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Am. J. Respir. Cell Mol. Biol. Vol. 20, pp. 631–642, 1999
Internet address: www.atsjournals.org
Cellular and Biochemical Response of the Human Lung after
Intrapulmonary Instillation of Ferric Oxide Particles
John C. Lay, William D. Bennett, Andrew J. Ghio, Philip A. Bromberg, Daniel L. Costa,
Chong S. Kim, Hillel S. Koren, and Robert B. Devlin
Center for Environmental Medicine and Lung Biology, University of North Carolina; Clinical Research Branch, Human
Studies Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency,
Chapel Hill; and Pulmonary Toxicology Branch, Experimental Toxicology Division, National Health and Environmental
Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Bronchoalveolar lavage (BAL) was used to sample lung cells and biochemical components in the lung air
spaces at various times from 1 to 91 d after intrapulmonary instillation of 2.6
m
m-diameter iron oxide par-
ticles in human subjects. The instillation of particles induced transient acute inflammation during the first
day post instillation (PI), characterized by increased numbers of neutrophils and alveolar macrophages as
well as increased amounts of protein, lactate dehydrogenase, and interleukin-8 in BAL fluids. This re-
sponse was subclinical and was resolved within 4 d PI. A similar dose-dependent response was seen in rats
1 d after intratracheal instillation of the same particles. The particles contained small amounts of soluble
iron (240 ng/mg) and possessed the capacity to catalyze oxidant generation
in vitro
. Our findings indicate
that the acute inflammation after particle exposure may, at least partially, be the result of oxidant genera-
tion catalyzed by the presence of residual amounts of ferric ion, ferric hydroxides, or oxyhydroxides asso-
ciated with the particles. These findings may have relevance to the acute health effects associated with in-
creased levels of ambient particulate air pollutants.
Lay, J. C., W. D. Bennett, A. J. Ghio, P. A.
Bromberg, D. L. Costa, C. S. Kim, H. S. Koren, and R. B. Devlin. 1999. Cellular and biochemical re-
sponse of the human lung after intrapulmonary instillation of ferric oxide particles. Am. J. Respir.
Cell Mol. Biol. 20:631–642.
Epidemiologic studies have shown consistently that a
modest rise in ambient air particle mass concentration,
measured either as total suspended particulate mass or as
the mass of particles
<
10
m
m mass median aerodynamic
diameter (PM10), is associated with exacerbation of respi-
ratory disease (1–3) and excess human mortality (4–6). In-
creased morbidity associated with particulate air pollution
is documented by increased numbers of hospital admis-
sions for respiratory tract disease (3), increased absences
of schoolchildren (7), and increased reporting of respira-
tory symptoms by persons keeping a daily diary of respira-
tory symptoms (1). These epidemiologic studies demon-
strate a positive association between elevations in ambient
particle concentrations and excess mortality (6, 8). At least
one study (6) suggests that it is fine particles (
<
2.5
m
m),
small enough to be deposited in the alveolar region, that
are probably responsible for these adverse health effects.
People most at risk of death are those with chronic car-
diopulmonary disease (especially smokers) or lung cancer
(6, 9).
The mechanism(s) of lung injury after exposure to air
pollution particles is not known. Injury has been postu-
lated to be mediated by ultrafine particles (10), biologic
agents (e.g., endotoxin) (11), acid aerosols (12), and poly-
aromatic hydrocarbons (13). Oxidant generation catalyzed
by metals associated with particles could also mediate lung
injury following exposure to particulate air pollutants. The
in vitro
generation of oxygen-derived free radicals by met-
als included in both emission source and ambient air pollu-
(
Received in original form March 4, 1998 and in revised form August 3, 1998
)
Disclaimer:
Although the research described in this article has been sup-
ported by the United States Environmental Protection Agency through
Cooperative Agreement CR824915 to the University of North Carolina
Center for Environmental Medicine and Lung Biology, it has not been
subjected to Agency review and therefore does not necessarily reflect the
views of the Agency and no official endorsement should be inferred. Men-
tion of trade names or commercial products does not constitute endorse-
ment or recommendation for use.
Address correspondence to:
John C. Lay, D.V.M., Ph.D., University of N.
Carolina, Ctr. for Environmental Medicine and Lung Biology, CB# 7310,
US EPA Human Studies Facility, Chapel Hill, NC 27599-7310.
Abbreviations:
alveolar macrophage(s), AM(s); bronchoalveolar lavage,
BAL; BAL fluid, BALF; ferric oxide, Fe
2
O
3
; ferric ion, Fe
3
1
; ferric chlo-
ride, FeCl
3
; Hanks’ balanced salt solution, HBSS; interleukin, IL; Limulus
amebocyte lysate, LAL; lactate dehydrogenase, LDH; leukotriene, LT;
superoxide anion, ; prostaglandin E
2
, PGE
2
; post instillation, PI; reac-
tive oxygen species, ROS; sterile physiologic saline solution, SPSS; thio-
barbituric acid, TBA.
O2
2
632
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999
tion particles has been documented (14–16). Iron is the
metal found in highest concentration among particulate
air pollutants studied, frequently in concentrations sever-
alfold greater than all others (17, 18).
We have previously reported our findings on the reten-
tion and clearance of particles in alveolar macrophages
(AMs) following intrapulmonary instillation of relatively
insoluble 2.6
m
m–diameter ferric oxide (Fe
2
O
3
) particles in
humans (19). Because we used bronchoalveolar lavage
(BAL) to monitor particle retention in the alveoli and air-
ways, we could also assess the lung’s response to the parti-
cles by quantifying changes in cell numbers and soluble
mediators of inflammation in BAL fluid (BALF). This re-
port examines cellular and biochemical changes associated
with a transient pulmonary inflammatory response in-
duced by the Fe
2
O
3
particles. To assess the effect of dose
(number of particles instilled per surface area of lung), we
also examined BALF after intratracheal instillation of
comparable doses of Fe
2
O
3
particles in rats. The Fe
2
O
3
particles used in this study were also compared with com-
mercially available Fe
2
O
3
particles for their capacity to
catalyze oxidant generation
in vitro
and to promote in-
flammation following intratracheal instillation in rats. Our
findings indicate that intrapulmonary deposition of these
particles induces transient mild acute inflammation that
may, at least partially, be the result of oxidant generation
catalyzed by the presence of residual amounts of ferric ion
(Fe
3
1
) or ferric oxyhydroxides associated with the parti-
cles. These findings may have relevance to the acute
health effects caused by exposure to ambient air particu-
late matter.
Materials and Methods
Study Population
The study population is the same as that reported previ-
ously (19), except that four additional subjects are in-
cluded in the group studied at 1 d post instillation (PI), giv-
ing a total of 34 healthy, nonsmoking volunteers (27 male,
7 female), 19.6 to 35.5 yr of age (mean age
5
25.8
6
4.3
yr). Potential subjects were excluded from participation if
they had a history of smoking, asthma, allergy, cardiac dis-
ease, chronic respiratory disease, recent acute respiratory
illness, or extensive exposure to pollutants. All potential
subjects underwent screening procedures, including com-
pletion of the Minnesota Multiphasic Personality Inven-
tory and medical history form, physical examination, chest
radiographs, pulmonary function tests, and routine hema-
tologic and serum chemistry tests. Subjects were randomly
assigned to groups. Subject groups were relatively matched
with regard to age except that the mean age of the group
lavaged 91 d PI (29.5
6
5.2 yr) was slightly greater than
that of the other groups. Three of the female subjects were
in the group lavaged at 1 d PI, two each were in the groups
lavaged at 4 and 91 d PI, and there were no female subjects
in the remaining groups (2 and 28 d PI). Subjects were in-
formed of the purposes of the study, the procedures of the
experiments, and the potential risk from participation, and
each subject signed a statement of informed consent. This
study was approved by the Committee on the Protection of
the Rights of Human Subjects of the University of North
Carolina School of Medicine (Chapel Hill, NC).
