An open-label trial of rituximab therapy in pulmonary alveolar proteinosis.

Rituximab, a monoclonal antibody directed against the B-lymphocyte antigen CD20, has shown promise in several autoimmune disorders. Pulmonary alveolar proteinosis (PAP) is an autoimmune disorder characterised by autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF). An open-label, proof-of-concept phase II clinical trial was conducted in 10 PAP patients. The intervention consisted of two intravenous infusions of rituximab (1,000 mg) 15 days apart. Bronchoalveolar lavage (BAL) fluid and peripheral blood samples were collected. The primary outcome was improvement in arterial blood oxygenation. Both arterial oxygen tension and alveolar-arterial oxygen tension difference in room air improved in seven out of the nine patients completing the study. Lung function and high-resolution computed tomography scans, which were secondary outcomes, also improved. Peripheral blood CD19+ B-lymphocytes decreased from mean ± sem 15 ± 2% to <0.05% (n = 10) 15 days post-therapy. This decrease persisted for 3 months in all patients; at 6 months, CD19+ B-cells were detected in four out of seven patients (5 ± 2%). Total anti-GM-CSF immunoglobulin (Ig)G levels from baseline to 6 months were decreased in BAL fluids (n = 8) but unchanged in sera (n = 9). In this PAP cohort: 1) rituximab was well-tolerated and effectively ameliorated lung disease; and 2) reduction in anti-GM-CSF IgG levels in the lung correlated with disease changes, suggesting that disease pathogenesis is related to autoantibody levels in the target organ.

Human alveolar macrophages and monocytes as a source and target for nitric oxide.

Nitric oxide (NO) is synthesized in the lung and this free radical participates in a wide array of regulatory, protective, and adverse interactions with cells. Both excess NO and its insufficiency have been implicated in the pathogenesis of numerous lung diseases with inflammatory components. Much of the available data concerning the source and regulation of NO production is derived from rodent systems. However, the requirements for NO production are more stringent in human monocytes/macrophages than in rodent systems. In contrast to rodent macrophages, human moncytes/macrophages generally do not respond to cytokine triggers with NO production [J. Leukocyte Biol. 58 (1995) 643, J. Exp. Med. 181 (1995) 735] and if NO is detected the levels are generally low [J. Leukocyte Biol. 58 (1995) 643]. The regulation of macrophage NO in the human appears to be a more selective and variable process than that seen in the rodent macrophages. In the human lung, the function of NO as toxic pro-inflammatory or protective anti-inflammatory agent is unresolved. While not a major source of NO in the human lung, the alveolar macrophage is an important producer of cytokines and this production may be modified by NO. Clear evidence of abnormalities in NO levels in the lungs of patients with asthma, bronchiectasis, viral infections, lung cancer and primary pulmonary hypertension (PPH) has been documented. Elevated inflammatory cytokines and oxidant production have been associated with all of these disease states. In terms of cytokine production, NO has been shown to decrease nuclear factor kappa B (NF-kappaB) activation. However, oxidants may interact with NO to form toxic compounds (e.g., NO combines with superoxide anion to form peroxynitrite). Furthermore, such reactions may decrease the availability of NO for blocking inflammatory cytokine production. Thus, available data suggests that a multiplicity of factors affect NO regulatory properties in inflammatory situations.

Nitric oxide synthase 2 through an autocrine loop via respiratory epithelial cell-derived mediator.

Respiratory epithelium expresses nitric oxide synthase 2 (NOS2) continuously in vivo; however, mechanisms responsible for its expression are only partially understood. We definitively identify an autocrine mechanism of induction and maintenance of NOS2 in human airway epithelial cells through the synthesis and secretion of a soluble mediator. Short exposure of human airway cells to interferon (IFN)-gamma leads to prolonged NOS2 expression. Transfer of the overlying culture medium (conditioned medium) induces NOS2 expression in other airway epithelial cells, suggesting the presence of an intermediary substance regulating NOS2 expression in an autocrine loop. Characterization of the soluble mediator reveals that it is stable and transferable in conditioned medium for up to 7 days. However, soluble mediator does not induce NOS2 mRNA in human alveolar macrophages, indicating that the response to soluble mediator is unique to human respiratory epithelium. Soluble mediator is heat labile but is not inactivated by acid treatment, unlike IFN-gamma itself. Importantly, IFN regulatory factor-1, which is critical for murine NOS2 expression, is expressed and activated by soluble mediator through the signal transducer and activator of transcription-1-dependent pathway. Based on these findings, we propose novel regulatory mechanisms for NOS2 expression in human airway epithelium.

NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response.

A wealth of evidence supports increased NO (NO.) in asthma, but its roles are unknown. To investigate how NO participates in inflammatory airway events in asthma, we measured NO. and NO. chemical reaction products [nitrite, nitrate, S-nitrosothiols (SNO), and nitrotyrosine] before, immediately and 48 h after bronchoscopic antigen (Ag) challenge of the peripheral airways in atopic asthmatic individuals and nonatopic healthy controls. Strikingly, NO(3)(-) was the only NO. derivative to increase during the immediate Ag-induced asthmatic response and continued to increase over 2-fold at 48 h after Ag challenge in contrast to controls [P < 0.05]. NO(2)(-) was not affected by Ag challenge at 10 min or 48 h after Ag challenge. Although SNO was not detectable in asthmatic airways at baseline or immediately after Ag, SNO increased during the late response to levels found in healthy controls. A model of NO. dynamics derived from the current findings predicts that NO. may have harmful effects through formation of peroxynitrite, but also subserves an antioxidant role by consuming reactive oxygen species during the immediate asthmatic response, whereas nitrosylation during the late asthmatic response generates SNO, safe reservoirs for removal of toxic NO. derivatives.

Our new understanding of pulmonary alveolar proteinosis: what an internist needs to know.

Pulmonary alveolar proteinosis (PAP; the accumulation of surfactant lipids and proteins in the alveoli) has a number of infectious and environmental causes but is usually idiopathic. The clinical presentation of PAP is nonspecific; thus, the diagnosis is frequently missed, leading to inappropriate therapy and unnecessary morbidity. Recent advances suggest that a deficiency in granulocyte-macrophage colony-stimulating factor (GM-CSF) activity may lead to this surfactant accumulation. Anti-GM-CSF antibodies have been found in PAP patients, fueling speculation that PAP may be an autoimmune disease. These findings are being translated into novel forms of therapy.

Interleukin 10 (IL-10)-mediated inhibition of inflammatory cytokine production by human alveolar macrophages.

Alveolar macrophages are an important source of inflammatory cytokines in the lung. IL-10 has been shown to inhibit inflammatory cytokine production by human alveolar macrophages, but mechanisms are unclear. The purpose of the present study was to investigate whether IL-10 modified cytokine production by interference with transcriptional pathways. Alveolar macrophages were obtained from healthy controls by fiberoptic bronchoscopy and incubated with LPS+/-IL-10. Results indicated that steady state mRNA levels of tumour necrosis factor-alpha (TNF) and interleukin 1-beta (IL-1) decreased in the presence of IL-10. Consequently, electrophoretic mobility shift assays were performed using end-labelled nuclear factor-kappa B (NF-kappa B) or activator protein-1 (AP-1) probe. NF-kappa B binding was decreased in extracts from macrophages incubated for 4 h with LPS+IL-10 in comparison to those incubated with LPS alone. IL-10 also inhibited TNF secretion and NF-kappa B activation induced by another stimulus, staphylococcal toxin. Supershift assays revealed the presence of both p50 and p65 subunits of NF-kappa B. AP-1 was not affected by IL-10. Further examination of mechanisms indicated that IL-10 delayed the LPS-mediated degradation of the inhibitor protein I kappa B, thus delaying the nuclear translocation of the p65 subunit. These observations provide the first evidence that IL-10 antagonizes cytokine transcription in human alveolar macrophages by impeding the nuclear translocation of NF-kappa B by delaying the degradation of I kappa B.

Differential induction of extracellular glutathione peroxidase and nitric oxide synthase 2 in airways of healthy individuals exposed to 100% O(2) or cigarette smoke.

