1 Department of Pediatrics and 2 Pulmonary, Critical Care, and Occupational Medicine Division, Department of Internal Medicine, The University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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To determine the role of tumor
necrosis factor (TNF)- and interleukin (IL)-1
in the lower
respiratory tract inflammatory response after inhalation of
lipopolysaccharide (LPS), we conducted inhalation exposure studies in
mice lacking expression of TNF-
and/or IL-1 receptor type 1 and in
mice with functional blockade of these cytokines using adenoviral
vector delivery of soluble receptors to one or both cytokines.
Alterations in airway physiology were assessed by pulmonary function
testing before and immediately after 4 h of LPS exposure, and the
cellular inflammatory response was measured by whole lung lavage and
assessment of inflammatory cytokine protein and mRNA expression. Airway
resistance after LPS exposure was similarly increased in all groups of
mice without evidence that blockade of either or both cytokines was
protective from this response. Additionally, all groups of mice
demonstrated significant increases in lung lavage fluid cellularity
with a complete shift in the population of cells to a predominantly
neutrophilic infiltrate as well as elevation in inflammatory cytokine
protein and mRNA levels. There were no significant differences between the groups in measures of lung inflammation. These results indicate that TNF-
and IL-1
do not appear to have an essential role in mediating the physiological or inflammatory response to inhaled LPS.
tumor necrosis factor-; interleukin-
; lipopolysaccharide; endotoxin; cytokines; asthma; airway inflammation
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INTRODUCTION |
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ENDOTOXIN OR LIPOPOLYSACCHARIDE (LPS) is present in varying concentrations in the air we breathe and both causes and exacerbates airway disease. Among workers exposed to organic dusts, the concentration of endotoxin in the bioaerosol is associated with the development and progression of airway disease (15, 20, 35). Exposure to dusts causes a variety of acute respiratory problems including wheezing, dyspnea, and decreased airflow (26). Recent reports (29, 30) have indicated that the concentration of endotoxin in the domestic setting is related to the clinical severity of asthma. In fact, among house dust mite-allergic asthmatic patients, the concentration of endotoxin (but not the concentration of the mite allergen Dermatophagoides pteronyssinus p I) was significantly associated with subjective and objective measures of asthma severity (30). In addition, endotoxin may play a role in airway disease caused by air pollution. Recent studies have shown that the particulate matter that is strongly associated with the progression of airway disease (9) is contaminated with endotoxin (2, 3). The concentration of endotoxin in particulate matter is directly related to the induction of growth factors (3) and the release of interleukin (IL)-6 (2) by monocytes in vitro. These findings suggest that endotoxin may contribute to the development and progression of airway disease in those exposed to organic dusts, allergens, and air pollution.
The components of the pulmonary inflammatory response to inhaled LPS
have been studied with inhalation studies in humans (5-7, 18, 46) and mice (7, 17, 38), demonstrating that
after a single exposure to LPS, neutrophils are rapidly recruited to the lung and proinflammatory cytokines [IL-1, tumor necrosis factor
(TNF)-
, and IL-6] and chemokines [IL-8 and macrophage inflammatory
protein (MIP)-2] are produced and released for up to 48 h
(7). These studies underline the potential importance of
TNF-
and IL-1
as pivotal cytokines in the initiation of the early
inflammatory process after endotoxin exposure. Both macrophages and
neutrophils appear to have important roles in this early inflammatory response. Macrophage activation occurs early, with an in vitro study
(8) demonstrating the release of the proinflammatory cytokines TNF-
, and IL-1
after macrophage stimulation with LPS. A
study in mice (21) demonstrated a dose-dependent influx of neutrophils to the alveoli after endotoxin inhalation that was partially inhibited by pretreatment with TNF-
antibodies. In a
sepsis model, both murine and human monoclonal TNF-
antibodies have
been shown to provide improved survival from endotoxemia or
Escherichia coli sepsis (11, 27, 44).
Additionally, these antibodies led to a reduction in circulating IL-1,
IL-6, and IL-8, suggesting an important role for TNF-
in the
amplification of the inflammatory response (12).