Study Design
Subjects were randomly assigned to one of five groups of
six subjects each (except that the group studied 1 d after
exposure included 10 subjects). Each subject underwent
two bronchoscopy procedures. During the first bronchos-
copy, Fe
2
O
3
microspheres suspended in nonpyrogenic,
sterile physiologic saline solution (SPSS) (Baxter Health-
care Products, Deerfield, IL) were instilled into an iden-
tified subsegment of the lingula that could readily be
wedged by advancing the bronchoscope. As a control,
SPSS (without particles) was instilled into a segment (me-
dial or lateral) of the right middle lobe. Subsequently, dur-
ing the second bronchoscopy procedure, particles, cells,
and soluble materials were recovered from the instillation
sites by BAL at a specified time PI (1, 2, 4, 28, or 91 d).
Each subject was thus lavaged only once, and none under-
went serial lavages.
Choice of Particles
Our choice of Fe
2
O
3
for studying retention and clearance
of insoluble particles in the lavagable AM compartment
was based on evidence that Fe
2
O
3
is nontoxic, noncarcino-
genic, and nonfibrogenic (20). Fe
2
O
3
has been used previ-
ously by many investigators for studying mucociliary clear-
ance in humans following inhalation of radiolabeled
particles (21–25). It also has been used as a control particle
for comparisons of the inhalation toxicity of different dust
particles (26) and has been used extensively as a carrier
particle for instillation studies (27–30) of the carcinogenic-
ity of polyaromatic hydrocarbons. Control groups given
Fe
2
O
3
alone showed no changes in longevity and no evi-
dence of carcinogenicity, chronic inflammation, or fibrosis
(26, 29, 30). In one study of hamsters (30), intratracheal in-
stillation of 50 mg Fe
2
O
3
resulted in no long-term adverse
health effects. Based on the amount of material per unit of
alveolar surface area (mg Fe
2
O
3
/m
2
), this dose in hamsters
is approximately 77 times the dose instilled in our human
subjects.
Particle Generation
Particles were generated from colloidal Fe
2
O
3
made via
the hydrolysis and hot dialysis of ferric chloride (FeCl
3
)
(31). Spherical Fe
2
O
3
particles (nonradioactive, 2.6
m
m
count median diameter,
s
g
5
1.3) (Figure 1) were gener-
ated in multiple batches as previously described (19). Just
before instillation, concentrated particles were suspended
in 2 to 3 ml SPSS and placed in an ultrasonic bath for 30
min. Particles were examined and counted in a hemacy-
tometer to assure the dispersion of clumps and to quantify
particle numbers. Finally, 3
3
10
8
particles were suspended
in 10 ml SPSS and transferred to a sterile syringe for instil-
lation. Some portion of these particles is lost to the syringe
and tubing during the instillation process (
see below
).
Sterilization and Testing for Endotoxin
Initial particle batches were sterilized by autoclaving at
121
8
C for 30 min. Later batches were sterilized by baking
the particles at 250
8
C for 3.5 h, which also insured the de-
Lay, Bennett, Ghio,
et al.
: Lung Cellular and Biochemical Response to Instilled Particles 633
struction of any endotoxin activity that might be present in
the particle suspension. Batches of all the particle suspen-
sions were tested for endotoxin activity using a gelation-
capillary method (Endotect; ICN Biomedical, Costa Mesa,
CA) or using a semiquantitative method (performed by
UNC Tissue Culture Facility), both of which are based on
the Limulus amebocyte lysate (LAL) assay. The capillary
LAL method detects endotoxin concentrations as low as
0.06 to 0.10 ng/ml and provides only a positive or negative
indication of the presence of endotoxin. The semiquantita-
tive LAL method is equally sensitive and provides an indi-
cation of the actual concentration of endotoxin present.
All particle suspensions tested by the capillary method
were negative. Particle suspensions tested by the semi-
quantitative LAL method were
<
0.06 endotoxin units/ml
(1 EU
5
0.1 ng endotoxin).
Particle Instillation and BAL
Bronchoscopy and BAL were performed as previously de-
scribed (32). Before bronchoscopy, all subjects were pre-
medicated intravenously with 0.6 mg atropine. The poste-
rior pharynx was anesthetized by gargling with a saline
solution containing 4% lidocaine, and the nasal passage
was anesthetized with a lubricating jelly containing 2%
lidocaine. The larynx, trachea, and bronchi were anesthe-
tized with topical 2% lidocaine instilled through a fiberop-
tic bronchoscope (Olympus BF, type 1T20D; Olympus,
Lake Success, NY) to control coughing.
To instill the particles into the distal airways and alve-
oli, the bronchoscope was passed to an identified subseg-
mental bronchus of the lingula but was not wedged. A
sterile Teflon catheter was passed through the biopsy
channel and then extended 4 to 5 cm beyond the tip of the
bronchoscope into a subsegment of the lingula. Subjects
were instructed to take deep, slow, regular breaths. A total
of 10 ml SPSS containing 3
3
10
8
Fe
2
O
3
microspheres was
slowly instilled through the catheter coincident with inspi-
rations to maximize aspiration of particles into the alveo-
lar region. This was followed by an additional 10 ml SPSS
from a different syringe (for a total of 20 ml) with the in-
tent of washing particles remaining in airways into the al-
veoli. A total of 20 ml SPSS (without particles) was in-
stilled, as described, into the medial segment of the right
middle lung lobe to serve as a control. To assess the num-
ber of particles that were lost to the syringe and catheter
during the instillation process, simulated instillations were
performed
in vitro
by injecting particle suspensions through
the catheter into a glass vial and counting the particles de-
posited in the vial. On the basis of these simulations, al-
most one-third of the particles (31.4
6
3.8%) were lost to
the syringe and catheter, so the actual number of particles
instilled into the lung is estimated to be about 2.1
3
10
8
particles (
<
5 mg Fe
2
O
3
particles).
Segmental BAL was performed at a specific interval PI
in the same lingular subsegment in which Fe
2
O
3
(or saline)
was previously instilled. The lavage of each segment com-
prised six washes using a total of 270 ml SPSS per segment.
The first washing was done with only 20 ml SPSS and was
considered to be enriched with materials from the bron-
chial airways (33). BALF from the first wash (bronchial
fraction) was kept separate from five subsequent washings
of 50 ml each. The control segment was lavaged in a simi-
lar manner.
Cell Preparation
BALF and cells were processed as previously described
(32). BALF was centrifuged at 250
3
g
for 10 min. The cell
pellets from the five 50-ml washes (alveolar fraction) were
combined. Supernatants from the first two 50-ml washes
were combined for analysis and the supernatants from the
remaining washes were discarded. Some of the fluid was
used immediately for biochemical assays, and the remain-
der was frozen at
2
70
8
C for additional assays to be per-
formed later. Cells from both the bronchial and alveolar
fractions were washed twice with RPMI 1640 (Sigma Chem-
ical Co., St. Louis, MO) and used immediately for phago-
cytic assays and superoxide anion ( ) assays.
Total cell counts were obtained by light microscopy us-
ing a hemacytometer. Cell viability was determined by ex-
clusion of Trypan blue dye. Differential cell counts were
obtained using slides prepared in a cytocentrifuge (Cytospin
3; Shandon, Inc., Pittsburgh, PA) at 500 rpm for 3 min.
Slides were stained with a modified Wright’s stain (Leuko-
stat Stain; Fisher Scientific, Fairlawn, NJ), and at least 300
cells were counted and evaluated.
Biochemical Assays
Lactate dehydrogenase (LDH) was measured using a kit
purchased from Sigma Chemical Co. The kit was modified
to allow 0.1 ml BALF to be assayed using a more concen-
trated substrate solution in a microtiter plate reader. LDH
activity was calculated as milliunits of LDH per milliliter.
produced by lavaged cells (from alveolar wash only)
was quantified by measuring the kinetics of ferricyto-
chrome-C reduction as described previously (32, 34). Briefly,
O2
2
O2
2
Figure 1. The particles used for instillation in human subjects
were smooth and spherical with a count median diameter of 2.6
mm and geometric standard deviation (sg) of 1.3. The particles
were generated from colloidal Fe2O3 made from the hydrolysis o
f
FeCl3. Bar in the top margin of the photograph 5 10 mm.