Reactive oxygen species (ROS) is increased in the airway during the inhalation of 100% O(2) or cigarette smoke and participates in the development of tracheobronchitis. We hypothesized that inhaled ROS upregulates local extracellular ROS scavenging systems or reactive molecules, e.g., nitric oxide (NO). Extracellular glutathione peroxidase (eGPx) is synthesized by airway epithelium and alveolar macrophages, secreted into the surface epithelial lining fluid, and functions as a first-line defense against inhaled ROS. NO, produced by NO synthase 2 (NOS2), combines rapidly with ROS to form reactive nitrogen species (RNS). In this study, human airway epithelial cells and alveolar macrophages from healthy individuals before and after exposure to 100% O(2) for 12 h, or from cigarette-smoking individuals, were evaluated for eGPx and NOS2 messenger RNA (mRNA) expression. Hyperoxia increased NOS2 mRNA in airway epithelial cells by 2.5-fold but did not increase eGPx mRNA. In contrast, cigarette smoke upregulated eGPx mRNA over 2-fold in airway epithelial cells and alveolar macrophages but did not affect NOS2 expression. In vitro exposure of respiratory epithelial cells to ROS or RNS also increased eGPx expression. These findings define distinct molecular responses in the airway to different inhaled ROS, which likely influences the susceptibility of the airway to oxidative injury.

Molecular mechanisms of increased nitric oxide (NO) in asthma: evidence for transcriptional and post-translational regulation of NO synthesis.

Evidence supporting increased nitric oxide (NO) in asthma is substantial, although the cellular and molecular mechanisms leading to increased NO are not known. Here, we provide a clear picture of the events regulating NO synthesis in the human asthmatic airway in vivo. We show that human airway epithelium has abundant expression of NO synthase II (NOSII) due to continuous transcriptional activation of the gene in vivo. Individuals with asthma have higher than normal NO concentrations and increased NOSII mRNA and protein due to transcriptional regulation through activation of Stat1. NOSII mRNA expression decreases in asthmatics receiving inhaled corticosteroid, treatment effective in reducing inflammation in asthmatic airways. In addition to transcriptional mechanisms, post-translational events contribute to increased NO synthesis. Specifically, high output production of NO is fueled by a previously unsuspected increase in the NOS substrate, l -arginine, in airway epithelial cells of asthmatic individuals. Finally, nitration of proteins in airway epithelium provide evidence of functional consequences of increased NO. In conclusion, these studies define multiple mechanisms that function coordinately to support high level NO synthesis in the asthmatic airway. These findings represent a crucial cornerstone for future therapeutic strategies aimed at regulating NO synthesis in asthma.

Eosinophils generate brominating oxidants in allergen-induced asthma.

Eosinophils promote tissue injury and contribute to the pathogenesis of allergen-triggered diseases like asthma, but the chemical basis of damage to eosinophil targets is unknown. We now demonstrate that eosinophil activation in vivo results in oxidative damage of proteins through bromination of tyrosine residues, a heretofore unrecognized pathway for covalent modification of biologic targets in human tissues. Mass spectrometric studies demonstrated that 3-bromotyrosine serves as a specific "molecular fingerprint" for proteins modified through the eosinophil peroxidase-H(2)O(2) system in the presence of plasma levels of halides. We applied a localized allergen challenge to model the effects of eosinophils and brominating oxidants in human lung injury. Endobronchial biopsy specimens from allergen-challenged lung segments of asthmatic, but not healthy control, subjects demonstrated significant enrichments in eosinophils and eosinophil peroxidase. Baseline levels of 3-bromotyrosine in bronchoalveolar lavage (BAL) proteins from mildly allergic asthmatic individuals were modestly but not statistically significantly elevated over those in control subjects. After exposure to segmental allergen challenge, lung segments of asthmatics, but not healthy control subjects, exhibited a >10-fold increase in BAL 3-bromotyrosine content, but only two- to threefold increases in 3-chlorotyrosine, a specific oxidation product formed by neutrophil- and monocyte-derived myeloperoxidase. These results identify reactive brominating species produced by eosinophils as a distinct class of oxidants formed in vivo. They also reveal eosinophil peroxidase as a potential therapeutic target for allergen-triggered inflammatory tissue injury in humans.

Pulmonary alveolar proteinosis is a disease of decreased availability of GM-CSF rather than an intrinsic cellular defect.

