1 Department of Internal Medicine, The University of Iowa College of Medicine, Iowa City, Iowa 52242-1081; and 2 Department of Bacteriology, The University of Wisconsin, William S. Middleton Memorial Veteran's Hospital, Madison, Wisconsin 53705-2254
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ABSTRACT |
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To further determine the importance of endotoxin
in grain dust-induced inflammation of the lower respiratory tract, we
evaluated the efficacy of pentaacylated diphosphoryl lipid A derived
from the lipopolysaccharide of
Rhodobacter
sphaeroides (RsDPLA) as a partial
agonist of grain dust-induced airway inflammation. RsDPLA is a
relatively inactive compound compared with lipid A derived from
Escherichia coli (LPS) and has been
demonstrated to act as a partial agonist of LPS-induced inflammation.
To assess the potential stimulatory effect of RsDPLA in relation to
LPS, we incubated THP-1 cells with RsDPLA (0.001-100 µg/ml), LPS
(0.02 µg endotoxin activity/ml), or corn dust extract (CDE; 0.02 µg
endotoxin activity/ml). Incubation with RsDPLA revealed a tumor
necrosis factor (TNF)- stimulatory effect at 100 µg/ml. In
contrast, incubation with LPS or CDE resulted in TNF-
release at
0.02 µg/ml. Pretreatment of THP-1 cells with varying concentrations
of RsDPLA before incubation with LPS or CDE (0.02 µg endotoxin
activity/ml) resulted in a dose-dependent reduction in the LPS- or
CDE-induced release of TNF-
with concentrations of RsDPLA of up to
10 µg/ml but not at 100 µg/ml. To further understand the role of
endotoxin in grain dust-induced airway inflammation, we utilized the
unique LPS inhibitory property of RsDPLA to determine the inflammatory
response to inhaled CDE in mice in the presence of RsDPLA. Ten
micrograms of RsDPLA intratracheally did not cause a significant
inflammatory response compared with intratracheal saline. However,
pretreatment of mice with 10 µg of RsDPLA intratracheally before
exposure to CDE (5.4 and 0.2 µg/m3) or LPS (7.2 and 0.28 µg/m3) resulted in significant
reductions in the lung lavage concentrations of total cells,
neutrophils, and specific proinflammatory cytokines compared with mice
pretreated with sterile saline. These results confirm the
LPS-inhibitory effect of RsDPLA and support the role of endotoxin as
the principal agent in grain dust causing airway inflammation.
endotoxin; lipopolysaccharide inhibition; asthma
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INTRODUCTION |
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OCCUPATIONAL AND ENVIRONMENTAL exposure to grain dust may cause a variety of acute respiratory symptoms, including chest tightness, wheezing, dyspnea, and transient declines in airflow (6). Epidemiologic studies in grain workers have demonstrated that chronic exposure to grain dust may lead to chronic respiratory symptoms, including chronic bronchitis, and progressive, irreversible airflow obstruction (1). Prior studies suggest an association between endotoxin and the physiological and inflammatory response to grain dust. In grain handlers, work-related respiratory symptoms were found to be strongly associated with the concentration of endotoxin in the bioaerosol (15). Inhalation of endotoxin and corn dust extract (CDE) produces similar symptoms, equivalent changes in airflow, and similar increases in bronchoalveolar lavage inflammatory cells and inflammatory cytokines (5).
To further investigate the relative importance of endotoxin in grain dust-induced airway inflammation, we have employed methods that attempted to remove endotoxin from CDE and demonstrated a relationship between the concentration of endotoxin in CDE and the magnitude of inflammation in the lung (4). However, these methods were limited by the inability to completely remove endotoxin from CDE and by the possibility of removal of other components in CDE that may promote lung inflammation. In this investigation, we propose to examine the relationship between endotoxin and grain dust-induced lung inflammation by specifically inhibiting endotoxin-mediated inflammatory events. Previous investigations have shown that the pentaacylated diphosphoryl lipid A derived from the lipopolysaccharide of Rhodobacter sphaeroides (RsDPLA) is a relatively inactive compound compared with lipid A derived from Escherichia coli (LPS; see Ref. 16). Moreover, it appears that RsDPLA acts as a partial agonist of LPS-induced inflammation (7, 17). In this investigation, we utilized the unique LPS inhibitory property of RsDPLA to determine the relative stimulatory effect of CDE in vitro and the inflammatory response in the lungs of mice after exposure to CDE in the presence of an endotoxin inhibitor. A priori, we hypothesize that the inflammatory response to CDE will be diminished in the presence of RsDPLA.