634
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999
cells (10
5
viable AMs) were incubated in Hanks’ balanced
salt solution (HBSS) in 96-well polypropylene microtiter
plates with and without stimulation by phorbol 12-myristate
13-acetate (PMA; Sigma) in the presence of 80
m
g of ferri-
cytochrome-C at 37
8
C for 40 min. Optical density (550 nm)
was read at intervals during the incubation, and activ-
ity was calculated as nanomoles per 100,000 cells per
hour. At 1 d PI, and to a lesser degree at 2 d PI, the BAL
cell mixture from particle-instilled lung segments contained
contaminating neutrophils in addition to the previously
stated number of AMs.
Biochemical assays were performed on BALF as de-
scribed previously (35). Total protein was assayed colori-
metrically using a COBAS FARA II integrated centrifu-
gal analyzer (Roche Diagnostic Systems, Branchburg, NJ)
and commercially available reagents (Coomassie blue;
Sigma). Interleukin (IL)-6 and IL-8 were quantified using
commercially available enzyme-linked immunosorbent as-
say (ELISA) kits (Quantikine immunoassay kits; R&D
Systems, Minneapolis, MN). Fibronectin and
a
1-antitryp-
sin levels were quantified with competitive ELISA assays
using antibodies and antigens purchased from Calbio-
chem, Inc. (La Jolla, CA).
Leukotriene C
4
(LTC
4
), LTD
4
, and LTE
4
were assayed
by radioimmunoassay (RIA) using a commercially avail-
able kit from Amersham (tritiated tracer, cross-reactivity:
100% with LTC
4
and LTD
4
, 41% with LTE
4
; Arlington
Heights, IL). Prostaglandin E
2
(PGE
2
) was assayed by RIA
(
125
I tracer, 100% cross-reactivity with PGE
2
, 3.7% cross-re-
activity with PGE
1
) using a commercially available kit from
New England Nuclear Research Products (Boston, MA).
Both of these arachidonic acid metabolites were measured
in lavage fluids after first being extracted and concentrated
(5
3
for bronchial wash, 10
3
for alveolar wash) on Sep-
Pack C
18
cartridges (Waters Associates, Milford, MA) (36).
Phagocytic activity of lavaged AMs (from alveolar frac-
tion only) was assayed by measuring uptake of
Candida al-
bicans
organisms (37).
C. albicans
(1
3
10
6
), labeled with
fluorescein isothiocyanate (35), were incubated (1:1 ratio)
with 1
3 106 AM in suspension in 1 ml RPMI 1640 tissue
culture medium (Sigma) at 378C for 60 min, with intermit-
tent shaking. Phagocytosis was stopped with the addition
of 3.5 ml ice-cold HBSS. The suspension was centrifuged
at 250 3 g for 10 min, and the cell pellet was suspended in
1 ml cold HBSS. Fluorescence associated with the cells
and C. albicans was measured using an Epics Profile II Flow
Cytometer (Coulter Corp., Hialeah, FL). AM were differ-
entiated from neutrophils and other cells on the basis of
their respective flow-cytometric properties. The percent-
age of AMs containing one or more fluorescent C. albicans
organism (phagocytic index) was determined as the percent-
age of AM with associated fluorescence following quenching
of extracellular fluorescence by addition of Trypan blue
dye to the cell suspension. The average number of C. albi-
cans organisms per phagocytic AM was calculated by di-
viding the average fluorescence per phagocytic AM by the
average fluorescence associated with C. albicans organisms.
Particle Instillation and BAL in Rats
Two different ancillary studies were performed in rats to
assess the capacity of the particles to induce inflammation
O2
2
O2
2
in the lungs of rats. In the first study, Fe2O3 particles were
instilled intratracheally to assess the effect of particle dose
on induction of inflammation when the doses (Fe2O3/m2
lung surface) were adjusted to compare to that used in hu-
mans. In the second study, the Fe2O3 particles used for in-
stillation in humans were compared with two commercial
Fe2O3 particles for their capacity to induce inflammation
in rat lung.
Study 1. Fe2O3 particles were instilled intratracheally
in a volume of 0.5 ml SPSS in halothane-anesthetized Fis-
cher 344 male rats (38) (250 g, 90 d old) assigned to four
groups of five rats each (low-, medium-, and high-dosage
levels and saline control group). The medium dose of par-
ticles instilled in the rats (77 3 106 particles) was calcu-
lated to approximate the dose instilled in the human sub-
jects (1.88 3 108 particles/m2), on the basis of estimated
alveolar surface area as indicated in Table 1. The estima-
tion of alveolar surface area was based on published allo-
metric comparisons of mammalian species (39), assuming
that the volume (and surface area) of the lingular subseg-
ment was 1/64 that of the total human lung.
At 24 h after instillation of particles, the rats were anes-
thetized with halothane and killed by exsanguination via
the abdominal aorta. The lungs were lavaged with SPSS
(29 ml/kg body weight) via an intratracheal cannula fixed
in place with a ligature using a syringe attached to the can-
nula. BALF and cells were then stored on ice until ana-
lyzed. Total cell counts were performed immediately using
an automated cell counter (Coulter). The BALF and cells
were then centrifuged at 250 3 g for 10 min and the super-
natant was decanted from the cell pellet. Total protein and
LDH activity in the supernatant were measured using the
COBAS FARA II integrated centrifugal analyzer with
commercially available reagents. Cytological slides were
prepared, and differential cell counts were performed as
previously described.
Study 2. The Fe2O3 particles used for instillation in hu-
mans were compared with two commercial Fe2O3 products
(Sigma; and Alfa Chemical Co., Ward Hill, MA) having
particle sizes of 0.2 mm diameter and 1 to 5 mm diameter,
respectively. Sixteen male Fischer 344 rats were divided
into four groups of four each. Rats in three of the groups
had 1 mg of particles from each of the three sources in-
stilled intratracheally in 0.5 ml sterile saline as described
previously. The control group received only sterile saline
without particles. At 24 h after instillation, the rats were
killed and their lungs lavaged, and total protein concentra-
TABLE 1
Dosage of intratracheally instilled Fe2O3 particles in rats
Group
Total
Particles
(106)
Particles/m2
Alveolar
Surface Area*
(108)
Total Mass
of Particles
(mg)
mg/m2
of Alveolar
Surface
Area
High 231 5.63 4.83 11.78
Medium 77 1.88 1.61 3.93
Low 7.7 0.188 0.16 0.39
Control 0 0 0 0
*Alveolar surface area estimated to be 0.41 m2 for a 250-g rat based on Ref.
39.
Lay, Bennett, Ghio, et al.: Lung Cellular and Biochemical Response to Instilled Particles 635
tion and percentage of neutrophils in the lavagate were
quantified as described previously.
Assessment of Solubility and Oxidant-Generating
Capacity of Particles
Soluble iron associated with the various Fe2O3 particles
was assessed using inductively coupled plasma emission
spectroscopy to measure iron extracted in distilled, deion-
ized water as previously described (14).
Oxidant generation catalyzed by particles used in this
study was compared with commercially available ferric ox-
ides using thiobarbituric acid (TBA)–reactive products of
deoxyribose. The pentose sugar 2-deoxy-D-ribose reacts
with oxidants to yield a mixture of products. On heating
with TBA at a low pH, these products form a pink chro-
mophore that can be measured by its absorbance at 532
nm. This chromophore is indistinguishable from a TBA–
malondialdehyde adduct. The reaction mixture containing
1.0 mM deoxyribose, 1.0 mM H2O2, 1.0 mM ascorbate, and
200 mg of either (1) the Fe2O3 particles used for instilla-
tion, (2) iron (III) oxide from Sigma, or (3) iron (III) oxide
from Alfa was incubated in saline at 378C for 60 min with
agitation and then centrifuged at 1,200 3 g for 10 min.
One milliliter each of 1.0% (wt/vol) TBA and 2.8% (wt/
vol) trichloroacetic acid were added to 1.0 ml of superna-
tant, heated at 1008C for 10 min, and cooled in ice, and the
chromophore concentration was determined in triplicate
specimens by its absorbance at 532 nm.
