1. Inflammatory Lung Injury after Bronchial Instillation
of Air Pollution Particles
ANDREW J. GHIO and ROBERT B. DEVLIN
Clinical Research Branch, Human Studies Division, National Health and Environmental Effects Research Laboratory,
Environmental Protection Agency, Research Triangle Park, North Carolina
PM levels in the Utah Valley. Alterations in composition were
Epidemiologic investigation has established an association between
also assumed. Associated with the closure of the mill, and both
exposure to particulate matter (PM) and human health in the Utah
Valley. Reduction of particle mass during the temporary closure of the reduction in total PM mass and changes in its composition,
a local steel mill was associated with diminished morbidity and were decreases in elementary school absences (3), bronchitis and
mortality. We tested the hypothesis that the biologic effect of PM asthma admissions for pre-school-age children (4), total respira-
would reflect findings of epidemiology with a greater injury after tory hospital admissions for pneumonia, pleurisy, bronchitis,
exposure to an equal mass of particles from those years in which and asthma (2), pulmonary function abnormalities (5), mortal-
the mill was in operation. Filters containing PM were collected ity (6), and age-adjusted death rates for malignant and nonma-
prior to closure of the steel mill, during the closure, and after its re-
lignant respiratory disease (7). Changes in total mass did not ac-
opening. Aqueous extracts of the filters were prepared. One of three
count for all variation in the biologic effects of PM in Utah Valley
extracts (500 g) was instilled through the bronchoscope into the
between those years before the closure of the steel mill, during
lungs of nonsmoking volunteers. Twenty-four hours later, the same
its shutdown, and following its reopening (6).
subsegment was lavaged. Exposure to aqueous extracts of PM col-
The closure and reopening of the steel mill allowed for an
lected before closure and after reopening of the steel mill pro-
examination of potential correlates between epidemiologic ob-
voked a greater inflammatory response relative to PM extract ac-
servations and measures of the biologic effect of PM with ex-
quired during the plant shutdown. This is the first demonstration
perimental human exposure. We tested the hypothesis that the
that pulmonary effects after experimental exposure of humans to
biological effects of PM collected from January to March of
PM can correlate with health outcomes observed in epidemiologic
1986, 1987, and 1988 and instilled into the human lung on an
studies of the same material under normal exposure conditions.
equal mass basis would reflect the findings of the epidemiologic
Findings suggest that mass may not be the most appropriate met-
investigation. We also explored the possibility that a metal-
ric to use in assessing health effects after PM exposure but rather
catalyzed oxidative stress could contribute to dissimilarities be-
specific components must be identified and assessed.
tween the effects of exposures to PM from Utah Valley ob-
Keywords: air pollution; lung diseases; inflammation; neutrophils; tained from January to March of 1986, 1987, and 1988.
bronchoscopy; oxidants; iron
METHODS
Ambient air pollution particles currently present a serious risk
to human health with significant increases in both morbidity and Corresponding to the times of previous epidemiologic investigation
mortality associated with their exposure. The World Health (2–7), filters containing PM10 were collected from January to March of
Organization estimates that inhalation of particulate matter 1986, 1987, and 1988 (n 34 each year; Lindon site, Utah Department of
(PM) in ambient air is responsible for 500,000 excess deaths Environmental Quality). Each filter was agitated in a 50-ml tube contain-
each year worldwide (1). However, there has been little de- ing 40 ml deionized water for 24 h. After centrifugation, the supernatant
fluid from all 34 filters of each year was pooled, lyophilized, weighed, and
scription of acute physiologic, biochemical, or molecular alter-
stored at 80 C. Each of the three extracts was treated with ultraviolet
ations after experimental exposure of humans to PM to cor-
radiation and autoclaved. Extracts were prepared in 1996 and stored at
roborate epidemiologic evidence of increased morbidity and
80 C until used in the investigation. Endotoxin levels of all three ex-
mortality. Furthermore, there is neither a consensus on a plau-
tracts were below detectable limits (E-toxin, Sigma, St. Louis, MO).
sible mechanism nor is there any agreement on which compo- The study population included 24 healthy, nonsmoking volunteers
nent of PM is responsible for biologic activity. (21 male, three female) with a mean age of 26.4 yr and a standard de-
The Utah Valley provided a unique opportunity to evaluate viation of 2.2 yr. The protocol and consent form were approved by the
the health effects of PM in humans. The area has had intermit- University of North Carolina School of Medicine Committee on the
tently high particle levels with the principal point source being a Protection of the Rights of Human Subjects. Prior to inclusion in the
study, subjects were informed of the procedures and potential risks,
steel mill. While operational, this plant contributed greater than
and each signed a statement of informed consent.
