Phagocytosis mediated by three distinct Fc gamma receptor classes on human leukocytes.

We have evaluated the capacity of the three major classes of human Fc gamma R to mediate phagocytosis by measuring the ability of adherent phagocytes to internalize erythrocytes coated with anti-Fc gamma R mAb. Five different cell types were studied, freshly purified monocytes, cultured monocytes, alveolar macrophages, freshly purified polymorphonuclear neutrophilic leukocytes, and PMNs cultured in IFN-gamma. Fc gamma RI and Fc gamma RII on whichever cells they were expressed were capable of phagocytosing anti-Fc gamma R mAb-coated erythrocytes. Furthermore, Fc gamma RIII on mononuclear phagocytes, which appears to be a conventional integral membrane protein that spans the lipid bilayer, was capable of phagocytosing anti-Fc gamma RIII-coated erythrocytes. However, Fc gamma RIII on neutrophils, a molecule linked to the membrane by a phosphatidylinositol-glycan moiety, although binding anti-Fc gamma RIII-coated erythrocytes vigorously was incapable of mounting a phagocytic response. This deficiency correlates with the limited capacity of Fc gamma RIII on neutrophils to mediate superoxide generation and antibody-dependent cell-mediated cytotoxicity and it may be related to the unique structural features of Fc gamma RIII.

The human lung mucosa drives differential Mycobacterium tuberculosis infection outcome in the alveolar epithelium.

Mycobacterium tuberculosis (M.tb) is deposited into the alveolus where it first encounters the alveolar lining fluid (ALF) prior contacts host cells. We demonstrated that M.tb-exposure to human ALF alters its cell surface, driving better M.tb infection control by professional phagocytes. Contrary to these findings, our results with non-professional phagocytes alveolar epithelial cells (ATs) define two distinct subsets of human ALFs; where M.tb exposure to Low (L)-ALF or High(H)-ALF results in low or high intracellular bacterial growth rates in ATs, respectively. H-ALF exposed-M.tb growth within ATs was independent of M.tb-uptake, M.tb-trafficking, and M.tb-infection induced cytotoxicity; however, it was associated with enhanced bacterial replication within LAMP-1+/ABCA1+ compartments. H-ALF exposed-M.tb infection of ATs decreased AT immune mediator production, decreased AT surface adhesion expression, and downregulated macrophage inflammatory responses. Composition analysis of H-ALF vs. L-ALF showed H-ALF with higher protein tyrosine nitration and less functional ALF-innate proteins important in M.tb pathogenesis. Replenishment of H-ALF with functional ALF-innate proteins reversed the H-ALF-M.tb growth rate to the levels observed for L-ALF-M.tb. These results indicate that dysfunctionality of innate proteins in the H-ALF phenotype promotes M.tb replication within ATs, while limiting inflammation and phagocyte activation, thus potentiating ATs as a reservoir for M.tb replication and survival.

Exposure to human alveolar lining fluid enhances Mycobacterium bovis BCG vaccine efficacy against Mycobacterium tuberculosis infection in a CD8+ T-cell-dependent manner.

Current tuberculosis (TB) treatments include chemotherapy and preventative vaccination with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In humans, however, BCG vaccination fails to fully protect against pulmonary TB. Few studies have considered the impact of the human lung mucosa (alveolar lining fluid (ALF)), which modifies the Mycobacterium tuberculosis (M.tb) cell wall, revealing alternate antigenic epitopes on the bacterium surface that alter its pathogenicity. We hypothesized that ALF-induced modification of BCG would induce better protection against aerosol infection with M.tb. Here we vaccinated mice with ALF-exposed BCG, mimicking the mycobacterial cell surface properties that would be present in the lung during M.tb infection. ALF-exposed BCG-vaccinated mice were more effective at reducing M.tb bacterial burden in the lung and spleen, and had reduced lung inflammation at late stages of M.tb infection. Improved BCG efficacy was associated with increased numbers of memory CD8+ T cells, and CD8+ T cells with the potential to produce interferon-γ in the lung in response to M.tb challenge. Depletion studies confirmed an essential role for CD8+ T cells in controlling M.tb bacterial burden. We conclude that ALF modifications to the M.tb cell wall in vivo are relevant in the context of vaccine design.