Statistical Evaluations
All values are expressed as means 6 standard error. Dif-
ferences between particle-instilled versus control seg-
ments and bronchial versus alveolar fractions for various
parameters were analyzed using Student’s t test for paired
samples (40). Comparisons of values for the various pa-
TABLE 2
Cell counts in BALF of individual subjects at
1 d PI: alveolar fraction
Neutrophils Macrophages
Subject Saline
(106 cells)Particles
(106 cells)Saline
(106 cells)Particles
(106 cells)
1 0.30 0.49 43.10 35.40
2 0.22 237.00 31.40 173.00
3 1.16 1.71 26.90 39.00
4 0.14 252.00 20.60 174.00
5 0.47 56.60 16.10 103.00
6 0.44 13.10 23.90 55.70
7 1.06 5.78 14.80 15.60
8 0.30 31.70 10.20 26.30
9 0.56 3.51 13.80 20.10
10 0.48 2.26 18.00 16.30
Mean 0.51 60.40 21.90 77.80
Standard error 0.11 31.20 3.10 22.90
TABLE 3
Total and differential cell counts: alveolar fraction*
Days PI
Total
Cells
(
3
107)AMs
(%)Lymphocytes
(%)
Polymorphonuclear
Leukocytes
(Neutrophils)
(%)Eosinophils
(%)Epithelial Cells
(%)
Saline
1 2.37 91.83 5.20 2.50 0.30 0.23
(0.32) (1.16) (0.80) (0.54) (0.10) (0.14)
2 2.93 94.20 4.80 0.80 0.27 0.00
(0.81) (1.28) (1.20) (0.39) (0.12) (0.00)
4 2.76 93.11 5.94 0.72 0.00 0.33
(0.51) (1.46) (1.33) (0.29) (0.00) (0.21)
28 2.22 91.80 6.20 1.53 0.07 0.47
(0.17) (2.34) (2.34) (1.04) (0.07) (0.33)
91 1.62 92.06 4.83 0.83 0.28 0.78
(0.21) (1.77) (1.69) (0.22) (0.10) (0.59)
Particles
1 13.20†67.68†4.80 27.08†0.30 0.13
(5.22) (6.69) (0.82) (6.69) (0.09) (0.07)
2 2.98 85.94 6.06 6.89 0.89 0.33
(0.78) (3.62) (0.71) (3.52) (0.32) (0.17)
4 3.15 91.50 7.00 0.89 0.33 0.39
(0.48) (0.51) (0.63) (0.27) (0.15) (0.22)
28 3.16 93.33 4.39 2.22 0.00 0.11
(0.34) (1.39) (1.31) (1.13) (0.00) (0.11)
91 2.29 93.22 5.89 0.72 0.22 0.11
(0.41) (2.62) (2.40) (0.23) (0.22) (0.07)
* Numbers in parentheses represent standard error.
† Significantly different from saline control values (P , 0.05).
636 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999
rameters at different times after instillation were assessed
by analysis of variance (ANOVA) and Scheffe’s post hoc
test. A P value of 0.05 was chosen as the level of signifi-
cance. Comparisons of results from the rat studies also
were performed using either ANOVA or t tests, whichever
was appropriate for a particular comparison.
Results
Cell Counts in BALF
Intrapulmonary instillation of Fe2O3 particles induced a
transient inflammatory response that was apparent in
most subjects (8 of 10) lavaged 1 d PI. Total and differen-
tial cell counts for alveolar and bronchial fractions are
listed in Tables 3 and 4, respectively. An influx of cells into
the particle-instilled segment was apparent at 1 d PI and
comprised markedly increased numbers of both AMs and
neutrophils (Figure 2). The mean number of AMs re-
turned to control levels by 2 d PI, and neutrophils were no
TABLE 4
Total and differential cell counts: bronchial fraction*
Days PI
Total
Cells
(3 105)AMs
(%)Lymphocytes
(%)
Polymorphonuclear
Leukocytes
(Neutrophils)
(%)Eosinophils
(%)Epithelial Cells
(%)
Saline
1 7.81 46.80 3.30 32.53 1.07 16.17
(1.75) (4.90) (0.71) (5.34) (0.32) (4.11)
2 20.70 73.87 4.93 14.33 1.60 5.27
(13.9) (8.55) (0.84) (6.80) (0.96) (3.39)
4 10.30 63.50 5.72 11.83 0.39 18.56
(2.91) (4.25) (0.81) (2.12) (0.16) (3.23)
28 11.92 54.07 2.07 5.67 0.33 37.80
(7.28) (2.93) (1.11) (2.03) (0.26) (5.28)
91 5.53 57.94 3.33 7.44 0.17 31.61
(6.98) (8.91) (0.86) (3.69) (0.07) (8.50)
Particles
1 21.30 32.71 3.50 47.13†1.17 15.25
(7.48) (3.22) (0.57) (5.44) (0.50) (3.73)
2 9.82 69.92 4.13 15.00 0.74 9.96
(2.80) (10.02) (0.81) (5.50) (0.13) (4.25)
4 14.30 55.17 4.72 20.06 0.89 18.89
(4.77) (3.48) (1.00) (4.89) (0.82) (3.51)
28 9.49 68.83 2.78 12.39 0.22 15.50
(1.11) (7.47) (1.06) (3.53) (0.11) (6.51)
91 8.23 49.56 1.78 8.72 0.28 39.44
(1.32) (8.64) (0.57) (2.93) (0.13) (6.07)
* Numbers in parentheses represent standard error.
† Significantly different from saline control values (P , 0.05).
Figure 2. The total number of cells recovered by BAL from the
alveolar region of the lingular (particle-instilled) segment was
markedly elevated at 1 d PI relative to the saline-instilled control
segment because of the influx of both neutrophils (left) and mac-
rophages (right) into the lavagable air spaces. The total numbers
of both cell types returned to near control levels by 2 d PI. In the
bronchial fraction, only neutrophils were elevated at 1 d PI. *Sig-
nificant increase relative to saline control (P , 0.05).
Lay, Bennett, Ghio, et al.: Lung Cellular and Biochemical Response to Instilled Particles 637
longer present at 4 d PI (Figure 2). A great deal of varia-
tion was observed at 1 d PI in the total number of neutro-
phils recovered in BALF (Figure 2 and Table 2). The per-
centage of neutrophils in alveolar BALF was elevated in
the lingular segment at 1 d PI (Table 3) and was still mar-
ginally elevated at 2 d PI, but was no longer elevated
above control values at 4 d PI and later. The percentage of
neutrophils in the bronchial fraction of the lingular lava-
gate also was elevated at 1 d PI but not at later times (Ta-
ble 4). As expected, the percentages of neutrophils present
in the bronchial fractions of both particle-instilled and
control segments were higher than in the alveolar fraction
throughout the 91-d study period. Neither cell viability nor
volume of BALF recovered from particle-instilled seg-
ments was different from that of control segments at any
of the sampling times.
Superoxide Generation by Lavaged Cells
generation by BALF cells from the alveolar fraction
was examined to help assess activation of AM by the in-
stilled particles. The proportional change in generation
by BAL cells following PMA stimulation appeared to be
slightly greater for cells from the particle-instilled segment
relative to the control at 1 to 4 d PI. This may at least par-
tially result from the presence of neutrophils in BALF at 1
O2
2
O2
2
and 2 d PI. These differences, however, were not statisti-
cally significant.
Phagocytosis Assays
Phagocytosis of C. albicans by AM from the alveolar frac-
tion was the other functional assay used to assess activa-
tion of AMs by the instilled particles. In nearly all in-
stances, for both particle-instilled and control segments, a
significantly greater phagocytic index and significantly
greater number of organisms per AM were measured for
opsonized than for unopsonized C. albicans. No significant
differences were detected between AMs from particle-
instilled versus control segments for either phagocytic in-
dex or the number of organisms phagocytized per AM.
These results suggest no apparent activation of AMs fol-
lowing uptake of Fe2O3 particles.
