Unstimulated Alveolar Macrophages Research Articles

Survey of the Transcription Factor Responses of Mouse Lung Alveolar Macrophages to Pneumocystis murina.

Pneumocystis jirovecii is a fungal pathogen that can cause life-threatening infections in individuals who are immunocompromised. Acquired via inhalation, upon entering the respiratory tract, the fungi first encounter innate immune cells such as alveolar macrophages (AMs). Relatively little is known about the AM cellular responses to the organism, and particularly transcription factor (TF) profiles leading to early host responses during infection. Utilizing the Mouse Transcription Factors RT2 Profiler™ PCR Array, we report an initial TF survey of these macrophage and Pneumocystis interactions. Expression levels of a panel of mouse TFs were compared between unstimulated and Pneumocystis murina-stimulated AMs. Interestingly, a number of TFs previously implicated in pathogen–host cell interactions were highly up- or downregulated, including hif1a and Pparg. qPCR experiments were further conducted to verify the results of these surveyed transcripts. Furthermore, with immunoblotting, we show that HIF-1A and PPAR-γ are indeed significantly upregulated and downregulated, respectively. Lastly, and importantly, we report that in the mouse model of Pneumocystis pneumonia (PCP), which mimics human Pneumocystis jirovecii pneumonia (PJP), qPCR analysis of Pneumocystis murina lungs also mimic the initial TF profile analysis, suggesting an importance for these TFs in immunocompromised hosts with Pneumocystis pneumonia. These data demonstrate the use of TF profiling in host AMs and Pneumocystis organism interactions that may lead to a better understanding of the specific inflammatory responses of the host to Pneumocystis pneumonia and may inform novel strategies for potential therapeutics.

The impact of alcohol use disorders on pulmonary immune cell inflammatory responses to Streptococcus pneumoniae.

Community-acquired pneumonia due to Streptococcus pneumoniae occurs commonly in alcohol use disorders (AUDs). Pneumonia in the AUD patient is associated with poorer outcomes, and specific therapies to mitigate disease severity in these patients do not exist. Numerous investigations have attributed increased severity of pneumonia in AUDs to aberrant function of the alveolar macrophage (AM), a lung immune cell critical in host defense initiation. No studies have examined the response of human AMs to S.pneumoniae in AUDs. We hypothesized that the inflammatory mediators released by AMs after S.pneumoniae stimulation would differ quantitatively in individuals with AUDs compared to non-AUD participants. We further postulated that AM inflammatory mediators would be diminished after exposure to the antioxidant, N-acetylcysteine (NAC). For comparison, responses of peripheral blood mononuclear cells (PBMCs) to pneumococcal protein were also examined. Otherwise healthy participants with AUDs and smoking-matched controls underwent bronchoalveolar lavage and peripheral blood sampling to obtain AMs and PBMCs, respectively. Freshly collected cells were cultured with increasing doses of heat-killed S.pneumoniae protein, with and without exposure to N-acetylcysteine. Cell culture supernatants were collected, and inflammatory mediators were measured, including interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. IFN-γ and IL-6 were significantly higher in unstimulated AM cell culture supernatants from subjects with AUDs. After stimulation with pneumococcal protein, a dose-response and time-dependent increase in pro-inflammatory cytokine production by both AMs and PBMCs was also observed; differences were not observed between AUD and non-AUD subjects. Addition of NAC to pneumococcal-stimulated AMs and PBMCs was generally associated with diminished cytokine production, with the exception of IL-1β that was elevated in AM culture supernatants from subjects with AUDs. Our observations suggest that AUDs contribute to basal alterations in AM pro-inflammatory cytokine elaboration, but did not support consistent differences in pneumococcal-stimulated AM or PBMC inflammatory mediator secretion that were referable to AUDs.

Carbon monoxide-releasing molecule, CORM-3, modulates alveolar macrophage M1/M2 phenotype in vitro.