Alveolar fluid neutrophil elastase activity in the adult respiratory distress syndrome is complexed to alpha-2-macroglobulin.

We characterized the elastase and antielastase activity of the alveolar fluid of seven patients with the adult respiratory distress syndrome (ARDS) and thirteen normal volunteers. Alpha-1-antitrypsin (A1AT) concentrations were 60-fold higher in ARDS as compared to normal lavage fluid (2,140 +/- 498 nM; 36.1 +/- 4.2 nM, respectively). ARDS fluid antineutrophil elastase activity was also considerably higher than that of normals (979 +/- 204 nM; 31.3 +/- 2.9 nM, respectively). Despite the antineutrophil elastase excess, 5 of 7 ARDS lavage samples contained elastase activity (mean, 6.1 +/- 2.4 pM) as assayed using low-molecular-mass substrate, while only 1 of 13 normal subjects had detectable elastase activity (0.2 pM) (P less than 0.01, compared with ARDS). That this activity was due to alpha-2-macroglobulin (A2MG)-complexed neutrophil elastase was evidenced by (a) the Sephadex G-75 elution profile; (b) the inactivity against insoluble [3H]elastin; (c) the inhibitory profile with phenylmethylsulfonyl fluoride, methoxy-succinyl-alanyl-alanyl-prolyl-valyl-chloromethylketone, ethylene diamine tetraacetic acid, and A1AT; and (d) the immobilization by A2MG antibody bound to polystyrene plates. Furthermore, in agreement with the predicted affinity of A1AT and A2MG for neutrophil elastase, the ratio of A2MG to A1AT in the fluid (0.57%) coincided with the ratio of the A2MG- to A1AT-complexed elastase (0.36%). These findings suggest that the net lung protease-antiprotease balance in ARDS is shifted largely in favor of the antiproteases (chiefly A1AT), and that the antiproteases, A1AT and A2MG, have similar affinities for neutrophil elastase in vivo.

Antineutrophil cytoplasmic antibodies induce monocyte IL-8 release. Role of surface proteinase-3, alpha1-antitrypsin, and Fcgamma receptors.

Cytoplasmic antineutrophil cytoplasmic antibodies (cANCA) that accompany the neutrophilic vasculitis seen in Wegener's granulomatosis (WG), are directed against proteinase-3 (PR-3), a serine proteinase which is located in azurophilic granules of neutrophils and monocytes. PR-3, when expressed on the surface of TNFalpha-primed neutrophils, can directly activate neutrophils by complexing cANCA and promoting concomitant Fcgamma receptor (FcgammaR) cross-linking. Although the neutrophil's pathogenic role in WG has been studied, the role of the monocyte has not been explored. The monocyte, with its ability to release cytokines and regulate neutrophil influx, also expresses PR-3. Therefore, the monocyte may play a significant role in WG via the interaction of surface PR-3 with cANCA, inducing cytokine release by the monocyte. To test this hypothesis, monocytes were studied for PR-3 expression and for IL-8 release in response to cANCA IgG. PBMC obtained from healthy donors displayed dramatic surface PR-3 expression as detected by immunohistochemistry and flow cytometry in response to 0. 5-h pulse with TNFalpha (2 ng/ml). Purified monoclonal anti-PR-3 IgG added to TNFalpha-primed PBMC induced 45-fold more IL-8 release than an isotype control antibody. Furthermore, alpha 1-antitrypsin (alpha1-AT), the primary PR-3 antiprotease, inhibited the anti-PR-3 induced IL-8 release by 80%. Importantly, Fab and F(ab')2 fragments of anti-PR-3 IgG, which do not result in Fcgamma receptor cross-linking, do not induce IL-8 release. As a correlate, IgG isolated from cANCA positive patients with WG induced six times as much PBMC IL-8 release as compared to IgG isolated from normal healthy volunteers. Consistent with PR-3 associated IL-8 induction, alpha1-AT significantly inhibited this effect. These observations suggest that cANCA may recruit and target neutrophils through promoting monocyte IL-8 release. This induction is mediated via Fcgamma receptor cross-linking and is regulated in part by alpha1-AT.