Biochemical Parameters in BALF
The results of biochemical assays of BALF from alveolar
and bronchial fractions are tabulated in Tables 5 and 6, re-
spectively. In the alveolar fraction, total protein, LDH,
and IL-8 were significantly elevated (P , 0.05) in BALF
from the particle-instilled segment relative to controls at
1 d PI. Mean levels of IL-6 and fibronectin appeared ele-
vated at 1 d PI; however, these differences were not statis-
tically significant because of large within-group variance
TABLE 5
Biochemical parameters in BALF: alveolar fraction*
Days PI
Total Protein
(
m
g/ml)LDH
(mU/ml)IL-6
(pg/ml)IL-8
(pg/ml)
Particles Saline Particles Saline Particles Saline Particles Saline
1 522.30†160.66 6.48†2.70 55.69 4.87 54.44†28.18
(285.8) (41.18) (1.38) (0.84) (30.81) (0.50) (8.59) (5.89)
2 166.55 173.36 5.31 2.07 5.53 4.68 29.13 13.72
(28.15) (60.30) (1.74) (0.45) (1.05) (0.70) (8.19) (3.47)
4 128.57 114.05 2.68 1.98 4.76 3.93 34.18 25.48
(17.61) (26.93) (0.66) (0.56) (0.50) (0.50) (10.50) (2.82)
28 246.7 201.1 2.39 2.56 3.65 4.15 26.57 37.33
(24.0) (21.1) (0.42) (0.39) (0.52) (0.64) (6.10) (14.81)
91 279.1†176.7 5.22 3.00 3.49 3.13 47.40 17.23
(48.0) (30.7) (0.87) (0.81) (0.36) (0.0) (10.15) (2.67)
PGE2
(pg/0.1 ml)LTC4/D4/E4
(pg/ml)a-1 Antitrypsin
(
m
g/ml)Fibronectin
(ng/ml)
Particles Saline Particles Saline Particles Saline Particles Saline
1 5.91 4.09 13.4 11.4 7.32 7.13 147.82 40.47
(2.03) (1.38) (3.7) (3.1) (1.47) (1.90) (67.95) (18.49)
2 7.61 4.39 8.1 8.3 17.13 14.11 61.67 35.14
(3.88) (2.76) (4.1) (2.8) (6.10) (3.56) (30.08) (16.09)
4 8.90 7.73 15.3 9.7 12.19 11.51 15.41 10.35
(2.19) (2.46) (6.5) (3.1) (5.32) (3.32) (8.90) (3.89)
28 0.13 0.20 11.2 20.1 4.51 3.35 59.52 44.53
(0.02) (0.06) (1.6) (6.8) (1.93) (1.63) (17.83) (14.59)
91 0.11 0.04 16.3 18.9 2.88 3.65 89.90 78.83
(0.05) (0.01) (3.8) (2.8) (1.08) (1.15) (18.88) (19.22)
* Numbers in parentheses represent standard error.
† Represents statistically significant elevation over saline control (paired Student’s t test, P , 0.05).
638 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999
(especially in the particle-instilled segments). Mean levels
of fibronectin, IL-8, and LDH also appeared elevated (al-
though not significantly) at 2 d PI. a1-antitrypsin was
slightly elevated in both particle-instilled and control seg-
ments at 2 and 4 d PI relative to that at 1 d PI. PGE2 ap-
peared elevated in both lingular and control segments at 1
to 4 d PI, relative to 28 and 91 d PI. In the bronchial frac-
tion, no significant differences were found between parti-
cle-instilled and control segments for any of the measured
parameters, although trends toward increased values in
particle-instilled segments were noted for some parame-
ters. The average IL-8 values tended to be higher in the
bronchial BALF of particle-instilled segments than in con-
trols at all time points, and IL-6 and PGE2 appeared to be
slightly elevated in particle-instilled segments at 1 and 2 d PI.
Dose-response study in rats. The total number of cells
recovered in BALF from particle-instilled rat lungs at 1 d
PI was markedly elevated in the two highest particle-dose
groups but was not changed in the low-dose group com-
pared with control animals. This increase in cell numbers
was due almost exclusively to the influx of neutrophils into
the lung spaces (Figure 3). The cellular influx was accom-
panied by dose-dependent elevations of total protein and
LDH in BALF from rats in the two highest particle-dose
groups (Figure 4).
Comparison with commercial Fe2O3 (rats). In compari-
son with saline alone and the two commercial Fe2O3 prod-
ucts, the particles used for instillation in the human sub-
jects induced significant elevations (P , 0.05) of neutrophil
numbers and total protein concentration in the BALF at 1
d following intratracheal instillation in rats (Figure 5).
Solubility and Oxidant-Generating Capacity
of Fe2O3 Particles
Water extraction of Fe2O3 particles generated for use in
this study demonstrated soluble iron at a concentration of
0.24 6 0.003 mg/mg of Fe2O3 sample. This translates to
0.036% of the total iron content of the particles assuming
no water of hydration associated with the Fe2O3. Soluble
iron was not detectable in the commercial Fe2O3 products.
The Fe2O3 particles used for instillation in the human sub-
jects produced significantly greater TBA absorbance at
532 nm (Figure 6) in an in vitro system, indicating signifi-
cantly greater oxidant-generating capacity than that of sa-
line or the commercial Fe2O3 samples examined. This
demonstrates that some portion of the iron was available
for electron transfer and therefore capable of participation
in Fenton-like reactions that could lead to lipid peroxida-
tion and generation of an inflammatory response.
Discussion
Our original and primary purpose for instilling particles in
the lungs of humans was to examine the retention, clear-
TABLE 6
Biochemical parameters in BALF: bronchial fraction*
Days PI
Total Protein
(
m
g/ml)LDH
(mU/ml)IL-6
(pg/ml)IL-8
(pg/ml)
Particles Saline Particles Saline Particles Saline Particles Saline
1 88.17 67.97 8.64 7.06 9.8 4.48 301.22 147.40
(21.29) (19.76) (1.92) (1.88) (4.55) (0.87) (173.14) (42.81)
2 50.10 30.70 8.88 2.74 6.10 3.45 123.00 42.75
(7.15) (6.40) (6.02) (0.90) (1.90) (0.35) (67.22) (15.75)
4 56.18 56.42 2.68 7.91 5.08 5.78 114.20 80.8
(26.58) (12.02) (0.66) (2.75) (1.48) (1.50) (76.22) (24.80)
28 122.13 89.67 5.65 4.52 4.51 3.65 74.57 42.05
(15.82) (26.29) (1.59) (1.32) (0.75) (0.52) (27.06) (9.04)
91 121.07 95.78 12.54 4.52 3.31 3.13 150.07 62.13
(23.73) (15.59) (1.55) (1.32) (0.18) (0.0) (36.52) (10.67)
PGE2
(pg/0.1 ml)LTC4/D4/E4
(pg/ml)a-1 Antitrypsin
(
m
g/ml)Fibronectin
(ng/ml)
Particles Saline Particles Saline Particles Saline Particles Saline
1 17.35 10.19 16.3 1.25 5.91 17.93 4.78 5.74
(3.73) (2.01) (15.0) (0.0) (2.40) (7.56) (1.71) (3.37)
2 25.05 7.87 1.25 22.9 7.9 10.70 4.78 0.73
(11.86) (0.03) (0.0) (21.7) (2.78) (7.00) (2.95) (0.18)
4 10.88 9.67 9.7 16.2 4.60 11.52 1.58 1.99
(3.89) (2.50) (8.4) (14.9) (2.19) (3.30) (0.87) (1.09)
28 0.17 0.17 21.2 38.0 0.84 0.78 19.43 10.93
(0.05) (0.05) (3.2) (9.0) (0.23) (0.49) (3.03) (3.86)
91 0.22 0.16 18.7 47.1 0.76 2.52 28.05 25.65
(0.14) (0.14) (2.9) (7.0) (0.10) (2.12) (3.81) (5.69)
*Numbers in parentheses represent standard error.
Lay, Bennett, Ghio, et al.: Lung Cellular and Biochemical Response to Instilled Particles 639
ance, distribution, and redistribution of inert and insoluble
particles within the lavagable AM compartment as a func-
tion of time. We have reported on the inhomogeneity of
particle distribution and disproportionate clearance of
particle-containing cells from the AM compartment fol-
lowing intrapulmonary instillation of these particles (19).