Alveolar macrophages are key contributors to both the promotion and resolution of inflammation in the lung and are categorized into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. The change in M1/M2 balance has been reported in various pulmonary diseases and is a target for therapeutic intervention. The aim of this study was to assess the modulation of M1/M2 phenotype in alveolar macrophages by water-soluble carbon monoxide-releasing molecule-3 (CORM-3). Rat alveolar macrophages (AM) (NR8383) in culture were stimulated with LPS (5ng/ml)/IFN-γ (10U/ml) or IL-4 (10ng/ml)/IL-13 (10ng/ml) to induce M1 and M2 phenotypes, respectively. Expression of M1 phenotype markers, iNOS and TNF-α, and M2 phenotype markers, CD206 and Ym-1, was assessed by western blotting after 1, 3, 6, or 24h in the absence or presence of CORM-3 (0.15mM) treatment. Inactive CORM-3 (iCORM-3) was used as a control. Treatment of naïve (unstimulated) AM with CORM-3 promoted progression of the M2 phenotype as evidenced by the increased expression of CD206 (at 1h; 1.8-fold) and Ym-1 (at 3h; 1.9-fold), respectively. Surprisingly, CORM-3 treatment also upregulated the expression of iNOS protein as assessed 6h following stimulation of AM with CORM-3 (2.6-fold). On the contrary, CORM-3 effectively reduced LPS/IFN-γ-induced expression of iNOS protein (0.6-fold); however, it had no effect on TNF-α expression. Finally, CORM-3 acutely (1-3h) upregulated CD206 (1.4-fold) and Ym-1 (1.6-fold) levels in IL-4-/IL-13-treated (M2-stimulus) macrophages. These findings indicate that CORM-3 modulates macrophage M1 and M2 phenotypes in vitro with respect to continuous suppression of iNOS expression in M1-polarized macrophages and transient (early-phase) upregulation of CD206 and Ym-1 proteins in M2-polarized macrophages.

The National Environmental Respiratory Center (NERC) experiment in multi-pollutant air quality health research: II. Comparison of responses to diesel and gasoline engine exhausts, hardwood smoke and simulated downwind coal emissions

The NERC Program conducted identically designed exposure–response studies of the respiratory and cardiovascular responses of rodents exposed by inhalation for up to 6 months to diesel and gasoline exhausts (DE, GE), wood smoke (WS) and simulated downwind coal emissions (CE). Concentrations of the four combustion-derived mixtures ranged from near upper bound plausible to common occupational and environmental hotspot levels. An “exposure effect” statistic was created to compare the strengths of exposure–response relationships and adjustments were made to minimize false positives among the large number of comparisons. All four exposures caused statistically significant effects. No exposure caused overt illness, neutrophilic lung inflammation, increased circulating micronuclei or histopathology of major organs visible by light microscopy. DE and GE caused the greatest lung cytotoxicity. WS elicited the most responses in lung lavage fluid. All exposures reduced oxidant production by unstimulated alveolar macrophages, but only GE suppressed stimulated macrophages. Only DE retarded clearance of bacteria from the lung. DE before antigen challenge suppressed responses of allergic mice. CE tended to amplify allergic responses regardless of exposure order. GE and DE induced oxidant stress and pro-atherosclerotic responses in aorta; WS and CE had no such effects. No overall ranking of toxicity was plausible. The ranking of exposures by number of significant responses varied among the response models, with each of the four causing the most responses for at least one model. Each exposure could also be deemed most or least toxic depending on the exposure metric used for comparison. The database is available for additional analyses.

Inadequate cytoplasmatic calcium signals in alveolar macrophages after cardiac surgery

Patients undergoing cardiac surgery have an elevated risk for pulmonary complications. A dysfunction of alveolar macrophages (AM) might promote postoperative infections. Therefore intracellular calcium [Ca(2+)](i) as an important second messenger in cellular signaling was assessed in AM. Twelve patients undergoing elective coronary artery bypass graft surgery (CABG) were enrolled in this clinical trial. After anesthesia induction and 2 h after cardiopulomary bypass (CPB) declamping, the bronchoalveolar lavage (BAL) fluid was collected preoperatively from the right middle lobe and postoperatively from the left lingula of the lung. Cell subpopulations and [Ca(2+)](i) signals were assessed via flow cytometry. To express the changes of [Ca(2+)](i) signals the Fluo4/FuraRed-Ratio was used. After surgery the [Ca(2+)](i) baseline in unstimulated AMs were significantly reduced (p < 0.001). A significant signal reduction after fMLP (p = 0.021) and C5a (p = 0.028) stimulation was found in FSC high AMs after surgery, even though all populations showed a trend of less responsiveness. We suggest that the reduced [Ca(2+)](i) signaling in postoperative AMs is caused by a reduced coupling to membrane channels. These preliminary data suggest an inadequate [Ca(2+)](i) signal of AM after surgery, which may contribute to a local immune dysfunction in the lung.