Modulation of alveolar macrophage-driven fibroblast proliferation by alternative macrophage mediators.

Tissue fibrosis results, in part, from an interaction between growth regulatory molecules released by mononuclear phagocytes and fibroblasts. In the chronic interstitial lung disorders, alveolar macrophages, the mononuclear phagocytes of the lung, are known to spontaneously release two growth factors for fibroblasts, fibronectin and alveolar macrophage-derived growth factor (AMDGF) that together stimulate nonreplicating lung fibroblasts to divide. In addition to these two primary growth promoting signals, alveolar macrophages are able to release other mediators that may have a potential role in modulating lung fibroblast replication in response to these primary signals, including interferon gamma (IFN gamma), prostaglandin E2 (PGE2), and interleukin 1 (IL-1). To evaluate this possibility, we examined the effect of each of these other mediators on lung fibroblast replication in response to fibronectin and AMDGF in serum-free, defined medium. IFN gamma had no effect on fibroblast replication. In contrast, PGE2 resulted in a dose-dependent inhibition of fibroblast replication in response to fibronectin and AMDGF with 50% of the maximum inhibition observed at a PGE2 concentration of less than 10 ng/ml. IL-1, while not active as a primary growth promoting signal, at concentrations of 4-10 U/ml, augmented fibroblast replication in response to fibronectin and AMDGF by 10 to 15%. Temporally, the growth augmenting effect of IL-1 occurred early in the G1 phase of the cell cycle. These data indicate that lung fibroblast replication in response to two of the primary growth promoting signals spontaneously released by alveolar macrophages in the interstitial lung disorders, while uninfluenced by IFN gamma, can be inhibited by PGE2 and modestly augmented by IL-1. Understanding the relevant fibroblast growth modulatory signals within the alveolar microenvironment in the chronic interstitial disorders may lead to rational therapeutic strategies designed to interrupt the fibrotic process.

Normal human alveolar macrophages obtained by bronchoalveolar lavage have a limited capacity to release interleukin-1.

Interleukin-1 (IL-1) is a mediator released by stimulated mononuclear phagocytes that is thought to play an important role in modulating T and B lymphocyte activation as well as in contributing to the febrile response and other inflammatory processes. Circulating mononuclear phagocytes, blood monocytes, readily release IL-1 when stimulated. However, the ability of lung mononuclear phagocytes, alveolar macrophages, to dispose of the large daily burden of inhaled antigens without stimulating an inflammatory response suggests that the release of IL-1 by alveolar macrophages may differ significantly from that of blood monocytes. To evaluate this hypothesis, normal autologous alveolar macrophages, obtained by bronchoalveolar lavage, were compared with blood monocytes for their ability to release IL-1 in response to a standard stimulus, lipopolysaccharide (LPS). Alveolar macrophages were found to be at least 1,000 times less sensitive to LPS than blood monocytes. Furthermore, alveolar macrophages released significantly less IL-1 than blood monocytes (26 +/- 11 vs. 128 +/- 21 U/10(6) cells X 24 h, respectively, after stimulation with 10 micrograms/ml of LPS, P less than 0.001). This difference was not due to the release of substances by macrophages, which inhibited lymphocyte proliferation in response to IL-1, or to degradation of IL-1 by macrophages. Culturing macrophages in the presence of indomethacin and dialysis of macrophage supernatants did not affect the difference, and culturing macrophages with monocytes did not decrease detectable IL-1 activity from the monocytes. The IL-1 produced by the two cell types was indistinguishable by anion-exchange chromatography, gel filtration, and isoelectric focusing. In addition, consistent with the findings for alveolar macrophages, macrophages generated by the in vitro maturation of blood monocytes were also deficient in their ability to release IL-1. These findings suggest that if the population of alveolar macrophages obtained by bronchoalveolar lavage represents the total in vivo population of alveolar macrophages, although normal human macrophages are capable of IL-1 release, they are relatively limited in this ability, and this limitation seems to be linked to the maturational state of the mononuclear phagocyte. These observations may explain, in part, the ability of alveolar macrophages to clear the airspaces of foreign antigens without extensive activation of other pulmonary inflammatory and immune effector cells.