Similarly, blockade of IL-1
by the IL-1 receptor antagonist
prevented death in both mouse and rabbit models of endotoxic shock
(32, 42), and the IL-1 receptor antagonist decreased
endotoxin-induced inflammation of the lower respiratory tract
(45). In addition to studies with monoclonal antibodies
and antagonists to alter the function of these two cytokines, early
work (33, 41, 43) with knockout mouse models has further
implicated the importance of TNF-
and IL-1
in the response to
LPS. Initially, a mutant mouse strain was developed with a disruption
in the gene for one (p55) of the two TNF-
receptors (TNFRs). This
receptor is known to be a key mediator of TNF-
activity, and these
mice demonstrated resistance to lethal doses of endotoxin given
systemically (33) and a modest reduction in lung
neutrophil recruitment after aerosolized LPS (41). In
additional studies, TNFRp55-deficient mice were unable to upregulate
CD14 expression in response to stimulation with recombinant TNF-
,
whereas mice deficient in the other TNFR, p75, had expression of CD14
similar to that of wild-type mice (43). More recently, a
TNF-
knockout that exhibits relative resistance to systemic toxicity
with LPS has been studied. IL-1 is also known to have two separate
receptors, with IL-1 receptor type 1 (IL-1R1) demonstrated to be
essential for many of the biological responses to IL-1. IL-1R1 knockout
mice display a highly reduced acute-phase response to turpentine but a
similar responsiveness to inflammation after intraperitoneal injection
of LPS as wild-type mice (24).
Based on these observations, we hypothesized that TNF- and/or
IL-1
was essential to the inflammatory response caused by inhaled
endotoxin. To further investigate the roles of TNF-
and IL-1
in
the pulmonary inflammatory response to inhaled LPS, we employed several
methods to block expression of these cytokines during inhalation
exposure studies in mice. In the initial experiments, we studied
blockade of one of these cytokines by both knockout mouse technology
and adenoviral vector delivery of a soluble receptor to bind the
circulating cytokine. We further studied simultaneous blockade of both
cytokines with similar techniques. Our results indicate that these
cytokines alone or in combination are not essential to the development
of lower respiratory tract inflammation or bronchial hyperreactivity
induced by endotoxin.
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METHODS |
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General protocol.
In all experiments, the general protocol included the comparison of a
control group of mice with an experimental group that lacked expression
of either TNF- or IL-1
(or IL-1R1) or a group lacking expression
of both genes. Mice underwent a 4-h aerosolized LPS exposure, and
pulmonary function testing was performed 1 day before this exposure
(baseline) and immediately after the exposure (postexposure). In a
separate group of mice, the inflammatory response to inhaled LPS was
evaluated by whole lung lavage and analysis of whole lung mRNA. The
inflammatory response was analyzed by comparing alterations in
cellularity and cytokine levels (TNF-
, IL-1
, MIP-2, and IL-6) in
whole lung lavage fluid and the relative abundance of mRNA of the same
cytokines in lung homogenate. In the systemic endotoxin exposures, mice
were injected intraperitoneally with LPS after baseline peripheral
white blood counts were obtained. These mice were observed for signs of
sepsis and were euthanized if they became moribund. Forty-eight hours
postinjection, the surviving mice were retested for peripheral white
blood cell and differential counts, and whole lung lavage was performed.
Mice.
Six- to ten-week-old B6/129 mice purchased from Jackson Laboratories
(Bar Harbor, ME) were used as control mice in the studies with knockout
mice. C57BL/6J mice were used in all of the studies with adenoviral
vectors. TNF- knockout mice were also purchased from Jackson
Laboratories, and IL-1R1 knockout mice created on a mixed B6/129
background were a gift from Mark Labow (Hoffman-LaRoche, Nutley, NJ).
Both knockout mice have a neomycin cassette inserted in place of the
disrupted gene. TNF-
knockout mice were bred with IL-1R1 knockout
mice, initially creating animals heterozygous for the double mutation.
These heterozygotes were intercrossed, and DNA from their progeny
(F2) was tested by PCR for the presence of homozygous
negative mice for both TNF-
and IL-1R1. Double-knockout mice
occurred in the expected frequency of ~1 in 16 and were interbred to
maintain the colony. All animals had free access to food and water
except during the 4-h endotoxin exposure period. Experimental protocols
were reviewed and approved by the Institutional Animal Care and Use
Committee at the University of Iowa (Iowa City, IA).