Chronic oral administration of dexamethasone to rats increases cytotoxicity, but not interleukin-1 elaboration, by alveolar macrophages

Chronic administration of corticosteroids has been used to induce pulmonary infection in animals. but the specific mechanisms by which corticosteroids modulate pulmonary host defence have not been clarified. Specifically, little is known about how corticosteroids influence the function of lung cells, such as alveolar macrophages. Cytotoxicity and interleukin-1 elaboration are two important mechanisms of macrophage defence against pathogens. To determine whether chronic administration of dexamethasone alters cytotoxicity and interleukin-1 elaboration by alveolar macrophages, we studied alveolar macrophages from rats treated with oral doses of dexamethasone for 2 weeks. We found that unstimulated alveolar macrophages from dexamethasone-treated rats exhibited increased cytotoxicity compared with alveolar macrophages from control rats. Moreover, alveolar macrophages from both groups of rats showed enhanced cytotoxicity after in vitro interferon-gamma and lipopolysaccharide treatment, in a dose-dependent manner. Although no spontaneous interleukin-1 elaboration was detected from alveolar macrophages from either group of rats, lipopolysaccharide increased interleukin-1 elaboration by alveolar macrophages from both groups of rats equivalently. These results indicate that chronic oral administration of dexamethasone to rats increases cytotoxicity, and does not alter the capacity of alveolar macrophages to elaborate interleukin-1. Therefore, chronic corticosteroid administration appears to produce selective alterations in these defence mechanisms of alveolar macrophages.

Encapsulation of Cryptococcus neoformans regulates fungicidal activity and the antigen presentation process in human alveolar macrophages.

Our previous studies have shown that unstimulated alveolar macrophages (AM) play a predominant role as antigen-presenting cells in Cryptococcus neoformans infections, while the function as effector cells seems to be of minor relevance. The present study focuses on the role of encapsulation of C. neoformans on fungicidal activity and the antigen presentation process of AM. Fungicidal activity in unstimulated AM occurs to a higher degree when the acapsular strain is employed, but this is impaired compared with other natural effectors, such as peripheral blood monocytes (PBM) and polymorphonuclear (PMN) cells. Cryptococcus-laden AM also induce a higher proliferative response in autologous CD4+ lymphocytes when the acapsular strain is used compared with encapsulated yeast. The enhanced blastogenic response is, in part, ascribed to an augmented IL-2 production by T cells. In addition, higher levels of interferon-gamma (IFN-gamma), but not IL-4, are produced by the responding T cells, when the acapsular strain is used compared with the encapsulated yeast. Moreover, IFN-gamma is able to induce fungicidal activity in AM against the encapsulated yeast and augments killing activity of the acapsular strain. This phenomenon is not mediated by nitric oxide production, but is correlated with an enhancement of fungicidal activity of cytoplasmic cationic proteases. We speculate that encapsulation of C. neoformans could down-regulate the development of the immune response mediated by Cryptococcus-laden AM at lung level.

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumour necrosis factor-alpha (TNF-alpha) activate human alveolar macrophages to inhibit growth of Mycobacterium avium complex.

We investigated the effects of certain macrophage-active cytokines on the phagocytosis and growth inhibition of Mycobacterium avium complex (MAC) by human alveolar macrophages (AM). We also evaluated the effects of pretreatment with each cytokine on the superoxide anion release (O2-) from AM. The cytokines that we used were recombinant GM-CSF, natural type TNF-alpha, recombinant interferon-gamma (IFN-gamma), and recombinant IL-2. We found that phagocytosis by the various cytokine-stimulated AM was similar to that of unstimulated AM. On the other hand, significant growth inhibition of MAC was observed in the macrophages treated with GM-CSF or TNF-alpha, while no growth inhibition of MAC was observed in the macrophages treated with IFN-gamma or IL-2. Pretreatment with all cytokines tested enhanced the O2- release from AM, but there was no correlation between the enhancement of O2- release and the growth inhibition of MAC. Thus, we concluded that GM-CSF or TNF-alpha could activate AM to inhibit growth of MAC, probably not through the enhanced production of reactive oxygen intermediates.