80% of industrially related PM in the valley (2). Because of a la-
The subjects underwent bronchoscopy. A sterile flexible catheter
bor dispute, the mill was shut down for 13 mo, from August 1,
was inserted through the biopsy channel of the bronchoscope and ex-
1986 to September 1, 1987 resulting in a substantial reduction of
tended 2.5 cm into the orifice of a segmental bronchus of the lingula.
Ten milliliters of sterile saline containing 500 g extract from either 1986
(n 8), 1987 (n 8), or 1988 (n 8) were instilled through the catheter
followed by 10.0 ml saline. Twenty milliliters of saline with no particles
(Received in original form November 24, 2000 and in revised form April 24, 2001)
were similarly instilled into a subsegment of the right middle lobe.
This report has been reviewed by the National Health and Environmental Effects
Twenty-four hours later, subjects underwent lavage of the same sub-
Research Laboratory, United States Environmental Protection Agency and ap-
segment in which the extract and the saline (control) had been placed.
proved for publication. Approval does not signify that the contents necessarily
Cells were counted by hemocytometer and differentials were deter-
reflect the views and policies of the Agency nor does mention of trade names or
mined. Lavage protein and albumin concentrations in the lavage fluid
commercial products constitute endorsement or recommendation for use.
were determined using the Pierce Coomassie Plus Protein Assay Re-
Correspondence and requests for reprints should be addressed to Andrew Ghio,
agent (Pierce Chemical Co., Rockford, IL) and an immunoprecipitin as-
Human Studies Facility, 104 Mason Farm Road, Chapel Hill, NC 27599-7315.
say (Diasorin, Stillwater, MN), respectively. Fibronectin and -1-anti-
E-mail: ghio.andy@epa.gov
trypsin were measured employing ELISAs developed using antibodies
Am J Respir Crit Care Med Vol 164. pp 704–708, 2001
to human fibronectin (Sigma) and antitrypsin (Calbiochem, La Jolla, CA).
Internet address: www.atsjournals.org

2. 705
Ghio and Devlin: Inflammation after Exposure to Pollution Particles
Figure 1A, B, and C. Total number of viable cells and
neutrophil counts in the lavage fluid after exposure of
healthy volunteers to PM extracts. There was an increase
in total viable cells in the lavage of subjects instilled with
any extract. Neutrophils were also elevated after exposure
of subjects to those extracts collected while the mill
was in operation (1986 and 1988). This was significant
whether the numbers of neutrophils were expressed as
an absolute number or a percentage. Asterisks denote a
significant difference in the post-hoc test (Scheffe’s) rel-
ative to the saline control with p 0.05.
Figure 2A, B, C, and D. Lung injury indices after expo-
sure of healthy volunteers to PM extracts. There was a
significant increase in all indices of lung injury in those
volunteers exposed to extracts collected while the steel
mill was functioning (1986 and 1988). Asterisks denote
a significant difference in the post-hoc test (Scheffe’s)
relative to the saline control with p 0.05.

3. 706 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 164 2001
The total number of viable cells in bronchoalveolar lavage
Concentrations of tissue factor and fibrinogen in the lavage fluid were
determined using an ELISA kit (R&D Systems, Minneapolis, MN) and fluid (BALF) after instillation of all extracts was increased rel-
an immunoprecipitin assay (Diasorin), respectively. IL-8, TNF, and ative to saline (Figure 1A). The percentage of neutrophils was
IL-1 were measured using ELISA methodology (R&D Systems). increased after instillation of PM extracts from 1986 and 1988
Fibrinogen was stained immunohistologically employing lavaged
relative to that from 1987 and saline (Figure 1C).
cells after cytocentrifugation. The primary antibody (rabbit antihu-
A neutrophilic influx into the lung can frequently be associ-
man -fibrinogen antibody; Sigma) was applied at a dilution of 1:100
ated with a lung injury presumed to result, in part, from a re-
in PBS. The counterstain employed was hematoxylin.
lease of proteases and endogenous oxidants from this cell.