Pulmonary defense and the human cathelicidin hCAP-18/LL-37.

Antimicrobial peptides form an important component of the innate immune system. The cathelicidin family, a key member of the antimicrobial peptide defenses, has been highly conserved throughout evolution. Though widespread in mammals, there is currently only one identified human example, hCAP-18/LL-37. The cathelicidins have been found to have multiple functions, in addition to their known antimicrobial and lipopolysaccharide-neutralizing effects. As a result, they profoundly affect both innate and adaptive immunity. Currently, antimicrobial peptides are being evaluated as therapeutic drugs in disease states as diverse as oral mucositis, cystic fibrosis, and septic shock. One such peptide, the cathelicidin hCAP-18/LL-37, is reviewed in detail in the context of its role in lung physiology and defense.

Processing proIL-1 beta decreases detection by a proIL-1 beta specific ELISA but increases detection by a conventional ELISA.

Recent investigations have noted that conventional IL-1 beta enzyme linked immunoassays (ELISA) may underestimate proIL-1 beta concentrations. In an attempt to circumvent this problem, we have devised a proIL-1 beta specific ELISA which sandwiches proIL-1 beta between a carboxy-terminus specific, capture antibody, and an amino-terminus specific, detection antibody. The amino-terminus specific antibody was generated against amino acids 3-21 of the intact proIL-1 beta molecule. This sandwich ELISA does not recognize mature 17 kDa IL-1 beta and is not inhibited by the coexistence of mature, 17 kDa IL-1 beta. We compared this proIL-1 beta specific ELISA (amino-terminus ELISA) to the conventional IL-1 beta ELISA (carboxy-terminus ELISA) on test samples that contained either proIL-1 beta or mature IL-1 beta. When purified proIL-1 beta is cleaved by increasing concentrations of IL-1 beta converting enzyme or porcine pancreatic elastase, there is a dose dependent increase in the signal from the conventional IL-1 beta ELISA and a concurrent decrease in signal from the proIL-1 beta specific ELISA. Furthermore, when the proIL-1 beta specific ELISA is used to quantify cellular sources of IL-1 beta, there is no detectable release of proIL-1 beta into the supernatants from either fresh blood monocytes or alveolar macrophages, despite detectable mature IL-1 beta. The cell associated compartment of IL-1 beta is largely proIL-1 beta and the relative amounts of IL-1 beta that remain intracellularly are quite large. Specifically, when assayed at 18 h, the proIL-1 beta ELISA detected 4.6 +/- 1.4 ng/ml per 10(6) monocytes and 13.8 ng/ml per 10(6) macrophages vs. 0.8 +/- 0.3 ng/ml/10(6) monocytes and 1.6 ng/ml/10(6) macrophages by conventional IL-1 beta ELISA. Thus, the 5-10-fold deficit in the detection of intracellular IL-1 beta by a conventional IL-1 beta ELISA, combined with the enhanced detection after enzymatic processing of proIL-1 beta, confirms that a proIL-1 beta specific ELISA is needed to accurately quantify proIL-1 beta.

Detection of soluble type II receptor in the presence of its natural ligand IL-1 beta. Quantification by sandwich ELISA.