PCR.
To determine the presence of the mutation for TNF- or IL-1R1,
mice underwent distal tail clipping for DNA extraction. Briefly, a
section of tail was incubated overnight with a buffer containing 500 µg/ml of proteinase K, 1% SDS, 0.05 M Tris-EDTA, and
0.1 M NaCl. After tissue digestion was complete, the sample was
incubated for 30 min at 37°C with RNase A and then precipitated with
ethanol. PCR was then performed in triplicate for each DNA
isolated with the neomycin primers
5'-GAAGCGGGAAGGGACTGGCTGCTA-3' (upper) and 5'-CGGGAGCGGCGATACCGTAAAGC-3' (lower), the IL-1R1 primers
5'-CCACATATTCTCCATCATCTCTGCTGGTA-3' (upper) and
5'-TTTCGAATCTCAGTTGTCAAGTGTGTCCC-3' (lower), and the TNF-
primers 5'-GCACAGAAAGCATGATCC-3' (upper) and
5'-TAGACAGAAGAGCGTGGTGG-3' (lower). PCRs were run at
95°C for 5 min and then for 30 cycles at 95°C for 30 s, at
60°C for 30 s, and at 72°C for 30 s. Mice found to
be positive for neomycin and negative for both TNF-
and IL-1R1
were marked as double knockouts.
Adenoviral vectors.
In a separate series of exposures, C57BL/6J mice were given intravenous
injections of 1 × 108 plaque-forming units of
replication-deficient adenoviral vectors containing either the human
55-kDa TNFR, a portion of the mouse IL-1R1, or both vectors 3 days
before exposure to endotoxin. Control mice received an adenoviral
vector containing LacZ. The adenoviral vectors were a gift from Dr. Jay
Kolls (Louisiana State University Medical College, New Orleans, LA) and
have been successfully used in the past (13, 22).
Preliminary studies determined that this dose of the vector resulted in
expression of the human TNF- receptor type 1 in mouse serum within
36 h, and expression persisted for at least 6 days. This
expression was measured by ELISA for human TNF receptor type 1 in serum
(R&D Systems).
Endotoxin exposures. Aerosol exposures were performed in a 20-liter exposure chamber with a Collison nebulizer delivering the endotoxin. The endotoxin concentrations generated by the aerosols during the exposure period were assayed with the chromogenic Limulus amebocyte lysate assay (QCL-1000, BioWhittaker, Walkersville, MD) with sterile pyrogen-free labware and a temperature-controlled microplate block and microplate reader (405 nm) as previously described (26). Briefly, four separate samples were taken during each 4-h exposure period by drawing air from the exposure chamber through 47-mm binder-free glass microfiber filters (EPM-2000, Whatman, Maidstone, UK) held within a 47-mm stainless in-line air-sampling filter holder (Gelman Sciences, Ann Arbor, MI). Endotoxin was extracted from the filters with pyrogen-free water at room temperature with gentle shaking. The extracts were then serially diluted and assayed for endotoxin. For the systemic endotoxin exposures, mice were injected intraperitoneally with 500 µg of LPS 3 days after adenoviral vector injection. LPS was purchased as lyophilized, purified E. coli 0111:B4 (Sigma, St. Louis, MO).
Assessment of pulmonary function.
Mice were placed in 80-ml whole body plethysmographs (Buxco
Electronics, Troy, NY) ventilated by bias airflow at 0.2 l/min. Changes
in airway resistance were measured as changes in enhanced pause
(Penh), where Penh = [(TE/40% Tr) (1 × Pef/Pif) × 0.67], where
TE is expiratory time, Tr is relaxation time,
Pef is peak expiratory flow, and Pif is peak
inspiratory flow. The validity of Penh as a measure of
bronchoconstriction has been examined (39). Pulmonary
function was measured at baseline and after inhaled methacholine
challenge with doses of 10, 15, 20, and 30 mg/ml delivered with a
DeVilbiss nebulizer.
Lung lavage and tissue processing.
Immediately after completion of the 4-h endotoxin exposure, animals
were killed by CO2 inhalation, and lavage was performed. The trachea was isolated and cannulated with PE-90 tubing, and 1-ml
aliquots of normal saline were infused into the lungs by gravity, with
a total volume of 6 ml infused. After lavage, the lungs were removed,
snap-frozen in liquid nitrogen, and stored at 70°C until further
use. The lavage fluid was centrifuged at 200 g for 5 min.