Granulocyte-macrophage colony stimulating factor (GM-CSF) deficient mice develop a pulmonary alveolar proteinosis (PAP) syndrome which is corrected by the administration/expression of GM-CSF. These observations implicate GM-CSF in the pathogenesis of human PAP. We hypothesized that human PAP may involve an intrinsic cellular defect in monocytes/macrophages with an inability to produce GM-CSF and/or respond to GM-CSF. Thus, we investigated the cytokine responses to GM-CSF and LPS from peripheral blood monocytes and alveolar macrophages from patients with idiopathic PAP and healthy controls. Macrophage inflammatory protein-1-alpha (MIP) was measured from GM-CSF-stimulated cells and GM-CSF was measured from LPS-stimulated cells by ELISA. The MIP and GM-CSF production by monocytes and alveolar macrophages did not differ between PAP patients and healthy controls. Growth of the GM-CSF-dependent human myeloid cell line TF-1 was inhibited by serum from all patients studied (n = 10) and all patients had anti-GM-CSF antibody in their serum. The BAL from PAP patients had less detectable GM-CSF by ELISA than healthy controls (P = 0.05); in contrast, the inhibitory cytokine, interleukin-10 (IL-10), was increased in PAP compared to controls (P = 0.04). IL-10 is a potent inhibitor of LPS-stimulated GM-CSF production from healthy control alveolar macrophages. These studies are the first to demonstrate that circulating monocytes and alveolar macrophages from PAP patients are able to synthesize GM-CSF and respond to GM-CSF, suggesting no intrinsic abnormalities in GM-CSF signaling. In addition, these observations suggest that PAP in a subset of patients is the result of decreased availability of GM-CSF due to GM-CSF blocking activity and reduced GM-CSF production by IL-10.

Exogenous granulocyte-macrophage colony-stimulating factor administration for pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by the accumulation of lipoproteinaceous material within the alveoli. Recent data suggest that granulocyte-macrophage colony- stimulating factor (GM-CSF) may be involved in the pathogenesis of PAP. To extend this understanding and clarify whether GM-CSF replacement confers benefit, we report the preliminary results for the first four patients in an open-label study of GM-CSF treatment for moderate exacerbation of PAP. All four patients had idiopathic PAP confirmed by open lung biopsy. Subcutaneous GM-CSF was self-administered once daily for 12 wk (dose escalation from 3 to 9 microg/kg/d). Response was assessed from symptom scores, arterial blood gas measurements, pulmonary function testing, and chest radiographs. Three of the four patients experienced symptomatic, physiologic, and radiographic improvement with GM-CSF. Responders experienced sufficient improvement in oxygenation as to eliminate the need for supplemental oxygen, and one patient was removed from the waiting list for lung transplantation. Improved oxygenation was not apparent until 8 to 12 wk after the start of therapy. Notably, expected increases in the peripheral white blood cell count did not occur, suggesting lack of a hematopoietic response to exogenous GM-CSF in PAP. We conclude that GM-CSF appears to benefit a subset of patients with adult PAP, and may represent an alternative to whole-lung lavage in treating the disease.

Increased alveolar macrophage nuclear factor-kappa B activation and macrophage inhibitory protein-1alpha levels in lung transplant patients.

BACKGROUND: Lung transplantation is increasingly used as the treatment for many end-stage pulmonary diseases. A major cause of morbidity and mortality in patients who undergo lung transplantation is rejection of the allograft. Proinflammatory macrophage-derived cytokines may sustain and/or enhance the immunological response to lung allograft antigens. Nuclear factor-kappa B (NF-kappaB) is a transcription factor that regulates the production of many of these cytokines and growth factors in alveolar macrophages (AMs). The aim of our study was to evaluate the activation of NF-kappaB in AMs and the levels of one of the proinflammatory cytokines whose production it controls, macrophage inhibitory protein-1alpha (MIP-1alpha), in AMs from transplanted lungs compared to those from healthy controls. METHODS: Twenty-eight (28) transplant recipients were included in the study. NFkappaB activation was evaluated by electrophoretic mobility shift assay of whole cell extracts and by immunohistochemical analysis on cytospin preparations. Concentrated bronchoalveolar lavage fluid was analyzed by enzyme-linked immunosorbent assay for MIP-1alpha levels. RESULTS: NF-kappaB was activated in alveolar macrophages from transplant patients as compared to healthy controls. MIP-1alpha levels in epithelial-lining fluid were elevated in transplant patients as compared to healthy controls. Increased MIP-1alpha levels correlated with viral infections in the transplant patients. Neither finding was found to correlate with acute rejection by transbronchial biopsy. CONCLUSIONS: These results demonstrate that NF-kappaB activation and MIP-1alpha levels are increased in transplanted lungs and may play a role in the inflammatory cytokine cascade that leads to the long-term tissue damage and allograft rejection in these patients.