Prostanoids Secreted by Alveolar Macrophages Enhance Ionic Currents in Swine Tracheal Submucosal Gland Cells

We examined the effect of substances released by swine alveolar macrophages (AMs) on ionic currents in airway submucosal gland cells (SGCs). AMs obtained by lavage were activated by 24-h zymosan exposure (0.1 mg/ml). Supernatant was collected and used to stimulate short-circuit current changes (DeltaI(SC)) in SGC monolayers in Ussing chambers. Dexamethasone (1 microM) or indomethacin (5 muM) during zymosan exposure of AMs reduced or abolished the supernatant-induced DeltaI(SC). Zymosan exposure induced a 5-fold increase in cyclooxygenase (COX)-2 but not COX-1 protein levels in AMs. Prostaglandin E(2) (PGE(2)) concentration in the supernatant from zymosan-activated AMs was 550 +/- 10 nM (n = 3) compared with 28 +/- 3 nM for unstimulated AMs (n = 3). PGE(2), applied serosally, induced DeltaI(SC) with an EC(50) of 15.5 +/- 1.3 nM (n = 4) and 3.6 +/- 1.8 microM (n = 3) when applied apically. Four types of endoprostanoid receptors (EP(1-4)) were detected in SGCs using Western blot. PGE(2)-induced DeltaI(SC) were inhibited by AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) but not by SC19220 (8-chloro-dibenzo[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-acetylhydrazide), suggesting that endoprostanoid (EP)(2) but not EP(1) receptors were activated by PGE(2). Pretreatment of SGCs with supernatant from zymosan-activated AMs, PGE(2), or forskolin enhanced the sensitivity to acetylcholine (ACh)-induced DeltaI(SC). PGE(2)-induced DeltaI(SC) were blocked by charybdotoxin (ChTX), chromanol 293B, or glibenclamide. ACh-induced DeltaI(SC) were only blocked by ChTX or glibenclamide. None of these blockers altered PGE(2) pretreatment-induced sensitization of ACh-induced DeltaI(SC). These results demonstrate that prostanoids released from activated AMs directly increase cystic fibrosis transmembrane conductance regulator and K(+) channel activity. ACh-induced DeltaI(SC) are also enhanced due to enhanced activation of Ca(2+)-activated K(+) channels (K(Ca)).

PCT-233, a novel modulator of pro- and anti-inflammatory cytokine production.

Plant extracts have been implicated in various immunoregulatory effects that are poorly understood. Thus, we investigated the modulatory activity of PureCell Complex (PCT)-233, an active molecular complex from mesophyll tissue of Spinacia oleacea on the inflammatory process. Alveolar macrophages (AM) were treated with PCT-233 and/or budesonide, a well-known anti-inflammatory agent, before or after being stimulated with lipopolysaccharides (LPS). Pro- and anti-inflammatory cytokine production, tumour necrosis factor (TNF) and interleukin (IL)-10, respectively, were measured in cell-free supernatants at different times after the treatment. PCT-233 increased unstimulated AM release of both TNF and IL-10, whereas heat- and light-inactivated PCT-233 stimulated only the release of TNF without affecting IL-10 production, suggesting that different mechanisms are involved in the modulation of TNF and IL-10 release by PCT-233. The presence of LPS did not modify PCT-233-stimulated TNF production, but the ratio TNF/IL-10 production by LPS-stimulated AM was reduced significantly in the presence of PCT-233. Pretreatment of AM with PCT-233 and budesonide before LPS stimulation reduced TNF production at both protein and mRNA levels, whereas IL-10 production was increased. Moreover, TNF/IL-10 ratio was reduced further with the combination PCT-233/budesonide. Interestingly, AM treatment with PCT-233 and budesonide 18 h after LPS stimulation did not modulate TNF release significantly but it did increase IL-10 production, and a synergistic effect was observed with the combination PCT-233/budesonide. These exciting data suggest that PCT-233 possesses some anti-inflammatory properties, even when added during the inflammatory process, and could potentiate the effect of other anti-inflammatory agents.