We chose Fe2O3 particles for these studies because they
are insoluble in aqueous solution at neutral pH (41) and
are nontoxic, noncarcinogenic, and nonfibrogenic (20). To
accomplish our goals in examining retention and clearance
of particles in lavaged AMs, it was necessary to instill a rel-
atively high concentration of particles into a localized re-
gion of the lung. This allowed adequate numbers of parti-
cles to be recovered by lavage for counting and evaluation
of their intracellular distribution. After making certain as-
sumptions regarding the size of the lung segment to be in-
stilled, we calculated that instillation of 3 3 108 particles
into a lingular subsegment should result in a concentration
of about 3.2 particles per AM within that region, based on
published allometric data (39). This would be true if all of
the particles reached the alveoli; it does not take into ac-
count that some particles are lost to the syringe and tubing
and that many of the particles may be cleared quickly by
mucociliary clearance. Thus, the actual number of parti-
cles reaching the alveoli is likely much lower. By our own
observations, the vast majority of lavaged AMs contained
Figure 5. Relative to saline alone and to commercial iron oxide
products purchased from Alfa Chemical Co. (A) and Sigma
Chemical Co. (B), the particles used for instillation in the human
subjects (C) induced significantly elevated neutrophil numbers
and total protein concentrations in BALF after intratracheal in-
stillation in rats. *Significant increase relative to saline alone and
to commercial iron oxides (P , 0.05).
Figure 6. Fe2O3 particles used in this study (C) had significantly
greater capacity for catalyzing the generation of ROS in vitro
than did saline controls or two commercial iron (III) oxides pur-
chased from Alfa Chemical Co. (A) and Sigma Chemical Co. (B).
*Significant increase relative to saline control and to commercial
iron oxides (P , 0.05).
Figure 3. Relative to saline controls, the high- and medium-dose
levels of the Fe2O3 particles used for instillation in human sub-
j
ects induced a marked increase in total cell numbers in the lungs
of rats 1 d after intratracheal instillation. The increase in lavaga-
ble cell numbers was due primarily to an influx of neutrophils.
*Significant increase relative to saline control (P , 0.05).
Figure 4. Total protein content and LDH activity in BALF from
rats increased in a dose-dependent manner 1 d after intratracheal
instillation of different amounts of the Fe2O3 particles used for
instillation in humans. Both parameters are significantly elevated
in the high- and medium-dose groups but are unchanged in the
low-dose group relative to saline controls. *Significant increase
relative to saline control (P , 0.05).
640 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999
no particles, and of those that had taken up particles, most
contained only one or two particles (19). Thus, it is proba-
ble that we underestimated the size of the lung compart-
ment into which the particles were instilled. Based on
these calculations and our own observations, it is unlikely
that a condition of particle “overload” (42) was achieved.
Intrapulmonary instillation of these Fe2O3 particles in-
duced an influx of both neutrophils and AMs, and eleva-
tion of LDH, total protein, and IL-8 in BALF at 1 d PI.
These changes were detectable only by BAL and were not
accompanied by overt clinical symptoms (fever, pain, cough-
ing, etc.). These changes indicate cell injury (LDH), al-
tered vascular permeability (total protein), and elabora-
tion of neutrophil chemoattractants (IL-8) by lung cells, and
are consistent with acute inflammation. Although transient
inflammation has been reported in a variety of species at 1
or 2 d after intratracheal instillation or inhalation of vari-
ous types of particles, including polystyrene (43), alumi-
num oxide (44), titanium dioxide (44, 45), carbon particles
(46), and Fe2O3 dust (47), we were nonetheless somewhat
surprised to see the marked neutrophil response to these
ostensibly inert particles at 1 d PI. In our study we did not
observe the sustained elevation in the number of lavagable
AMs that was described following instillation of carbon
particles in mice (46).
Functional differences between AMs from particle-
instilled and control lung segments were negligible as as-
sessed by differences in phagocytic capability and ability
to generate . Our inability to detect differences may
stem from the fact that only a small percentage of the AMs
actually phagocytized particles. The slightly lower phago-
cytic index measured in both groups at 1 d PI is inter-
preted to be the result of the pronounced influx of AMs,
many of which were immature cells with monocyte-like
morphology and limited capacity for phagocytosis.
A likely mechanism underlying the acute inflammatory
response to the particles used in this study is that of oxi-
dant generation and subsequent cell injury (lipid peroxida-
tion) by reactive oxygen species (ROS) (48). The particles
used in this study were generated from colloidal Fe2O3
made from the hydrolysis and hot dialysis of ferric chlo-
ride (FeCl3). This process results in the slow formation of
Fe2O3 with some portion first forming the intermediate
species ferric hydroxide (Fe[OH]3, unstable), ferrihydrite
(Fe5HO8 · 4 H2O), or ferric oxyhydroxide (FeOOH) be-
fore transformation to Fe2O3 (31, 49). We have established
that the particles instilled in the human subjects were ca-
pable of catalyzing the generation of ROS in vitro and that
a small amount of soluble iron (240 ng soluble Fe/mg par-
ticles) was associated with the particles. This is approxi-
mately 0.036% of the total iron content of the particles.
We interpret these findings to indicate incomplete conver-
sion of some small portion of the soluble Fe31 ion to sta-
ble, insoluble Fe2O3 during the hydrolysis of FeCl3. This
soluble Fe31 (14), and possibly also residual intermediate
iron oxide species (50, 51), may support electron transfer
and participate in Fenton-type chemistry resulting in oxi-
dant generation, lipid peroxidation, and inflammation.
The studies in rats demonstrated a dose-dependent re-
sponse to the particles generated from FeCl3 and also
showed that the inflammatory response was limited to the
O2
2
laboratory-made particles. Fe2O3 particles bought com-
mercially had no detectable soluble iron component, were
much less capable of oxidant generation, and did not in-
duce an inflammatory response when instilled into the
lungs of rats. These ancillary studies support our interpre-
tation that it was catalytically active iron that was respon-
sible for the inflammation seen following instillation of the
particles in the human volunteers. Although disparities in
iron species and catalysis of oxidants are associated with
the potential of the particles to induce an in vitro release
of pertinent mediators and an in vivo inflammatory re-
sponse, it is possible that size, shape, and surface charge of
the particles also contribute to these differences.
This interpretation of our results is consistent with the
hypothesis advanced previously (14–16) that transition
metals associated with airborne particles may underlie
some of the acute effects associated with particulate air
pollution. The inflammatory response following exposure
to the iron-containing particle in our study is comparable
to that seen following exposure to other metal chelates
(52). This inflammatory response is assumed to result from
an oxidant-sensitive activation of specific promoters by
the iron-containing particle. This presumably would in-
clude nuclear factor-kB (53), whose activation results in
increased release of several cytokines such as tumor necro-
sis factor and ILs (54, 55), which function as chemotactic
agents for inflammatory cells. Tissue injury subsequently
results from exposure to either (1) the free radicals cata-
lyzed by the metal reacting with critical molecules in the
lung environment, or (2) proteases and endogenous oxi-
dants generated by the recruited inflammatory cells.
Other possible mechanisms that may have contributed
to the inflammatory response include release of neutrophil
chemoattractants by AMs subsequent to the phagocytic
stimulus (56), complement activation (57), or “accidental”
(premature fusion of lysosome with phagosome) release of
lysosomal contents (hydrolases, proteases, ROS, other
products) following the sudden introduction of large num-
bers of particles and subsequent phagocytic activity. The
lack of response to the instilled commercial iron oxides
(no soluble iron) in rats in the present study suggests that
these alternate mechanisms did not play a significant role
in inducing inflammation.
We instilled 5 mg of Fe2O3 into one subsegment of the
lingula lobe of the lung, which we estimated to have a sur-
face area of about 1.6 m2. However, because only 0.036%
of the total iron content that was instilled was soluble, the
volunteers received a dose of 0.78 mg of soluble iron per
square meter of lung surface. A person breathing ambient
air containing 100 mg/m3 of fine particles would deposit
approximately 450 mg per 24 h, assuming a tidal volume of
0.5 liters, a ventilation rate of 15 breaths per min, and dep-
osition fraction of about 40% (58). Ionizable iron makes
up 1 to 2% of ambient PM material by weight and as much
as 4 to 5% of specific emission sources such as oil fly ash
(14). Thus, the 450 mg of particles retained in the lung in a
24-h period may contain as much as 4.5 to 22.5 mg soluble
iron or 0.04 to 0.22 mg/m2 lung surface, assuming 102 m2 of
alveolar surface area for the total lung (39). This suggests
that the amount of soluble iron instilled into the volun-
teers was not too dissimilar to what might be inhaled in a
Lay, Bennett, Ghio, et al.: Lung Cellular and Biochemical Response to Instilled Particles 641
heavily polluted urban area during a 24- to 48-h period.