Nitric oxide regulation of asthmatic airway inflammation with segmental allergen challenge.

BACKGROUND: Despite evidence of increased nitric oxide (NO) in asthmatic compared with healthy individuals, the role of NO in airway inflammation is unclear. OBJECTIVE: The purpose of the study was to determine the in vivo effects of localized allergen challenge on airway NO levels and transcription factor activation. METHODS: In this study localized allergen challenge was used as a model of asthmatic exacerbation to determine the relationship of NO to airway inflammation. RESULTS: With allergen challenge, asthmatic patients had a rise in airway NO levels, whereas NO levels in healthy controls did not change. The increased NO in asthma with allergen challenge compared with healthy control subjects was associated with an increase in inflammatory cytokines (GM-CSF and macrophage inflammatory protein-1) in epithelial lining fluid and eosinophilic infiltrate in bronchoalveolar lavage fluid (BAL) and biopsy specimens. To investigate the mechanisms of cytokine gene expression, activation of the transcription factors activator protein-1 and nuclear factor-kappaB (NF-kappaB) in cells from BAL were evaluated. Activator protein-1 was not activated before or after local allergen challenge. In contrast, NF-kappaB activation was less in BAL cells from asthmatic patients with increased NO in comparison with controls. CONCLUSION: Our studies are the first to suggest an inverse correlation between NF-kappaB and airway NO in a localized segmental allergen challenge model in allergic asthmatic patients. The current study demonstrates that activation of the inflammatory response (eg, cytokines, cellular infiltrate) in allergic asthmatic patients is temporally associated with increased airway NO. We propose that NO that is up-regulated by cytokines is part of an autoregulatory feedback loop (ie, allergen challenge stimulates inflammatory cytokine production, which in turn stimulates NO production, and NO down-regulates cytokine production).

Nitric oxide blocks nuclear factor-kappaB activation in alveolar macrophages.

Nitric oxide (NO) is an important endogenous regulatory molecule implicated in both proinflammatory and antiinflammatory processes in the lung. Previously, we demonstrated that in human alveolar macrophages (AM), NO decreased inflammatory cytokine production, including that of interleukin-1beta, tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha. One mechanism by which NO could regulate such diverse cytokine production is through effects on the transcription factor nuclear factor-kappaB (NF-kappaB), which controls the expression of the genes for these inflammatory cytokines and growth factors. We therefore investigated whether NO affects NF-kappaB activation in AM in vitro and in vivo. In vitro studies with AM showed that NF-kappaB activation by lipopolysaccharide (LPS) is decreased by NO in a dose-dependent manner. NO prevented an LPS-mediated decrease in the NF-kappaB inhibitory protein IkappaB-alpha. In asthma, airway NO levels are increased, whereas in primary pulmonary hypertension (PPH), airway NO levels are lower than in healthy lungs. In vivo investigations were conducted with freshly isolated AM from healthy controls, asthmatic individuals, and PPH patients. Healthy individuals had airway NO levels of 8 +/- 2 ppb (mean +/- SEM), which is associated with low NF-kappaB activation. Asthma patients with airway NO levels > 17 ppb showed minimal NF-kappaB activation, whereas asthmatic individuals with NO levels

Role of bronchoscopy in asthma research.