The supernatant was then decanted and stored at
70°C until further
use. The cell pellet was resuspended in Hanks' balanced salt solution,
and a small aliquot was used for counting the cells with a
hemacytometer. Another aliquot of the cell suspension was spun onto a
slide with a cytocentrifuge (Shanden, Southern Sewickley, PA). The
cytospun cells were stained with Diff-Quik stain set (Harleco,
Gibbstown, NY) and air-dried, and a coverslip was applied for counting
with light microscopy.
RNA isolation and RNase protection assay. Total RNA was isolated from lung tissue as previously described (4, 19). Briefly, frozen lung was homogenized in RNAzol (guanidinium thiocyanate-phenol-sodium acetate) with a tissue tearor (Biospec Products). Chloroform extraction was performed, and the RNA was precipitated in isopropanol and washed with 80% ethanol. The yield and purity of the RNA were determined by measuring absorbance at 260 and 280 nm. RNase protection assays were performed with the Riboquant kit (PharMingen, San Diego, CA) according to manufacturer's instructions with custom-probe template sets by PharMingen. Briefly, 10 µg of total RNA were hybridized overnight with a 32P-labeled antisense riboprobe to multiple cytokines and chemokines at 56°C. The nonhybridized single-strand RNA was digested with a mixture of RNase A and T1, and the remaining protected fragments were extracted with phenol-chloroform-isoamyl alcohol and ethanol precipitated. The hybridization products were separated on a 5% acrylamide-8 M urea gel run at 65 W for ~1 h. The gel was wrapped, dried, and exposed to a phosphorimager screen overnight. The relative abundances were quantitated with ImageQuant software for the phosphorimager (Sunnyvale, CA).
Cytokine protein analysis.
Commercially available kits (R&D Systems) were used according to the
manufacturer's instructions to determine concentrations of murine
TNF-, IL-1
, MIP-2, and IL-6 in the lavage fluid. In all cases, a
monoclonal antibody was used as a capture reagent in a standard
sandwich ELISA. Standard curves were derived from known concentrations
of the recombinant specific cytokine supplied by the manufacturer.
Statistical analysis.
The primary comparison under investigation was the effect of blockade
of TNF-, IL-1
, or both cytokines concurrently on the pulmonary
inflammatory response to inhaled endotoxin. Specifically, comparisons
evaluated differences in whole lung lavage fluid cellularity, cytokine
expression, mRNA abundance, and airway resistance. Statistical comparisons were made with nonparametric statistics, specifically the
Mann-Whitney U-test (34).
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RESULTS |
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Airway physiology.
Pulmonary function testing performed before LPS exposure demonstrated
no differences between control mice and mice that received adenoviral
vectors expressing TNFR or IL-1R1 in measures of airway resistance at
baseline or after inhalation of increasing concentrations of
methacholine. After inhalation of LPS, airway resistance increased in
all groups (compared with preexposure pulmonary function
testing), including control mice and those receiving the
adenoviral vectors expressing either the TNFR, the IL-1R1, or both
vectors. However, there were no differences between any of the groups,
indicating that cytokine blockade did not offer protection against the
development of increased airway resistance after LPS inhalation (Fig.
1). Single-knockout mice lacking either
TNF- or IL-1R1 and double-knockout mice lacking expression of both
TNF-
and IL-1R1 demonstrated increases in airway resistance similar
to those in B6/129 control mice after exposure to aerosolized LPS (Fig.
2).
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Lung inflammation.