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METHODS |
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Chemicals. Lyophilized E. coli 0111:B4 LPS was purchased from Sigma Chemical, St. Louis, MO. Corn dust used in this study was obtained from the air filtration system at an eastern Iowa grain handling facility. Sterile, pyrogen-free water and sterile, pyrogen-free normal saline (PFS) were purchased from Baxter Medical Laboratories, Deerfield, IL. Hanks' balanced salt solution (HBSS) was purchased from the University of Iowa tissue culture facility.
LPS and CDE preparation.
LPS solution was prepared by mixing lyophilized
E.
coli LPS in HBSS. CDE was prepared by
mixing 3.0 g of dust in 30 ml of HBSS (0.1% solution) by methods
previously described (4). Both LPS and CDE solutions were filter
sterilized through a 0.22-µm filter (Acrocap Low Protein Binding
Filter; Gelman Sciences, Ann Arbor, MI) and then were used immediately
or were stored at 70°C.
RsDPLA preparation. R. sphaeroides (ATCC 17023) was grown photoheterotrophically for 12-14 days and was harvested by centrifugation. LPS was extracted from R. sphaeroides by the phenol-chloroform-petroleum ether method of Galanos et al. (3). RsDPLA was prepared by treating R. sphaeroides LPS with 0.02 M sodium acetate (pH 2.5) for 70 min at 100°C (10). Crude RsDPLA was dissolved in chloroform-methanol and was purified by DEAE-cellulose column chromatography. RsDPLA was eluted with a linear ammonium acetate gradient and was characterized by analytical thin-layer chromatography, gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy (9). The RsDPLA was in the free acid form, was solubilized in 0.5% triethylamine salt, and was further diluted in saline before use in the experiments.
Endotoxin assay. Endotoxin concentrations were measured in the LPS and CDE solutions using the Limulus amebocyte lysate (LAL) assay (QCL-1000; Whittaker Bioproducts, Walkersville, MD). Measurement of airborne endotoxin concentrations generated from the various LPS and CDE solutions during animal exposure studies was carried out using methods previously described (14). The sensitivity of the LAL for aerosolized LPS is 0.25 ng/m3.
Mice. Male 6-wk-old inbred mice (C3Heb/FeJ) purchased from Jackson Laboratory (Bar Harbor, ME) were used in all exposure studies. They were housed in our institution's rodent vivarium, fed a normal diet (Formulab Chow no. 5008; PMI, Richmond, IN), provided water ad libitum, maintained on wood chip bedding (Northeastern Product, Warrensburg, NY), and used within 2 wk. Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Iowa.
Exposure apparatus and exposure protocol. A series of four mouse inhalation exposure experiments was performed to aerosols of LPS and CDE, the methods of which we have previously described (14). Exposures were performed in a 20-liter exposure chamber with a PITT no. 1 nebulizer with solutions for inhalation exposure supplied by a syringe pump. In each experiment, groups of 20 mice underwent inhalation exposure to LPS or CDE solutions over 4 h. Thirty minutes before inhalation challenge, each mouse underwent general anesthesia (Metofane; Pitman-Moore, Mundelein, IL) followed by instillation of equivalent volumes (40 µl) of RsDPLA (10 µg) or PFS into the hypopharynx through a 24-gauge Jelco intravenous catheter (Johnson and Johnson, Arlington, TX). This dose of RsDPLA was chosen after performing dose-response experiments with mice that demonstrated that 10 µg of RsDPLA do not cause changes in lung lavage cellularity compared with PFS. After exposure, animals were killed by cervical dislocation, the diaphragm was punctured to deflate the lungs, and whole lung lavage was performed.
Lung lavage. Mice tracheae were isolated and cannulated with PE-90 tubing, and whole lung lavage was performed in situ. One-milliliter aliquots of PFS were infused into the trachea at 25 cmH2O pressure head and collected, and the process was repeated six times. Lung lavage fluid was processed by our standard method (13).
In vitro endotoxin biological activity.