Over the past 15 years, much has been learned about the presence of airway inflammation in asthma through the use of investigative bronchoscopy. It has become quite clear that inflammation is present even in mild asthma. In addition to the eosinophils, T-lymphocytes and a variety of cytokines have been identified to play a prominent role in asthmatic inflammation. The concept of delayed asthmatic response after allergen exposure and its relationship to cellular inflammation and airway hyper-reactivity has become more clearly established. Our understanding of asthmatic airway inflammation, however, is incomplete. As interesting as the database has been so far, investigative FB has not defined a unique profile for patients with asthma. Specifically, lavage or endobronchial biopsy has not identified parameters that help in the diagnosis, assessment of disease severity, prognosis, or likelihood to respond to specific therapies. Also, the exact relationship between parameters in lavage compared with mucosal biopsy and how these are related to airway hyper-reactivity and the clinical syndrome of asthma remains poorly understood. In this regard, it must be confessed that currently FB with lavage and biopsy in asthmatics needs to be considered as a research tool for specimen retrieval to help characterize and express inflammation. Although these techniques have contributed immensely to our understanding of asthma pathogenesis, presently these techniques do not have any practical role or clinical usefulness.

Differential regulation of human blood monocyte and alveolar macrophage inflammatory cytokine production by nitric oxide.

BACKGROUND: Nitric oxide (NO) has been associated with airway inflammation in asthma. Our previous work suggests that NO functions in an anti-inflammatory capacity through downregulation of stimulated cytokine secretion by normal human alveolar macrophages. Functional differences between alveolar macrophages and blood monocytes are thought to be related to maturation. OBJECTIVE: The purpose of this study was to determine the effect of NO on stimulated cytokine production by monocytes from asthmatics and normal healthy controls. METHODS: Monocytes and alveolar macrophages were obtained from normal volunteers (n = 13) and asthmatics with atopy (n = 7). Monocyte and alveolar macrophage cultures were stimulated with 0.5 microgram/mL lipopolysaccharide +/- 1.0 mM DETA NONOate (releases NO in culture with t1/2 = 20 hours at 37 degrees C) and incubated for 24 hours. Cell-free supernatants were collected and assayed by ELISA for tumor necrosis factor-alpha (TNF) and granulocyte macrophage colony stimulating factor (GM-CSF). RESULTS: Nitric oxide did not inhibit TNF production in monocytes of asthmatics and normals (mean +/- SEM % TNF stimulation = 19.6 +/- 9.7). Similar to previous results, NO did inhibit alveolar macrophages (% TNF suppression = 60.6 +/- 4.4). To determine whether this differential effect of NO on the two cell populations was related to maturation, monocytes were matured by culture for 7 days. The in vitro matured monocytes demonstrated 51.7 +/- 7.9% suppression of TNF. For each cell population, the responses of the asthmatics and healthy controls were not different. The differential effect is not cytokine specific since similar results were obtained with GM-CSF. CONCLUSION: These results demonstrate a differential effect of NO on monocyte and alveolar macrophages cytokine regulation and this effect may be related to the state of maturation.

Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension.

Primary pulmonary hypertension (PPH) is a rare and fatal disease of unknown etiology. Inflammatory oxidant mechanisms and deficiency in nitric oxide (NO) have been implicated in the pathogenesis of pulmonary hypertension. In order to investigate abnormalities in oxidants and antioxidants in PPH, we studied intrapulmonary NO levels, biochemical reaction products of NO, and antioxidants (glutathione [GSH], glutathione peroxidase [GPx], and superoxide dismutase [SOD]) in patients with PPH (n = 8) and healthy controls (n = 8). Intrapulmonary gases and fluids were sampled at bronchoscopy. Pulmonary hypertension was determined by right-heart catheterization. NO and biochemical reaction products of NO in the lung were decreased in PPH patients in comparison with healthy controls (NO [ppb] in airway gases: control, 8 +/- 1; PPH, 2.8 +/- 0. 9; p = 0.016; and NO products [microM] in bronchoalveolar lavage fluid [BALF]: control, 3.3 +/- 1.05; PPH, 0.69 +/- 0.21; p = 0.03). However, GSH in the lungs of PPH patients was higher than in those of controls (GSH [microM] in BALF: 0.55 +/- 0.04; PPH, 0.9 +/- 0.1; p = 0.015). SOD and GPx activities were similar in the two groups (p >/= 0.50). Biochemical reaction products of NO were inversely correlated with pulmonary artery pressures (R = -0.713; p = 0.047) and with years since diagnosis of PPH (R = -0.776; p = 0.023). NO reaction products are formed through interactions between oxidants and NO, with the end products of reaction dependent upon the relative levels of the two types of molecules. The findings of the study therefore show that NO and oxidant reactions in the lung are related to the increased pulmonary artery pressures in PPH.