The type II interleukin-1 receptor (IL-1R II) is a newly described 60-68 kDa protein expressed on monocytes, neutrophils, and lymphocytes. It is hypothesized that a 45 kDa soluble form of the IL-1R II attenuates the proinflammatory effects of IL-1 by preventing its binding to the type I IL-1 receptor. However, very little information exists regarding the detection of soluble IL-1R II. Specifically, there are no reports to date characterizing IL-1R II detection by enzyme-linked immunoassay in the presence of IL-1 beta or characterizing IL-1 beta detection in the presence of IL-1R II. This study addresses the detection and quantitation of IL-1R II and IL-1 beta by a number of sandwich ELISA formats and characterizes the sensitivity of detection in the presence of competitive cytokines. We generated two distinct IL-1R II sandwich ELISAs that can detect receptor down to a level of 50 pg/ml. One, M22/R2, detects only unbound IL-1R II and the other, M2/R2, detects both bound and unbound IL-1R II. In this context, a 4:1 molar ratio of IL-1 beta to IL-1R II interferes with the IL-1R II detection by the M22/R2 but not the M2/R2 ELISA. Conversely, IL-1R II at physiologically relevant concentrations interferes with the detection of IL-1 beta by three distinct IL-1 beta ELISA formats. Taken together, these studies suggest that when measuring samples that may contain both IL-1 beta and IL-1R II, careful attention must be given to assay specificity.

Sandwich ELISA formats designed to detect 17 kDa IL-1 beta significantly underestimate 35 kDa IL-1 beta.

The IL-1 beta precursor (proIL-1 beta) represents a significant component of total IL-1 beta production in certain cell types such as keratinocytes, fibroblasts and alveolar macrophages. It has been presumed that immunodetection systems for the mature 17 kDa IL-1 beta can be used interchangeably for the 35 kDa intracellular proIL-1 beta. However, during attempts to purify alveolar macrophage proIL-1 beta, we found that conventional enzyme-linked immunoassays (ELISAs) (using antibodies directed against the 17 kDa mature IL-1 beta) underestimated the amounts of 35 kDa proIL-1 beta by at least ten-fold compared to detection by Western blot techniques. This difference was due to the fact that ELISAs, with an antigen capture format (i.e., that use more than one epitope), can more readily see these distinct epitopes on mature or partially processed IL-1 beta than on the proIL-1 beta molecule. This problem does not occur with the Western blot technique, either because only one antibody is needed and hence there is no stearic blockade of a second epitope or because it denatures 35 kDa proIL-1 beta during the immobilization step, presumably better exposing epitopes as expressed on mature 17 kDa IL-1 beta. The problem with the ELISA can be partially corrected by proteolytic removal of the aminoterminus of 35 kDa proIL-1 beta with neutrophil elastase. More accurate determinations of proIL-1 beta by ELISA can be made by using 35 kDa proIL-1 beta as the reference standard (when the 35 kDa proIL-1 beta is free of molecular weight IL-1 beta). These data suggest that there are conformational differences between the carboxyterminus of 35 kDa proIL-1 beta and mature 17 kDa IL-1 beta which may affect immunodetection when using antibodies directed against mature 17 kDa IL-1 beta.

Acute occlusion of a mainstem bronchus by a rapidly expanding foreign body.

We report a case of an 85-year-old woman who presented with acute respiratory failure, caused by aspirating a sucralfate tablet that totally occluded her left main-stem bronchus. Acute respiratory failure resolved after bronchoscopic removal of the markedly expanded tablet. To our knowledge, the acute obstruction of a main-stem bronchus by an aspirated foreign body has not been previously described in adults. We believe that the unusual properties of sucralfate tablets (rapid expansion and mucosal binding) were contributing factors in this patient.

HIV-associated bronchiolitis obliterans organizing pneumonia.