Chemiluminescence of pulmonary macrophages from patients with inactive sarcoidosis, in different radiological stages.

Luminol-amplified chemiluminescence was generated by alveolar macrophages, harvested from the bronchoalveolar lavages of 16 patients with different radiological stages of non-invasive (asymptomatic) sarcoidosis. None of the patients received any steroid therapy during this study. The mean duration of the disease in these patients was 8 months, with a duration time range of 6-14 months. Six patients were in radiological stage 1, five in radiological stage 2 and five in radiological stage 3. Alveolar macrophages from bronchoalveolar lavages of eight healthy non-smoking volunteers were used as controls. All alveolar macrophages were stimulated by phorbol myristate acetate. A significant decrease was recorded in the intensity of chemiluminescence generated by the phorbol-ester-stimulated alveolar macrophages obtained from patients with sarcoidosis of radiological stages 1 and 2, as compared to the cells collected from healthy individuals (controls). No decrease was recorded in the chemiluminescence generated by stimulated alveolar macrophages collected from patients with radiological stage 3, or from unstimulated alveolar macrophages of any patient. These results provide us with an indicative tool, which might enable us to differentiate, on a functional basis, between the activities of alveolar macrophages in non-active sarcoidosis.

INCREASED SPONTANEOUS INTERLEUKIN-10 RELEASE FROM ALVEOLAR MACROPHAGES IN ACTIVE PULMONARY SARCOIDOSIS

The study was designed to determine whether alveolar macrophages (AM) in acute pulmonary sarcoidosis release in vitro the anti-inflammatory cytokine interleukin (IL)-10. To learn more about the coherence between IL-10 and proinflammatory cytokines in active sarcoidosis, the release of interferon (IFN)- γ, macrophage inhibitory protein (MIP)-1 α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied and additionally compared to normal controls and patients with pneumonia and interstitial lung fibrosis. AM were obtained by bronchoalveolar lavage from 13 patients with active sarcoidosis, 8 patients with interstitial lung fibrosis, 10 patients with bacterial pneumonia, and 14 normal controls. The spontaneous and stimulated (tumor necrosis factor [TNF]- α, IL-1 β) cytokine release was measured in the supernatant of cultured AM by enzyme-linked immunosorbent assay (ELISA). Unstimulated AM from sarcoidosis patients released more IL-10, IFN- γ, MIP-1 α, and GM-CSF than normal controls and patients with pneumonia and interstitial lung disease. Stimulation with TNF- α or IL-1 β increased the MIP-1 α and GM-CSF release from AM of normal controls and patients with pneumonia and interstitial lung disease: however, no further enhancement of MIP-1 α and GM-CSF production was observed in AM from sarcoidosis patients. Exogenous IL-10 reduced the spontaneous and stimulated MIP-1 α and GM-CSF release in sarcoidosis to a lesser extent than in controls and patients with fibrosis and pneumonia. The up-regulated IL-10 in active pulmonary sarcoidosis may be a compensatory response to the enhanced expression of proinflammatory cytokines in order to down-regulate the inflammatory process. The results suggest an involvement of the anti-inflammatory cytokine IL-10 in the immunopathogenesis of sarcoidosis.

Arachidonic acid differentially affects basal and lipopolysaccharide-induced sPLA(2)-IIA expression in alveolar macrophages through NF-kappaB and PPAR-gamma-dependent pathways.