Lung lavage performed on control B6/129 mice before exposure to
endotoxin demonstrated ~10,000-20,000 cells/ml lavage fluid that
are >95% macrophages (data not shown). Immediately after inhalation
challenge with aerosolized LPS, whole lung lavage demonstrated a
>10-fold increase in the total cell number and a complete shift in the
population of cells to a predominantly neutrophilic infiltrate. There
were no significant differences in the total cell counts between the
control, TNF- knockout, IL-1R1 knockout, and TNF-
, IL-1R1
double-knockout mice (Fig. 3); although
the TNF-
single-knockout and the TNF-
, IL-1R1 double-knockout
mice did have slightly fewer cells per milliliter, this difference was
not significant. All four groups had similar total neutrophil
percentages postexposure, ranging from 81 to 85% (Fig. 3). The results
of lung lavage fluid cellularity in the C57BL/6J mice that received
adenoviral vector delivery of the TNFR, the IL-1R1, or both the TNFR
and IL1R1 again demonstrated a marked increase in total lavage fluid
cellularity post-LPS exposure, with a shift in the population of cells
to predominantly neutrophils and no significant decrease in total cell
count or change in the percentage of neutrophils compared with control
mice receiving the LacZ vector (Fig. 4).
The lung lavage fluid was also assayed after exposure to aerosolized
LPS as an additional marker of the pulmonary inflammatory response for
the presence of several inflammatory cytokines, including levels of
TNF-
, IL-1
, MIP-2 (the murine homolog of IL-8, a neutrophil chemokine), and IL-6. Except for the absence of TNF-
in the lavage fluid from the TNF-
knockout mice, there were no significant differences between the control and knockout mice in terms of cytokine
protein expression in the lavage fluid (data not shown). In the mice
that received adenoviral vector delivery of either TNFR, IL-1R1, or
both, the lavage cytokine data demonstrated that the vectors did
function as expected to significantly decrease the abundance of the
each of their respective cytokines compared with the control LacZ
vector (Fig. 5, A and
B). Expression of IL-6 was unchanged among the four
groups; however, the mice receiving either the IL-1R1 vector alone or
in combination with the TNFR vector did demonstrate a significant
reduction in lavage fluid MIP-2 concentration. (Fig. 5, C
and D).
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Systemic LPS exposure. Baseline peripheral white blood cell counts were determined in a subset of C57BL/6J mice 3 days after adenoviral vector delivery of either a LacZ control vector or the TNFR and IL-1R1 vectors combined and before systemic endotoxin administration. The average total white blood cell count at baseline was 5,527 ± 356 cells/mm3; there were no differences between the mice that received the control vector versus those that received the TNFR and IL-1R1 vectors. Five hours after intraperitoneal administration of 500 µg of LPS, peripheral blood counts were obtained in a subset of mice and demonstrated a dramatic decline in the peripheral white blood cell count in both mice that received the control vector and those that received the combination of TNFR and IL-1R1 vectors (909 and 895 cells/mm3, respectively). Mice that received the TNFR and IL-1R1 vectors had significantly reduced mortality after intraperitoneal LPS compared with mice that received the LacZ control vector (43 and 79%, respectively, after 48 h; P = 0.05; n = 14/group). In addition, among mice that survived the endotoxemia, recovery of the peripheral white blood cells at 48 h was improved in the group that received adenoviral delivery of TNFR plus IL-1R1 versus those that received LacZ (4,460 and 2,180 cells/mm3, respectively; P < 0.05), with a greater predominance of neutrophils in the white blood cell population from the mice that had adenoviral delivery of TNFR plus IL-1R1 (67 and 40.6%, respectively; P < 0.05).
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DISCUSSION |
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Our results indicate that TNF- and/or IL-1
does not appear
to be essential to the physiological or inflammatory response to
inhaled LPS. Simultaneous blocking of both of these cytokines did not
result in a significant reduction in the cellular inflammatory infiltrate, and there was no inhibition of the development of bronchial
hyperresponsiveness. A potential criticism in using knockout mouse
models is the likelihood that these mice develop compensatory signaling
mechanisms in response to their deficiencies. We have addressed this
concern by using adenoviral vector delivery of soluble cytokine
receptors as an alternate method to block these cytokines. Both
experimental approaches have resulted in consistent findings and
suggest that TNF-
and IL-1
are not individually or
collectively essential to the airway response to inhaled LPS.