Oxidant generation by the three extracts was measured using thiobar-
Compared with saline controls and extract from 1987, BALF
bituric acid (TBA) reactive products of deoxyribose as an end point.
protein and albumin concentrations were greater after instilla-
Aliquots of the aqueous extracts were each agitated in 1.0 N HCl
(1.0 mg/1.0 ml) for 1 h at room temperature and centrifuged, and the tion of PM extracts from 1986 and 1988 obtained when the
supernatant was removed for analysis. Metals were individually ana- steel mill was in operation (Figures 2A and B). Elevations in
lyzed employing inductively coupled plasma emission spectroscopy concentrations of the glycoproteins fibronectin and -1-anti-
(ICPES, Model P40; Perkin Elmer, Norwalk, CT).
trypsin in BALF are also reflective of a lung injury. Elevations
Data are expressed as mean values SE. Differences were deter-
of both were significantly greater after instillations of extracts
mined using one-way analysis of variance (ANOVA) and two-way
from filters obtained during 1986 and 1988 relative to that
ANOVA (8).
from 1987 and the saline control (Figure 2C and D).
The equilibrium between mechanisms of thrombosis and fi-
RESULTS
brinolysis that normally exists in the alveolar epithelial lining
The total mass of PM sequestered on the filters selected for fluid can be disrupted in an injury (e.g., adult respiratory distress
this study was 2,770, 1,324, and 2,132 mg from 1986, 1987, and syndrome and asbestos exposures) (9, 10). A subsequent activa-
1988, whereas the soluble component recovered accounted for tion of coagulation pathways with fibrin formation in the lung
429, 343, and 422 mg, respectively. The levels of ambient PM can amplify an inflammatory response by providing a chemotac-
in the Utah Valley were not notably different from those years tic signal for neutrophils. Staining of alveolar macrophages recov-
immediately preceding the study. ered in BALF for fibrinogen demonstrated an increased binding
Figure 3A, B, C, and D. Activation of coagulation in the lungs of healthy volunteers exposed to filter extracts. Immunohistochemistry demonstrates
an increased concentration of fibrinogen in those cells collected from subjects instilled with a PM extract (1986) (B) relative to those exposed to sa-
line (A). Tissue factor and fibrinogen were both decreased in those subjects instilled with extracts collected while the steel mill was in operation
(1986 and 1988) relative to saline. Asterisks denote a significant difference in the post-hoc test (Scheffe’s) relative to the saline control with p
0.05.

4. 707
Ghio and Devlin: Inflammation after Exposure to Pollution Particles
Figures 4A, B, and C. Concentrations of proinflamma-
tory mediators in the BALF of healthy volunteers ex-
posed to PM extracts. Concentrations of the cytokines
were elevated after exposure to extracts obtained
from Utah Valley while the mill was in operation (1986
and 1988) relative to saline instillation. Asterisks de-
note significant difference in the post-hoc test (Scheffe’s)
relative to the saline control with p 0.05.
of the antibody after exposure to an extract relative to saline tion (13). The in vitro oxidant generation by each PM extract
(Figure 3A and B). There were also decreases in the concentra- (500 g) was measured as the thiobarbituric acid (TBA) reac-
tions of tissue factor and fibrinogen in the BALF after instillation tive products of deoxyribose. Extracts from 1986 and 1988
of extracts from 1986 and 1988 relative to both 1987 and saline produced a significantly greater oxidative stress compared with
(Figure 3C and D). This decrease likely reflects a deposition of fi- extract from 1987 and saline (Figure 5). DMTU diminished
brinogen within the alveolar compartment after exposure to the the absorbance of TBA-reactive products while the metal che-
extracts with clearance through binding by the macrophages (11). lator deferoxamine inhibited all oxidant generation by the
An influx of neutrophils into a tissue can be directed by numer- three extracts, suggesting that metals included in the PM could
ous chemotactic mediators. Interleukin (IL)-8, tumor necrosis fac- participate in the catalysis of reactive oxygen intermediates
tor (TNF), and IL-1 are among those cytokines with such a ca-
pacity and their in vitro expression by respiratory cells can be
effected by particle exposure (12). Concentrations of IL-8, TNF,
and IL-1 in BALF were significantly elevated in those subjects in-
stilled with extracts from particles collected while the mill was op-
erating relative to that acquired during shutdown (Figures 4A–C).