Furthermore, patients with airways disease (e.g., chronic
obstructive pulmonary disease) have enhanced deposition
of particles in their airways (59–61), so that in terms of
dose per surface area, the deposited dose is even more
comparable to the amount instilled into the volunteers in
this study. Having made these arguments, we also recog-
nize that the route of exposure (intrapulmonary instilla-
tion) is not a normal mode of entry and that the dose of
particles instilled is unlikely to occur except in the most
polluted environment over a period of time. Thus, they
may have limited relevance to actual ambient exposures.
The mean percentage of neutrophils in BALF 24 h af-
ter instillation of particles in this study was considerably
higher than that found 1 h (35) or 18 h (32) after a 2-h ex-
posure to 0.4 ppm ozone (27% neutrophils for particles
versus about 10% for ozone). This indicates that exposure
to particles and associated soluble iron may have the po-
tential for inducing an inflammatory response in the lung
whose severity is similar to or greater than that induced by
ozone. Additional studies are required to determine what
dose of inhaled particles might be necessary to produce
such a response. Ambient air particles with relatively high
levels of soluble iron might reasonably be expected to pro-
duce a greater inflammatory response than those with a
low level of soluble iron.
Conclusions
This is the first study in humans to use bronchoscopy and
intrapulmonary instillation to assess the lung’s response to
an instilled burden of respirable-sized particles. We have
demonstrated a transient acute pulmonary inflammatory
response to Fe2O3 particles at 1 d after instillation, which is
likely due to ROS induced by residual soluble Fe31 or cat-
alytically active residual intermediate iron oxide species
associated with the particles. These findings are consistent
with the hypothesis that transition metals associated with
airborne particles underlie some of the acute effects attrib-
uted to particulate air pollution.
Acknowledgments: The authors acknowledge and thank Lisa Dailey, Jackie
Quay, Joleen Soukup, Alex Chall, Debra Levin, Maryann Bassett, Jim Leh-
mann, and John McGee, and doctors Tim Gerrity, Brian Boehlecke, Mike
Madden, Frank Biscardi, Mark Robbins, Greg Bottei, and Kirby Zeman for
their efforts or advice in performing various aspects of this work.
References
1. Schwartz, J., D. Wypij, D. W. Dockery, J. Ware, S. Zeger, J. D. Spengler,
and B. Ferris, Jr. 1991. Daily diaries of respiratory symptoms and air pollu-
tion: methodological issues and results. Environ. Health Perspect. 90:181–
187.
2. Chestnut, L. G., J. Schwartz, D. A. Savitz, and C. M. Burchfiel. 1991. Pul-
monary function and ambient particulate matter: epidemiological evi-
dence from NHANES 1. Arch. Environ. Health 46:135–144.
3. Pope, C. A., III. 1991. Respiratory hospital admissions associated with PM10
pollution in Utah, Salt Lake, and Cache Valleys. Arch. Environ. Health
46:90–97.
4. Schwartz, J., and A. Marcus. 1990. Mortality and air pollution in London: a
time series analysis. Am. J. Epidemiol. 131:185–194.
5. Pope, C. A., III, J. Schwartz, and M. R. Ransom. 1992. Daily mortality and
PM10 pollution in Utah Valley. Arch. Environ. Health 47:211–217.
6. Dockery, D. W., C. A. Pope, III, J. D. Spengler, J. H. Ware, M. E. Fay, B. G.
Ferris, Jr., and F. E. Speizer. 1993. An association between air pollution
and mortality in six U.S. cities. N. Engl. J. Med. 329:1753–1759.
7. Ransom, M. R., and C. A. Pope, III. 1992. Elementary school absences and
PM10 pollution in Utah Valley. Environ. Res. 58:204–219.
8. Schwartz, J. 1991. Particulate air pollution and daily mortality in Detroit.
Environ. Res. 56:204–213.
9. Utell, M. J., and M. W. Frampton. 1995. Particles and mortality: a clinical
perspective. Inhalation Toxicology 7:645–655.
10. Oberdörster, G., J. Ferin, and B. E. Lehnert. 1994. Correlation between
particle size, in vivo particle persistence, and lung injury. Environ. Health
Perspect. 102:173–179.
11. Anto, J. M., and J. Sunyer. 1990. Epidemiologic studies of asthma epidemics
in Barcelona. Chest 90:185s–190s.
12. Schlesinger, R. B., and J. A. Graham. 1992. Health effects of atmospheric
acid aerosols: a model problem in inhalation toxicology and air pollution
risk assessment. Fundam. Appl. Toxicol. 18:17–24.
13. Diaz-Sanchez, D., A. R. Dotson, H. Takenaka, and A. Saxon. 1994. Diesel
exhaust particles induce local IgE production in vivo and alter the pattern
of IgE messenger RNA isoforms. J. Clin. Invest. 94:1417–1425.
14. Pritchard, R. J., A. J. Ghio, J. R. Lehman, D. W. Winsett, J. S. Tepper, P.
Park, M. I. Gilmour, K. L. Dreher, and D. L. Costa. 1996. Oxidant genera-
tion and lung injury after particulate air pollutant exposure increases with
the concentrations of associated metals. Inhalation Toxicology 8:457–477.
15. Ghio, A. J., J. Stonehuerner, R. J. Pritchard, C. A. Piantadosi, D. R. Quig-
ley, K. L. Dreher, and D. L. Costa. 1996. Humic-like substances in air pol-
lution particulates correlate with concentrations of transition metals and
oxidant generation. Inhalation Toxicology 8:479–494.
16. Dreher, K. L., R. H. Jaskot, J. R. Lehmann, J. H. Richards, J. K. McGee,
A. J. Ghio, and D. L. Costa. 1997. Soluble transition metals mediate resid-
ual oil fly ash induced acute lung injury. J. Toxicol. Environ. Health 50:
285–305.
17. Jacob, D. J., and E. W. Gottlieb. 1989. Chemistry of a polluted cloudy
boundary layer. J. Geophys. Res. 94:12975–13002.
18. Munger, J. W., D. J. Jacob, J. M. Waldman, and M. R. Hoffmann. 1983.
Fogwater chemistry in an urban atmosphere. J. Geophys. Res. 88:5109–
5121.
19. Lay, J. C., W. D. Bennett, C. S. Kim, R. B. Devlin, and P. A. Bromberg.
1998. Retention and intracellular distribution of instilled iron oxide parti-
cles in human alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 18:687–
695.
20. Stokinger, H. E. 1984. A review of world literature finds iron oxides noncar-
cinogenic. Am. Ind. Hyg. Assoc. J. 45:127–133.
21. Wilkey, D. D., P. S. Lee, F. J. Hass, T. R. Gerrity, D. B. Yeates, and R. V.
Lourenco. 1980. Mucociliary clearance of deposited particles from the hu-
man lung: intra- and inter-subject reproducibility, total lung clearance, and
model comparisons. Arch. Environ. Health 35:294–303.
22. Ilowite, J. S., G. C. Smaldone, R. J. Perry, W. D. Bennett, and W. M. Foster.
1989. Relationship between tracheobronchial particle clearance rates and
sites of initial deposition in man. Arch. Environ. Health 44:267–273.
23. Lourenco, R. V., M. F. Klimek, and C. J. Borowski. 1971. Deposition and
clearance of 2m particles in the tracheobronchial tree of normal subjects—
smokers and nonsmokers. J. Clin. Invest. 50:1411–1420.
24. Morrow, P. E., F. R. Gibb, and K. M. Gazioglu. 1967. A study of particulate
clearance from the human lungs. Am. Rev. Respir. Dis. 96:1209–1221.
25. Bennett, W. D., W. F. Chapman, J. C. Lay, and T. R. Gerrity. 1993. Pulmo-
nary clearance of inhaled particles 24 to 48 hours post deposition: effect of
beta-adrenergic stimulation. J. Aerosol Med. 6:53–62.