However, several lines of evidence indicate that TNF- and/or IL-1
should play significant roles in LPS-induced airway inflammation. The
early localization of both TNF-
and IL-1
in the lung after inhalation of endotoxin is well established. Lung histology studies including immunohistochemical staining (37) and in situ
hybridization (48) have indicated that the macrophage and
the neutrophil are actively involved in the de novo synthesis of these
proinflammatory cytokines/chemokines. mRNA analysis of murine lungs
after inhalation challenge with corn dust extract, LPS, or saline
indicates that the mRNAs for TNF-
, IL-1
, IL-1
, MIP-2 (murine
homologue of IL-8), MIP-1
, MIP-1
, IL-6, transforming growth
factor-
1, and interferon-
are upregulated immediately after
inhalation of either corn dust extract or LPS, and for some cytokines,
this persists for up to 24 h. Although these data demonstrate the
presence of these cytokines during the initial phases of neutrophil
recruitment, it does not establish a causal link. The results presented
here suggest that these cytokines are a nonessential participant in the
inflammatory response rather than having a role in initiating and
perpetuating the neutrophil inflammatory process.
The findings in this investigation are certainly unexpected in view of
the previous work studying the role of TNF- and IL-1
in other
model systems of endotoxin exposure (11, 27, 28). Importantly, the
major focus of many of the previous studies that blocked
expression of TNF-
and/or IL-1
was on the systemic response to
endotoxemia rather than on the pulmonary inflammatory process. Similarly, we demonstrated that blockade of expression of these cytokines in systemic endotoxemia reduces mortality despite the lack of
effect after aerosol exposure to LPS. In many cases, even when the
alveolar compartment has been studied, it is after intravenous or
intraperitoneal exposure to endotoxin (28, 47). The one study (41) that investigated the air space response to
inhaled LPS demonstrated only a modest reduction in recruitment of
neutrophils in TNFRp55-deficient mice (41). The
compartmentalized nature of the host response to endotoxin has been
demonstrated by comparison of lung cytokine release after intravascular
versus alveolar endotoxin challenge in isolated rabbit lungs. TNF-
is liberated into the airways after intravascular challenge but at a
markedly decreased level compared with TNF-
levels after aerosolized
endotoxin challenge (14). The movement of
polymorphonuclear neutrophils from the pulmonary circulation to the air
space requires a complex series of interactions that is likely to be
very different from the scenario that occurs in the systemic
circulation and underscores the importance of using inhalation exposure
as the mechanism for studying the response of the lower respiratory
tract to endotoxin.
There are a number of other potential mediators that may be more
essential to the inflammatory response to inhaled LPS. Several families
of cellular adhesion molecules expressed on both the vascular
endothelium and neutrophils have been implicated in pulmonary neutrophil recruitment after inflammatory stimuli.
2-Integrin expression by circulating neutrophils is
induced in many inflammatory states, and blockade of subunits of these
molecules has been demonstrated to reduce pulmonary leukocyte
infiltration in both an allergic lung inflammation model
(36) and certain bacterial pneumonitis models (10,
23). Similarly, studies using monoclonal antibody blockade of
intercellular adhesion molecule-1 that is expressed on both the
vascular endothelium, and certain epithelial cells have shown reduced
neutrophil recruitment in phorbol ester-induced lung inflammation
(1), immune complex-mediated lung injury (31), and several model systems of ischemia-reperfusion
(16, 25, 40, 49). The potential role of these families of
adhesion molecules as well as of many others in endotoxin-induced
airway disease has not been studied.
The present investigation demonstrates that neither TNF- nor IL-1
are essential triggers of the pulmonary inflammatory response to
inhaled LPS. These studies suggest that the well-documented upregulation of both protein and mRNA for TNF-
or IL-1
that occurs shortly after exposure to endotoxin is a concurrent finding with
the onset of neutrophil recruitment and the development of bronchial
hyperreactivity but is not the cause of these manifestations. Because
these results differ strikingly from what has been demonstrated in
systemic models of endotoxin exposure, further studies will be needed
to determine whether the pulmonary circulation is dependent on an
alternate group of cytokines or chemokines or a completely separate
class of molecules for the initiation of neutrophil recruitment and the
increases in airflow obstruction.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Environmental Health Sciences Grants ES-07498 and ES-09607; National Heart, Lung, and Blood Institute Grant HL-62628; National Institute of Child Health and Human Development Child Health Research Center Grant HD-27748; and Department of Veterans Affairs Merit Review.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. G. Moreland, Division of Pediatric Critical Care, Dept. of Pediatrics, The Univ. of Iowa, Iowa City, IA 52242 (E-mail: jessica-moreland{at}uiowa.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 February 2000; accepted in final form 9 August 2000.
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