Exposures were repeated in a smaller number of volun-
teers (n 2/filter extract) but at a lower mass (100 g in-
stilled). Lavage collected 24 h later again demonstrated an in-
flammatory injury after instillation of that extract collected
while the steel mill was functioning (Table 1).
An inflammatory response can be coordinated through an
oxidant-sensitive activation of specific transcription factors.
These factors influence cytokine and other mediator expres-
sion by binding promoter sites and affecting mRNA transcrip-
Figure 5. Thiobarbituric (TBA) reactive products of deoxyribose after
TABLE 1. NEUTROPHILS, PROTEIN, AND IL-8 IN LAVAGE FLUID
exposure to PM extracts. The absorbance of oxidized products of
AFTER INSTILLATION OF LOWER MASS (100 g) OF EXTRACT
deoxyribose was significantly greater after incubation with extracts
1986 1987 1988 collected while the steel mill was functioning (1986 and 1988). The
absorbance of oxidized products after incubations with all three ex-
Neutrophils, % 37.0 6.6 15.7 2.7 31.5 6.2
tracts was significantly greater than that demonstrated by saline. Both
Protein, g/ml 175 29 76 14 153 32
dimethylthiourea (DMTU) and deferoxamine diminished the absor-
IL-8, pg/ml 56.5 17.4 18.8 10.1 61.0 10.5
bance of oxidized products of TBA.

5. 708 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 164 2001
lar and tissue exposure to metal-catalyzed oxidants can promote
TABLE 2. METAL CONCENTRATIONS IN THE THREE PM EXTRACTS
FROM UTAH VALLEY (NG METAL/MG EXTRACT) activation of both cell signaling and transcription factors, which
influence release of inflammatory mediators. The product of this
1986 1987 1988
cascade of reactions is an inflammatory injury.
Iron 82.2 14.8 257.5
The possibility that the disparate effects of PM extracts ob-
Copper 402.8 29.1 471.8
served in this study resulted from physical or chemical differ-
Zinc 1276.5 20.2 690.2
ences other than metal content and catalyzed oxidants cannot
Lead 186.6 5.7 286.7
be excluded. Because a soluble extract was studied and not the
Nickel 17.6 3.8 11.0
original particles themselves, the influence of size, shape, and
Vanadium 6.0 7.4 37.7
insoluble components in modulating health effects in the Utah
Valley remain unknown.
This investigation took advantage of an unusual situation to
through their support of electron transport. Subsequently, metals
demonstrate for the first time a correlation between findings of
in the PM extracts were quantified. Concentrations were in-
previous epidemiologic studies and the biologic effects of PM in
creased in the PM extracts from 1986 and 1988 obtained while
humans. In addition, the results of this study indicate that equal
the steel mill was in operation relative to values from 1987
masses of PM can induce disparate lung injuries, suggesting that
procured during its closure (Table 2).
particle components may be relevant in assessing health effects
after their exposure. Specifically, metals can participate in the
DISCUSSION
biologic effects of PM collected from the Utah Valley.
Volunteers instilled with aqueous extracts of PM filters while
Acknowledgment : The authors want to thank Judy Richards, Jacqueline
the steel mill was open had significant increases in both lung Carter, Jacqueline Stonehuerner, Lisa Dailey, Rob Silbajoris, and Joleen
inflammation and injury in contrast to those volunteers in- Soukup for technical assistance and Mary Ann Bassett, Debbie Levin, Linda
stilled with an equal mass of PM extract from filters collected Laetz, and Susan Derrenbacher for both facilitating bronchoscopies and
providing medical care to the volunteers while in the EPA Medical Station.
while the mill was closed. The increased biologic effect of the
PM extract collected while the mill was open is analogous to
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