TNF-alpha and IL-1beta are not essential to the inflammatory response in LPS-induced airway disease

Jessica G. Moreland1, Robert M. Fuhrman1, Christine L. Wohlford-Lenane2, Timothy J. Quinn2, Erin Benda2, Jonathan A. Pruessner1, and David A. Schwartz2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To determine the role of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in the lower respiratory tract inflammatory response after inhalation of lipopolysaccharide (LPS), we conducted inhalation exposure studies in mice lacking expression of TNF-alpha 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-alpha and IL-1beta do not appear to have an essential role in mediating the physiological or inflammatory response to inhaled LPS.

tumor necrosis factor-alpha ; interleukin-beta ; lipopolysaccharide; endotoxin; cytokines; asthma; airway inflammation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta , tumor necrosis factor (TNF)-alpha , 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-alpha and IL-1beta 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-alpha , and IL-1beta 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-alpha antibodies. In a sepsis model, both murine and human monoclonal TNF-alpha 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-alpha in the amplification of the inflammatory response (12). Similarly, blockade of IL-1beta 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-alpha and IL-1beta 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-alpha receptors (TNFRs). This receptor is known to be a key mediator of TNF-alpha 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-alpha , whereas mice deficient in the other TNFR, p75, had expression of CD14 similar to that of wild-type mice (43). More recently, a TNF-alpha 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-alpha and/or IL-1beta was essential to the inflammatory response caused by inhaled endotoxin. To further investigate the roles of TNF-alpha and IL-1beta 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha or IL-1beta (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-alpha , IL-1beta , 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha , IL-1beta , 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-alpha , IL-1beta , 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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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-alpha or IL-1R1 and double-knockout mice lacking expression of both TNF-alpha 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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Fig. 1.   Airway resistance measured as enhanced pause (Penh) before exposure to lipopolysaccharide (LPS) and immediately after 4-h exposure to aerosolized LPS at baseline (no methacholine) and gradually increasing doses of methacholine in mice receiving adenoviral (Ad) vectors containing either LacZ (control), tumor necrosis factor (TNF)-alpha receptor (TNFR), interleukin-1 receptor type 1 (IL-1R1), or both TNFR and IL-1R.



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Fig. 2.   Airway resistance measured as Penh immediately after 4-h exposure to aerosolized LPS at baseline (no methacholine) and after gradually increasing doses of methacholine in B6/129 control, IL-1R1 knockout (KO), TNF KO, and TNF, IL-1R1 double-KO mice.

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-alpha knockout, IL-1R1 knockout, and TNF-alpha , IL-1R1 double-knockout mice (Fig. 3); although the TNF-alpha single-knockout and the TNF-alpha , 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-alpha , IL-1beta , MIP-2 (the murine homolog of IL-8, a neutrophil chemokine), and IL-6. Except for the absence of TNF-alpha in the lavage fluid from the TNF-alpha 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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Fig. 3.   Concentration of total cells (left) and polymorphonuclear neutrophils (PMNs; right) in whole lung lavage fluid after 4-h exposure to aerosolized LPS in B6/129 control, IL-1R1 KO (-/-), TNF-alpha KO, and TNF-alpha , IL-1R1 double-KO mice.



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Fig. 4.   Concentration of total cells (left) and PMNs (right) in whole lung lavage fluid after 4-h exposure to aerosolized LPS in mice receiving adenoviral vectors containing either LacZ (control), TNFR, IL1R1, or both TNFR and IL-1R1.



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Fig. 5.   Concentration of specific cytokines [TNF-alpha (A), IL-1beta (B), macrophage inflammatory protein (MIP)-2 (C), and IL-6 (D)] in lung lavage fluid after 4-h exposure to aerosolized LPS in mice receiving adenoviral vectors containing either LacZ (control), TNFR, IL1R1, or both TNFR and IL-1R1. * P < 0.05 compared with LacZ control.

RNase protection assays were performed to determine whether the marked upregulation in mRNA abundance of the inflammatory cytokines (TNF-alpha , IL-1beta , and MIP-2) that occurred after inhaled LPS exposure was reduced by blockade of TNF-alpha , IL-1beta , or both. After a 4-h exposure to LPS, there was a dramatic increase in mRNA abundance for each of these cytokines compared with that in unexposed mice. There were no differences observed between control mice and knockout mice in mRNA abundance for any of the inflammatory cytokines studied, except for the presence of increased RNA expression of TNF-alpha in the TNF-alpha -deficient mice because the knockout mice only have a deletion of a central portion of the coding sequence and a nonfunctional mRNA is detected by the probe (data not shown). mRNA abundance of TNF-alpha , IL-1, and MIP-2 were all similar in control mice versus mice receiving either one or both of the adenoviral vectors containing the TNFR or IL-1R1. IL-6 could not be detected in any significant amount in any of the groups. A representative RNase protection assay is shown in Fig. 6; although MIP-2 expression was slightly diminished after the addition of adenoviral vectors with TNFR, IL-1R1, or both in the assay shown, this difference was not consistent over multiple experiments.


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Fig. 6.   Representative RNase protection assay demonstrating relative abundance of TNF-alpha , IL-1beta , and MIP-2 mRNAs in whole lungs from unexposed mice and after 4-h exposure to aerosolized LPS in mice receiving adenoviral vectors containing either LacZ (control), TNFR, IL1R1, or both TNFR and IL-1R1. L32, which encodes a ubiquitously expressed ribosome subunit protein, is shown for comparison of loading.

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).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our results indicate that TNF-alpha and/or IL-1beta 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-alpha and IL-1beta are not individually or collectively essential to the airway response to inhaled LPS.

However, several lines of evidence indicate that TNF-alpha and/or IL-1beta should play significant roles in LPS-induced airway inflammation. The early localization of both TNF-alpha and IL-1beta 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-alpha , IL-1alpha , IL-1beta , MIP-2 (murine homologue of IL-8), MIP-1alpha , MIP-1beta , IL-6, transforming growth factor-beta 1, and interferon-gamma 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-alpha and IL-1beta 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-alpha and/or IL-1beta 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-alpha is liberated into the airways after intravascular challenge but at a markedly decreased level compared with TNF-alpha 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. beta 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-alpha nor IL-1beta 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-alpha or IL-1beta 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

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