A man with serologic evidence of HIV infection and a depressed T-helper:suppressor ratio developed fever, pulmonary infiltrates, and respiratory failure. Bronchoalveolar lavage and transbronchial biopsy failed to reveal an infectious cause; however, an open lung biopsy demonstrated classic bronchiolitis obliterans organizing pneumonia. The patient responded completely to corticosteroids. To the best of our knowledge, this represents a previously undescribed and readily treatable cause of respiratory failure in patients with HIV infections.

BAL and serum IgG levels in healthy asymptomatic HIV-infected patients.

OBJECTIVES: To determine if the increased susceptibility to bacterial infection in asymptomatic HIV-infected patients is associated with decreased total IgG or IgG2 levels in lung epithelial lining fluid. BACKGROUND: A decrease in lung IgG levels or subtypes has been proposed as contributing to the increased risk of bacterial lung infections in HIV-infected patients. Previous studies measuring lung lavage IgG concentrations have been inconsistent. METHODS: Twenty-three HIV patients and 25 control subjects underwent BAL. Both patient groups were of similar age, and had similar pulmonary function studies and body mass index. Smokers were equally represented in both groups, and the majority of subjects in both groups were male. Total IgG and IgG2 levels in lavage fluid were assayed in both cohorts and compared using a two-tailed Student's t test. RESULTS: The lung lining fluid IgG level in HIV-infected patients was 0.19 +/- 0.13 microg/microg of protein (mean +/- SD) vs 0.11 +/- 0.09 microg/microg of protein in control subjects (p < 0.05). The IgG(2) level in HIV patients was 0.034 +/- 0.038 microg/microg of protein and 0.014 +/- 0.01 microg/microg of protein in control subjects (p = 0.054). Lavage IgG levels reflected serum IgG values (correlation coefficient, 0.56; p < 0.001) but did not correlate with lung immunoglobulin-producing cells. CONCLUSIONS: The increased susceptibility to bacterial pneumonia in asymptomatic HIV-infected individuals is neither explained by depressed total IgG levels nor a deficiency in IgG(2) levels in the lungs. The strong correlation between serum and lavage IgG levels suggests that lavage IgG derives from serum.

Effect of O2 partial pressure on the myeloperoxidase pathway of neutrophils.

Neutrophils recruited to different tissues undergo respiratory burst activity at widely different PO2 levels. The present study investigated the in vitro effects of PO2 on neutrophil oxidative metabolism. When neutrophils were stimulated with either zymosan or phorbol myristate acetate (PMA) under different PO2's (0-700 Torr), hexose monophosphate shunt activity, H2O2, and hydroxyl radical (OH.) production were directly related to the level of PO2. Neutrophils functioned surprisingly well at PO2's as low as 10 Torr, where metabolic burst activity was prolonged and usually exceeded 50% of maximal values. The production of neutrophil stable oxidants and hypochlorous acid (HOCl) by zymosan-stimulated neutrophils was also directly related to PO2. In contrast, the production of stable oxidants and HOCl by PMA-stimulated neutrophils was significantly higher at 10 Torr compared with 700 Torr. The decrease in stable oxidant production by PMA-stimulated neutrophils at elevated PO2's was explained by both increased destruction of stable oxidant products and by decreased availability of the precursor HOCl. Superoxide dismutase and the OH. scavenger benzoate partially prevented the fall in stable oxidants at elevated PO2's. Measurements of stable oxidants in PMA-stimulated supernates generated at 10 and 700 Torr correlated with the ability of these supernates to decrease the elastase inhibitory capacity of the serum antiprotease alpha 1-antitrypsin. These findings suggest that different PO2's alter the magnitude and pattern of neutrophil oxidative metabolism.

Effect of dimethylthiourea on the neutrophil myeloperoxidase pathway.