26. Wright, J. L., N. Harrison, B. Wiggs, and A. Churg. 1989. Quartz but not
iron oxide causes air-flow obstruction, emphysema, and small airways le-
sions in the rat. Am. Rev. Respir. Dis. 138:129–135.
27. Saffiotti, U., F. Cefis, and L. H. Kolb. 1968. A method for the experimental
induction of bronchogenic carcinoma. Cancer Res. 28:104–124.
28. Stenbäck, F., J. Rowland, and A. Sellakumar. 1976. Carcinogenicity of
benzo(a)pyrene and dusts in the hamster lung (instilled intratracheally
with titanium oxide, aluminum oxide, carbon and ferric oxide). Oncology
33:29–34.
29. Schreiber, H., D. H. Martin, and N. Pazmino. 1975. Species differences in
the effect of benzo(a)pyrene-ferric oxide on the respiratory tract of rats
and hamsters. Cancer Res. 35:1654–1661.
30. Saffiotti, U., R. Montesano, A. R. Selladumar, F. Cefis, and D. G. Kaufman.
1972. Respiratory tract carcinogenesis in hamsters induced by different
numbers of administrations of benzo(a)pyrene and ferric oxide. Cancer
Res. 32:1073–1081.
31. Neidle, M., and J. Barab. 1917. Studies in dialysis: II. The hot dialysis of the
chlorides of ferric iron, chromium, and aluminum, and the rapid prepara-
tion of their colloidal hydrous oxides. J. Am. Chem. Doc. 39:71–81.
32. Koren, H. S., R. B. Devlin, D. E. Graham, R. Mann, M. P. McGee, D. H.
Horstman, W. J. Kozumbo, S. Becker, D. House, W. F. McDonnell, and
P. A. Bromberg. 1989. Ozone-induced inflammation in the lower airways
of human subjects. Am. Rev. Respir. Dis. 139:407–415.
33. Rennard, S. I., M. Ghafouri, A. B. Thompson, J. Linder, W. Vaughan, K.
Jones, R. F. Ertl, K. Christensen, A. Prince, M. Stahl, and R. A. Robbins.
1990. Fractional processing of sequential bronchoalveolar lavage to sepa-
rate bronchial and alveolar samples. Am. Rev. Respir. Dis. 141:208–217.
34. Pick, E., and Y. Keisari. 1981. Superoxide anion and hydrogen peroxide
production by chemically elicited peritoneal macrophages—induction by
multiple nonphagocytic stimuli. Cell Immunol. 59:301–318.
35. Devlin, R. B., W. F. McDonnell, S. Becker, M. C. Madden, M. P. McGee, R.
Perez, G. Hatch, D. E. House, and H. S. Koren. 1996. Time-dependent
642 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 20 1999
changes of inflammatory mediators in the lungs of humans exposed to 0.4
ppm ozone for 2 hours: a comparison of mediators found in bronchoalveo-
lar lavage fluid 1 and 18 hours after exposure. Toxicol. Appl. Pharmacol.
138:176–185.
36. Matthay, M. A., W. L. Eschenbacher, and E. J. Goetzl. 1984. Elevated con-
centrations of leukotriene D4 in pulmonary edema of patients with adult
respiratory distress syndrome. J. Clin. Immunol. 4:479–483.
37. Bjerknes, R. 1984. Flow cytometry assay for combined measurement of
phagocytosis and intracellular killing of Candida albicans. J. Immunol.
Methods 72:229–241.
38. Costa, D. L., J. R. Lehmann, W. M. Harold, and R. T. Drew. 1986. Tran-
soral tracheal intubation of rodents using a fiberoptic laryngoscope. Lab.
Anim. Sci. 36:256–261.
39. Stone, K. C., R. R. Mercer, P. Gehr, B. Stockstill, and J. D. Crapo. 1992. Al-
lometric relationships of cell numbers and size in the mammalian lung.
Am. J. Respir. Cell Mol. Biol. 6:235–243.
40. Snedecor, G. W., and W. G. Cochran 1967. Statistical Methods. Iowa State
University Press, Ames, IA.
41. Lide, David R., editor. 1996. Handbook of Chemistry and Physics. CRC
Press, Boca Raton.
42. Morrow, P. E. 1988. Possible mechanisms to explain dust overloading of the
lungs. Fundam. Appl. Toxicol. 10:369–384.
43. Lehnert, B. E., Y. E. Valdez, and S. H. Bomalaski. 1985. Lung and pleural
“free-cell responses” to the intrapulmonary deposition of particles in the
rat. J. Toxicol. Environ. Health 16:823–839.
44. Lindenschmidt, R. C., K. E. Driscoll, M. Perkins, J. Higgins, J. K. Maurer,
and K. A. Belfiore. 1990. The comparison of a fibrogenic and two nonfib-
rogenic dusts by bronchoalveolar lavage. Toxicol. Appl. Pharmacol. 102:
268–281.
45. Oberdörster, G., J. Ferin, R. Gelein, S. C. Soderholm, and J. Finkelstein.
1992. Role of the alveolar macrophage in lung injury: studies with ultrafine
particles. Environ. Health Perspect. 97:193–199.
46. Bowden, D. H., and I. Y. R. Adamson. 1978. Adaptive responses of the pul-
monary macrophagic system to carbon: I. Kinetic studies. Lab. Invest.
38:422–429.
47. Kavet, R. I., J. D. Brain, and D. J. Levens. 1978. Characteristics of pulmo-
nary macrophages lavaged from hamsters exposed to iron oxide aerosols.
Lab. Invest. 38:312–319.
48. Winterbourn, C. C. 1995. Toxicity of iron and hydrogen peroxide: the Fen-
ton reaction. Toxicol. Lett. 82/83:969–974.
49. Schwertmann, U., and R. M. Cornell. 1991. Iron Oxides in the Laboratory.
VCH, Weinheim, Germany.
50. Wardman, P., and L. P. Candeias. 1996. Fenton chemistry: an introduction.
Radiat. Res. 145:523–531.
51. Goldstein, S., D. Meyerstein, and G. Czapski. 1993. The Fenton reagents.
Free Radic. Biol. Med. 15:435–445.
52. White, D. A., M. G. Krfis, and D. E. Stover. 1987. Bronchoalveolar lavage
cell populations in bleomycin lung toxicity. Thorax 42:551–552.
53. Hayashi, T., Y. Ueno, and T. Okamato. 1993. Oxidoreductive regulation of
nuclear factor kB. J. Biol. Chem. 268:11380–11388.
54. DeForge, L. E., A. M. Preston, E. Takeuchi, J. Kenney, L. A. Boxer, and
D. G. Remick. 1993. Regulation of interleukin 8 gene expression by oxi-
dant stress. J. Biol. Chem. 268:25568–25576.
55. Simeonova, P. P., and M. I. Luster. 1995. Iron and reactive oxygen species in
the asbestos-induced tumor necrosis factor-a response from alveolar mac-
rophages. Am. J. Respir. Cell Mol. Biol. 12:676–683.
56. Adamson, I. Y. R., and D. H. Bowden. 1982. Chemotactic and mitogenic
components of the alveolar macrophage response to particles and neutro-
phil chemoattractant. Am. J. Pathol. 109:71–77.
57. Warheit, D. B., L. H. Overby, G. George, and A. R. Brody. 1988. Pulmo-
nary macrophages are attracted to inhaled particles through complement
activation. Exp. Lung Res. 14:51–66.
58. Stahlhofen, W., G. Rudolf, and A. C. James. 1989. Intercomparison of ex-
perimental regional aerosol deposition data. J. Aerosol Med. 2:285–308.
59. Bennett, W. D., K. L. Zeman, C. Kim, and J. Mascarella. 1997. Enhanced
deposition of fine particles in COPD patients spontaneously breathing at
rest. Inhalation Toxicology 9:1–14.
60. Isawa, T., K. Wasserman, and B. V. Taplin. 1970. Lung scintigraphy and
pulmonary function studies in obstructive disease. Am. Rev. Respir. Dis.
102:161–172.
61. Lin, M. S., and D. A. Goodwin. 1976. Pulmonary distribution of an inhaled
radioaerosol in obstructive pulmonary disease. Radiology 118:645–651.