To measure the in vitro cellular response to RsDPLA, LPS, and CDE,
THP-1 cells (American Type Culture Collection, Rockville, MD), a human
monocytic leukemia cell line, were used (18). Cells (passages
20-24)
were grown in pyrogen-free 10% RPMI 1640 medium in a 24-well cell
culture plate (Costar, Cambridge, MA) at a density of 1 × 106 cells/well at 37°C, 5%
CO2 in 1 ml of medium. For
dose-response curve experiments, the cells were incubated with RsDPLA
at varying concentrations (0, 0.001, 0.01, 0.1, 1.0, 10, and 100 µg/ml) in HBSS. For coincubation studies with LPS or CDE, RsDPLA
(0-100 µg/ml) was introduced 30 min before treatment with LPS or
CDE (endotoxin concentration 0.02 µg/ml). After 24 h, the cultures then underwent freeze-thaw cycling two times at 20°C
followed by centrifugation (2,500 g)
for 5 min. The supernatants were collected and assayed for tumor
necrosis factor (TNF)-
.
Cytokine analyses.
Commercially available kits (R&D Systems, Minneapolis, MN) were used to
determine concentrations of murine interleukin (IL)-1, IL-6,
macrophage inflammatory protein-2 (MIP-2), TNF-
, and human TNF-
. In all cases, a monoclonal antibody was used as a
capture reagent in a standard sandwich enzyme-linked immunosorbent
assay (ELISA). The assays were performed according to the
manufacturer's instructions, and standard curves were derived using
known concentrations of the recombinant specific cytokine supplied by
the manufacturer. All assays were performed using the standard diluent
supplied in the ELISA kit.
Statistical analysis.
Two primary comparisons were made in this study:
1) comparison of the in vitro
TNF- production of THP-1 cells exposed to LPS and CDE solutions
after pretreatment with RsDPLA and
2) comparison of the in vivo
inflammatory response as assessed by both whole lung lavage fluid in
mice exposed to LPS or CDE after pretreatment with RsDPLA or sterile
saline intratracheally. Statistical comparisons for continuous data
were made using the Mann-Whitney
U-test (11).
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RESULTS |
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The in vitro stimulatory effect of RsDPLA on THP-1 cells was determined
by performing dose-response experiments in which THP-1 cells were
incubated with increasing concentrations of RsDPLA (0.001-100 µg/ml)
for 24 h. The supernatant fluid was assayed for TNF- (Fig.
1A).
At relatively low concentrations of RsDPLA (
10 µg/ml), very little
TNF-
stimulatory effect was observed compared with higher
concentrations of RsDPLA (100 µg/ml), which caused substantially
greater stimulatory release of TNF-
in a dose-dependent fashion.
Although higher concentrations of RsDPLA caused a stimulatory effect on
THP-1 cells, this effect was achieved at a considerably higher
concentration compared with LPS, which stimulates TNF-
release at
concentrations as low as 0.01 µg/ml (Fig.
1B).
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Because of its relative nonstimulatory effect at low concentrations,
further studies were carried out to determine whether RsDPLA could
display inhibitory properties when incubated with cells before LPS or
CDE stimulation. Incubation of THP-1 cells with LPS (Fig.
2A) or
CDE (Fig. 2B) alone (endotoxin
activity 0.02 µg/ml) resulted in the stimulation of TNF-
production and release from these cells. When THP-1 cells were
pretreated with 10-fold increasing concentrations of RsDPLA
(0.001-100 µg/ml) before stimulation with LPS or CDE, there was
a similar pattern in the magnitude of TNF-
release from THP-1 cells
for each concentration of RsDPLA. At lower concentrations of RsDPLA
(0.001-1 µg/ml), there was a dose-dependent reduction in LPS and
CDE stimulatory release of TNF-
, which was maximal in both instances
at 1 µg/ml. However, this inhibitory effect was not
apparent at 100 µg/ml, where RsDPLA stimulates THP-1 cells to produce
and release TNF-
(Fig. 1A).
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RsDPLA was tested for its in vivo ability to stimulate lung inflammation. Mice underwent intratracheal instillation of RsDPLA at doses of 0.1, 1, 10, and 100 µg or sterile saline, and these doses of RsDPLA (except 100 µg) were not found to stimulate neutrophil recruitment in the lung (data not shown). Because of the lack of neutrophil recruitment in the lung lavage fluid after intratracheal instillation of RsDPLA at 10 µg, this dose was chosen for subsequent experiments to determine whether RsDPLA had inhibitory properties when given intratracheally before inhalation exposure to LPS or CDE.