The sulfur-centered compound dimethylthiourea (DMTU) affords antioxidant protection in animal models of acute lung injury, an effect that has been attributed to its OH. scavenging properties. Although DMTU can also react with H2O2 in certain experimental systems, the effect of DMTU on the neutrophil myeloperoxidase (MPO) pathway has not been studied. DMTU (1-10 mM) completely blocked stable oxidants and hypochlorous acid formation by phorbol myristate acetate- and zymosan-stimulated neutrophils. DMTU also provided complete inhibition when incubated with cell-free supernatants after the formation of the MPO products. DMTU prevented the oxidative inactivation of alpha 1-antitrypsin by neutrophil-stable oxidants. Evidence that DMTU was oxidized by the MPO products was obtained by titration of oxidized DMTU with reduced glutathione. Surprisingly, supernatants from cells incubated with DMTU (10 mM) consumed two- to threefold higher amounts of reduced glutathione than supernatants from cells incubated with taurine (15 mM). Metabolic studies with stimulated neutrophils and experiments with the MPO enzyme system in a cell-free system suggested that DMTU acts by scavenging the products of the MPO pathway rather than by blocking H2O2 production in the intact cell. These findings demonstrate that DMTU blocks the neutrophil MPO pathway in addition to its known ability to scavenge other reactive O2 species. The capacity of DMTU to scavenge MPO products may explain some of its protective effects in acute lung injury.

The pathogenesis of sepsis. Factors that modulate the response to gram-negative bacterial infection.

Gram-negative bacteria gain access to the bloodstream by evading host defenses. Once in circulation, lipopolysaccharide interacts with the host receptor CD14 and initiates the host's immune response. Lipolysaccharide stimulates the host to produce a cascade of mediators that activate and target leukocytes, opsonize the bacteria, and induce fever to defend against the invading bacteria. Unregulated release of these mediators, however, leads to the production of vasoactive substances, activation of the clotting cascade, and diminution of cardiac performance, which leads to the sepsis syndrome. This article discusses the pathogenic events that lead to sepsis syndrome and reviews critical steps in regulating these inflammatory mediators to allow the host to recover from gram-negative bacteremia.

Induction of interleukin-8 release by lung epithelium with cystic fibrosis epithelial lining fluid is marginally affected by inhibitors of interleukin-1beta.

Interleukin-1beta (IL-1beta) and neutrophil elastase (NE) are present in the epithelial lining fluid (ELF) of patients with cystic fibrosis (CF). Both factors activate surrounding cells including lung epithelial cells, causing release of IL-8, a potent chemoattractant for neutrophils. Previous studies showed up-regulation of IL-8 release by lung epithelial cells as a function of NE in CF; however, few studies addressed the relationship between IL-1beta and activation of lung epithelial cells in CF lungs. Confluent layers of A549 cells, a type II-like human lung epithelial cell line, were incubated overnight with IL-1beta (0-5 ng/ml) or NE (100 nM), and supernatants were analyzed for IL-8 by enzyme-linked immunosorbent assay (ELISA). Both IL-1beta and NE led to a significant increase in IL-8: 12.8 +/- 2.8 ng/ml and 0.8 +/- 0.3 ng/ml, respectively. Next, bronchoalveolar lavage (BAL) samples were obtained from one healthy adult volunteer and six patients with CF and measured for IL-8 and IL-1beta concentrations by ELISA. Both IL-8 (range 169.00 +/- 56.57 to 1742.04 +/- 338.98 pg/ml) and IL-1beta (range 0-24.26 +/- 0.52 pg/ml) were detected in CF specimens, whereas neither was detected in the volunteer's specimen. Normal and CF BALs then were incubated overnight at a 1:10 dilution with confluent A549 cells. Analysis by ELISA of cell-free supernatants revealed increased IL-8 production from cells stimulated with CF BALs only. Similar experiments were performed with BAL supernatants that had been incubated with soluble IL-1 type II receptor, soluble IL-1 receptor antagonist, or a peptide inhibitor of NE. Addition of IL-1 inhibitors had a marginal effect on the amount of IL-8 release after incubation with CF BAL samples, whereas inhibition of NE had no effect. Our results indicate that other factors present in ELF in CF account for IL-8 release from lung epithelial cells.