Constitutive NF-kappaB levels in human alveolar macrophages from normal volunteers.

Alveolar macrophages regulate the inflammatory and immune responses within the lung through cytokine production. Nuclear factor kappa B (NF-kappaB), a transcription factor, controls the synthesis of cytokines such as interleukin 1beta, tumour necrosis factor alpha, and interleukin 8. In quiescent cells, NF-kappaB is located in the cytosol as a dimer of protein components (p50, p65) bound to an inhibitor (IkappaB). Upon activation, NF-kappaB translocates to the nucleus and binds to DNA. To determine the constitutive level of NF-kappaB activation in non-smoking normal volunteers, immunohistochemical analysis of alveolar macrophages from 29 subjects was performed with antibody directed against the p65 component of NF-kappaB. These results were confirmed in four subjects by electrophoretic mobility shift assay (EMSA). A human monocytic cell line, THP-1 with and without endotoxin stimulation was used as positive and negative controls, respectively. The mean number of positive cells was 4.1%+/-0.8. EMSA performed on whole cell extracts from four normal volunteers demonstrated minimal constitutive binding compared to the positive control. Supershift assay revealed the presence of the p65 dimer. By both immunohistochemistry and EMSA, alveolar macrophages from healthy non-smoking individuals demonstrate minimal NF-kappaB activation. Immunohistochemistry is a sensitive and quantifiable technique requiring only a minimal number of cells, and this technique may be useful in monitoring small changes in NF-kappaB activation in inflammatory diseases of the lung.

Inhaled corticosteroids and beta-agonists inhibit oxidant production by bronchoalveolar lavage cells from normal volunteers in vivo.

To study the anti-inflammatory mechanisms of inhaled corticosteroids and beta-agonist therapies, we evaluated basal and stimulus-induced superoxide production by human airway inflammatory cells obtained by bronchoalveolar lavage from normal volunteers before and after 3 weeks of an inhaled corticosteroid (flunisolide) and beta-agonist (metaproterenol). Assay of superoxide production by the bronchoalveolar lavage cells was performed in the presence of media alone or media containing phorbol ester by optical density determination of reduced ferricytochrome c at 550 nm. Interleukin-1 beta released from unstimulated cells and cells stimulated with lipopolysaccharide was quantitated by enzyme immunoassay. Interestingly, phorbol ester-stimulated superoxide production was strikingly inhibited (P < 0.05) by inhaled therapies, while stimulus induced Interleukin-1 beta production was not significantly affected (P = 0.12). Suppression of oxidant production by airway inflammatory cells may be a major mechanism for the beneficial anti-inflammatory effects of inhaled corticosteroids and beta-agonists.

Perflubron decreases inflammatory cytokine production by human alveolar macrophages.

OBJECTIVE: To determine whether inflammatory cytokine production by stimulated human alveolar macrophages is affected by perflubron exposure. DESIGN: Controlled laboratory investigation of alveolar macrophage function in vitro. SETTING: Research laboratory. SUBJECTS: Cultured alveolar macrophages obtained by bronchoalveolar lavage from eleven normal volunteers. INTERVENTIONS: Endotoxin-stimulated alveolar macrophages were treated with perflubron. MEASUREMENTS AND MAIN RESULTS: Alveolar macrophages were stimulated for 1 hr with lipopolysaccharide and then treated with perflubron for 23 hrs. Cell-free supernatants were collected and cytokines were assayed by enzyme-linked immunosorbent assay. Tumor necrosis factor-alpha, interleukin-1, and interleukin-6 were stimulated by lipopolysaccharide (endotoxin) and all of these cytokines were significantly (p < .05) inhibited by perflubron. Cell viability was not affected by perflubron. Basal cytokine concentrations from unstimulated alveolar macrophages were not altered by perflubron. CONCLUSIONS: Exposure of stimulated human alveolar macrophages to perflubron in vitro decreases cytokine production. This observation suggests that perflubron may have anti-inflammatory activity.