After intratracheal instillation of RsDPLA or HBSS, groups of 10 mice
underwent inhalation challenge to LPS or CDE at various airborne
endotoxin levels. At high airborne levels of endotoxin (LPS: 7.2 µg/m3; CDE: 5.4 µg/m3), mice pretreated with
HBSS developed increasing concentrations of total cells, predominantly
neutrophils, as well as elevated concentrations of TNF-, IL-1
,
IL-6, and MIP-2 in lung lavage fluid after exposure to both LPS (Fig.
3A) and
CDE (Fig. 3B). In mice pretreated
with RsDPLA, statistically significant reductions in both total cell,
neutrophil, IL-1
, and IL-6 concentrations were observed after
exposure to LPS or CDE compared with HBSS-pretreated mice exposed to
the same bioaerosols (P < 0.05 for
all comparisons made). At high airborne levels of endotoxin, no
differences in lung lavage TNF-
or MIP-2 were detected between
RsDPLA- and HBSS-treated mice after exposure to either LPS or CDE.
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Mice pretreated with HBSS followed by exposure to low airborne levels
of endotoxin (LPS: 0.28 µg/m3;
CDE: 0.2 µg/m3) developed
increased lung lavage concentrations of total cells, neutrophils,
TNF-, IL-1
, IL-6, and MIP-2 (Fig. 4,
A and
B), but the increases in cellularity
and cytokine concentrations were not as great compared with
the high endotoxin exposures (Fig. 3,
A and
B). After exposure to LPS, mice
pretreated with RsDPLA demonstrated significant reductions in lavage
concentrations of total cells, neutrophils, TNF-
, IL-6, and MIP-2
compared with control mice pretreated with HBSS. After exposure to CDE,
mice pretreated with RsDPLA also demonstrated significant reductions in
lavage concentrations of total cells, neutrophils, TNF-
, and MIP-2,
but no differences in lavage concentrations of IL-1
or IL-6 were
detected between treatment groups.
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DISCUSSION |
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Our study results confirm previous observations that RsDPLA is less
potent than LPS at stimulating TNF- release but is capable of
inhibiting LPS-mediated TNF-
release (17). Likewise, RsDPLA is
capable of inhibiting CDE-mediated production and release of TNF-
,
suggesting a similar mechanism by which LPS and CDE stimulate THP-1
cells to produce and release TNF-
. Furthermore, when administered to
mice intratracheally at a dose of 10 µg, RsDPLA alone does not cause
significant lung inflammation. However, when 10 µg of RsDPLA were
administered before inhalation of either LPS or CDE, this dose was
capable of inhibiting LPS- and CDE-mediated lung inflammation, again
suggesting a common mechanism by which LPS and CDE cause lung
inflammation. Our results suggest that CDE causes lung inflammation
through endotoxin-mediated inflammatory events, which are inhibited by
RsDPLA.
The results of this study add further support to the growing evidence that endotoxin is one of the primary agents in grain dust causing airway inflammation. In previous epidemiologic studies, we have found that the concentration of endotoxin in the bioaerosol is strongly associated with respiratory symptoms, airflow obstruction, and longitudinal declines in airflow among grain handlers and other farmers (12, 15). In addition, we have previously investigated the relationship between endotoxin and grain dust-induced airway disease using a murine model of acute grain dust inhalation challenge. We have demonstrated a clear dose-response relationship between the concentration of endotoxin in the CDE bioaerosol and the magnitude of lung inflammation and cytokine release in lung lavage fluid (14). A second approach involved the determination of whether host susceptibility to endotoxin affected the inflammatory response to CDE (14). Utilizing strains of mice that are genetically endotoxin hyporesponsive (C3H/HeJ) or endotoxin sensitive (C3Heb/FeJ), we previously demonstrated that when exposed to an identical CDE exposure, mice that are genetically hyporesponsive to endotoxin develop significantly less lung lavage inflammatory cells and cytokines compared with endotoxin-sensitive mice. Furthermore, when endotoxin-sensitive mice were made endotoxin tolerant (acquired endotoxin hyporesponsiveness) through serial injections with LPS, these mice developed significantly less airway inflammation compared with control mice after the same CDE exposure. A third approach examining the relationship between endotoxin and grain dust-induced lung inflammation involved determining whether endotoxin removal from CDE altered the magnitude of CDE-induced lung inflammation. In these experiments, we demonstrated that, using three different methods of endotoxin removal from biological solutions, the magnitude of the inflammatory response to CDE was related to the endotoxin concentration in the CDE (4). The results from these previous investigations support the findings from our current study. In the present investigation, by utilizing the unique properties of RsDPLA that act as a specific inhibitor of endotoxin, we have demonstrated that the inflammatory response to CDE is substantially reduced when this agent is given intratracheally before exposure to CDE. In aggregate, we have demonstrated convincing evidence linking endotoxin with the acute inflammatory response to CDE by demonstrating a dose-response relationship between endotoxin concentration in the CDE bioaerosol and the magnitude of lung inflammation, by altering the host's ability to respond to endotoxin, by altering the concentration of endotoxin in CDE, and by selectively inhibiting endotoxin-mediated inflammatory events.
