
anisms of lung inflammation and injury
discussed earlier pertain in human popula-
tions, subjects with chronic lung inflamma-
tion (alveolitis) should be at increased risk.
This group would include workers and
others with allergic alveolitis and patients
(such as those with idiopathic pulmonary
fibrosis) who are subject to recurrent epi-
sodes of inflammatory infiltration of the
distal lung with leukocytes and macro-
phages.
Summary
Inhalation of O3 by experimental animals
causes pulmonary edema after exposures of
short duration and results in peribronchio-
lar and centriacinar pulmonary fibrosis af-
ter longer exposures. This discussion has
stressed possible linkages between these
outcomes, including the probable central
role of the alveolar macrophage as an effec-
tor cell. The phenomenon and the under
lying mechanisms of pathogenesis of pul-
monary edema are probably well enough
known for these observations to be extrap-
olated to assessment of human risks upon
exposure to a given concentration of 03, and
sensitive clinical methods applicable to hu-
mans are available to compare directly with
such estimates. However, the reversibility of
such changes and whether increased fluid in
the bronchial and alveolar airspaces is
"damage" in the regulatory sense (that is, "an
adverse health effect") are unknown.
At the other end of the process, much is
being learned about the biochemistry of
collagen in the fibrotic lung. Little or noth-
ing is known of the cellular mechanisms
responsible for excessive synthesis and dep-
osition of fibrotic collagen, or whether
such changes are reversible. As far as O3 is
concerned, nothing is known of the corre-
sponding cellular and biochemical mecha
. · .
nlsms in Humans.
The events linking acute and chronic
responses of the lung to O3 are poorly
understood and should be a primary focus
of future research, in cell biology as well as
in biochemistry. Most of the methodology
necessary for such studies in animals is
probably available.
Other topics of interest are progressive
versus self-limited injury to the lung, re-
versibility of damage, and the potential for
synergism between mixtures of air pollut-
ants, especially O3 and respirable aerosols
or particulates. Recent studies in various
animal models have suggested that certain
combinations of agents, which alone cause
limited or undetectable lung injury, can
cause progressive pulmonary fibrosis.
These models have certain features in
common with the continuing changes ob-
served in lungs of rats and monkeys ex-
posed to O3 followed by periods where
they breathe only filtered pure air. Rats
exposed to O3 in combination with various
acidic aerosols, which by themselves appar-
ently cause no lung damage at the concen-
trations tested, have suffered enhanced
acute lung damage. The significance of
these observations to potential chronic, ir-
reversible changes in rat lungs, or to poten-
tial human health effects, remains to be
determined.
OCR for page 434
434
Biochemical and Cellular Interrelationships
Summary of Research Recommendations
Acute Phase of Injury
In the acute injury phase, the greatest need is for a better
understanding of the pathophysiological significance of apparent
alterations in broncho-alveolar epithelial permeability in animals
and humans acutely exposed to O3.
HIGH PRIORITY
Recommendlation 2 To facilitate interpretation of human studies, sensitive ravage-based
assays of protein content need to be correlated with diethylenetri
aminepentaacetic (DTPA) acid aerosol transport assays in animals.
MEDIUM PRIORITY
Recommendlation 1 Using experimental animals, anatomic regions in the lung asso
ciated with tracer transport from blood to ravage fluid, and vice
versa, after O3 exposure, should be localized and correlated with
bulk movement of albumin into airspaces.
Recommendation 3 Human studies using DTPA aerosols or other tracers in conjunction
with concentration/response assays for edema should be undertaken.
LOW PRIORITY
Recommendation4 Animal studies should be done to determine the role of free
radicals and active oxygen species in lung injury.
Middle Phase of Injury
The greatest needs for research in the middle phase of injury/
fibrosis are for a better understanding of the basis of cellular
changes that perpetuate and amplify inflammation and cellular
damage in lungs of animals exposed to O3.
HIGH PRIORITY
Recommendation 5 Studies of the linkages between epithelial cell damage and repair
and changes in populations of pulmonary macrophages and inter
stitial cells should be undertaken in whole animals.
Recommendlation 6 Basic research in cell culture systems should be performed to
examine the biochemical basis of cell-cell communication and the
molecular nature of various mediators, released by leukocytes and
macrophages from damaged lungs, that enhance or prolong the
cellular inflammatory response.
MEDIUM PRIORITY
Recommendation 7 Mediators possibly released by damaged lung epithelial cells or
derived from damaged matrix components (arachidonic acid
OCR for page 435
Jerold A. Last
435
metabolites, chemotactic factors, cytokines), which maintain and
amplify lung injury after acute cellular or organ damage should be
characterized.
Recommendation 8 Examination of factors released by inflammatory cells that
modulate collagen synthesis or fibroblast proliferation, especially
in response to 03, might help to define the mechanisms under
lying the transition from the damaged, inflamed lung to the fibrotic
lung.
Late Phase of Injury
In the fibrotic phase, or late stages of injury, the greatest need is
to understand whether the increased collagen synthesis observed in
lungs of rats and monkeys acutely exposed to O3 results in
pulmonary fibrosis.
HIGH PRIORITY
Recommendation9 The reversibility of excess collagen deposition in the fibrotic
lung, as reflected by increased hydroxyproline content of the lung,
should be determined.
Recommendation 11 The relationship between the presence of specific abnormal
collagen cross-links in the lung and the reversibility of fibrosis
should be examined.
.
Recommendation 12 The response of the developing lung versus the mature lung in
animals exposed to O3 should be studied to ascertain whether the
developing lung is more susceptible to damage, fibrosis, or change.
MEDIUM PRIORITY
Recommendation 10 In addition to collagen, the content and structure of other
extracellular matrix components in the fibrotic lung should be
examined.
Other Considerations
H I G H P R I O R I T Y
Recommendation 13 Detailed study of lung structure and biochemistry over long
postexposure periods should be made to allow better understand
ing of progression of injury after exposure to O3 is terminated.
Recommendation 14 A rational basis should be developed for prediction of synergistic
or antagonistic interactions of pollutant mixtures by systematically
examining binary and ternary combinations of the pollutants. The
possibility that agents known to affect epithelial cell turnover might
interact synergistically with O3 deserves special attention.
Recommendation 15 Greater use should be made of animal models of susceptible
populations.
OCR for page 436
436
Biochemical and Cellular Interrelationships
Recommendation 16 Descriptive, subjective pathology is of little or no value. Long
term (lifetime) exposures of rodents under realistic protocols with
sophisticated assays at termination are required, with morphomet
ric and detailed biochemical examination of structural components
at the least. Such studies should also examine tumor incidence and,
to be meaningful, would require very large groups of animals.
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Representative terms from entire chapter:
collagen synthesis