The results of our study are the first to demonstrate the ability of
RsDPLA to inhibit endotoxin-mediated lung inflammation using an
inhalational model of acute lung inflammation. Previous investigations
have demonstrated that RsDPLA is capable of blocking the induction of
TNF- from a macrophage cell line treated with LPS in a
concentration-dependent manner (17). Pretreatment of mice with RsDPLA
intravenously blocked the rise in serum TNF-
normally observed in
mice after intravenous injection with LPS (8). Furthermore, RsDPLA was
protective in preventing endotoxin-mediated death in mice, possibly due
to induction of early endotoxin tolerance (19). Our present
investigation supports the role of RsDPLA as an inhibitor of
CDE-induced lung inflammation by acting as a competitive agonist of
endotoxin-mediated induction of specific proinflammatory cytokines in
vitro. The mechanism by which RsDPLA acts to reduce lung inflammation
due to grain dust in our study is not entirely clear but may be due to
direct inhibition of endotoxin binding to cell receptors in the lung or
possibly secondary to the induction of early endotoxin tolerance.
Cytokines undoubtedly play a major role in the inflammatory response to
inhaled grain dust. In humans and mice, we have demonstrated that
TNF-, IL-6, IL-8 (humans), and MIP-2 (mice) are released within
hours of the exposure and may persist for up to 48 h after the
inhalation challenge (2). Interestingly, mice pretreated with RsDPLA
followed by inhalation challenge with either LPS or CDE at high
endotoxin concentrations did not develop significant reductions in lung
lavage concentrations of TNF-
despite reductions in lavage
cellularity, IL-1
, and IL-6 levels (Fig. 3,
A and
B). In contrast, TNF-
and MIP-2
concentrations in lung lavage fluid were significantly reduced in mice
pretreated with RsDPLA exposed to lower concentrations of endotoxin in
LPS or CDE (Fig. 4, A and
B). This observation may be
attributed to the possibility that the airborne levels of endotoxin in
the high-endotoxin LPS or CDE exposures exceeded the inhibitory
capacity of RsDPLA used in the study. In addition, because RsDPLA was
administered intratracheally and not by aerosolization, the
distribution of RsDPLA in the lung may have differed between these
routes of administration, affecting the efficacy of RsDPLA in
inhibiting LPS- or CDE-induced TNF-
and MIP-2 release at the higher
endotoxin concentrations. Finally, the complex combination of cytokine
gradients may play an important role in recruitment of neutrophils
after different types of inhalation challenges.
In conclusion, our results confirm prior observations demonstrating the relative nontoxic properties of RsDPLA, including intratracheal lung administration. In addition, we have demonstrated that RsDPLA inhibits grain dust-induced lung inflammation, supporting the hypothesis that endotoxin is the primary component in grain dust responsible for the development of airway inflammation.
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ACKNOWLEDGEMENTS |
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We appreciate and acknowledge the support of Dr. Peter Thorne and the Inhalation Toxicology Facility in the Environmental Health Science Research Center (ES 05605).
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FOOTNOTES |
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This study was supported by grants from the National Institute of Occupational Safety and Health (OH-00134-02), National Institutes of Health (ES-00203, ES-06537, and GM-50870), and the Medical Research Service of the Department of Veterans Affairs.
Address for reprint requests: D. A. Schwartz, Pulmonary Diseases, Critical Care, and Occupational Medicine Division, Dept. of Internal Medicine, The Univ. of Iowa College of Medicine, Iowa City, IA 52242-1081.
Received 15 October 1996; accepted in final form 7 September 1997.
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