Secretory type IIA phospholipase A(2) (sPLA(2)-IIA) is a critical enzyme involved in inflammatory diseases. We have previously identified alveolar macrophages (AMs) as the major pulmonary source of lipopolysaccharide (LPS)-induced sPLA(2)-IIA expression in a guinea pig model of acute lung injury (ALI). Here, we examined the role of arachidonic acid (AA) in the regulation of basal and LPS-induced sPLA(2)-IIA expression in AMs. We showed that both AA and its nonmetabolizable analog, 5,8,11,14-eicosatetraynoic acid (ETYA), inhibited sPLA(2)-IIA synthesis in unstimulated AMs. However, only AA inhibited sPLA(2)-IIA expression in LPS-stimulated cells, suggesting that this effect requires metabolic conversion of AA. Indeed, cyclooxygenase inhibitors abolished this down-regulation. Prostaglandins PGE(2), PGA(2), and 15d-PGJ(2) also inhibited the LPS-induced sPLA(2)-IIA expression. Nuclear factor-kappaB (NF-kappaB) was found to regulate sPLA(2)-IIA expression in AMs. Both AA and ETYA inhibited basal activation of NF-kappaB but had no effect on LPS-induced NF-kappaB translocation, suggesting that suppression of sPLA(2)-IIA synthesis by AA in LPS-stimulated cells occurs via a NF-kappaB-independent pathway. 15-Deoxy-Delta(12,14)-PGJ(2) and ciglitazone, which are, respectively, natural and synthetic ligands for peroxisome proliferator-activated receptor-gamma (PPAR-gamma), inhibited LPS-induced sPLA(2)-IIA synthesis, whereas PPAR-alpha ligands were ineffective. Moreover, electrophoretic mobility shift assay showed PPAR activation by AA and PPAR-gamma ligands in LPS-stimulated AMs. Our results suggest that the down-regulation of basal sPLA(2)-IIA expression is unrelated to the metabolic conversion of AA but is dependent on the impairment of NF-kappaB activation. In contrast, the inhibition of LPS-stimulated sPLA(2)-IIA expression is mediated by cyclooxygenase-derived metabolites of AA and involves a PPAR-gamma-dependent pathway. These findings provide new insights for the treatment of ALI.

Alveolar macrophage deactivation in murine septic peritonitis: role of interleukin 10.

Sepsis predisposes the host to a number of infectious sequelae, particularly the development of nosocomial pneumonia. Mechanisms by which sepsis results in impairment of lung antibacterial host defense have not been well defined. Alveolar macrophages (AM) represent important immune effector cells of the lung airspace. In this study, we examined the effects of cecal ligation and puncture (CLP) on murine AM function ex vivo, including the expression of proinflammatory cytokines and AM phagocytic activity. AM were harvested from mice subjected to a sham operation and CLP 24 h after laparotomy, adherence purified, and challenged with lipopolysaccharide (LPS) or left unstimulated. Both unstimulated and LPS-stimulated AM from mice subjected to CLP (CLP mice) produced significantly smaller amounts of proinflammatory cytokines tumor necrosis factor alpha and interleukin (IL-12) and C-X-C chemokines KC and macrophage inflammatory protein 2 than similarly treated AM from animals subjected to a sham operation. Furthermore, AM isolated from CLP mice displayed a marked impairment in phagocytic activity, as determined by flow cytometry, with this defect persisting to 48 h post-CLP. Induction of peritoneal sepsis syndrome resulted in a time-dependent increase in IL-10 in plasma and peritoneal fluid. Interestingly, the impairment in AM proinflammatory-cytokine production and phagocytic activity observed in AM from CLP mice was partially reversed by the in vivo neutralization of IL-10 prior to AM harvest. These observations suggest that abdominal sepsis syndrome results in significant impairment in AM effector cell function, which is mediated, in part, by sepsis-induced expression of IL-10.

Phagocytosis of particulate air pollutants by human alveolar macrophages stimulates the bone marrow.

Epidemiologic studies have shown an association between the level of ambient particulate matter < 10 microm (PM(10)) and cardiopulmonary mortality. We have shown that exposure of rabbits to PM(10) stimulates the bone marrow. In this study, we determined whether human alveolar macrophages (AMs) that phagocytose atmospheric PM(10) produce mediators capable of stimulating the bone marrow. AMs incubated with PM(10) for 24 h produced tumor necrosis factor-alpha in a dose-dependent manner (86.8 +/- 53.29 pg/ml with medium alone; 1,087.2 +/- 257.3 pg/ml with 0.1 mg/ml of PM(10); P < 0.02). Instillation of the supernatants from AMs incubated with 0.1 mg/ml of PM(10) into the lungs of rabbits (n = 6) increased circulating polymorphonuclear leukocyte (PMN) and band cell counts as well as shortened the PMN transit time through the bone marrow (87.9 +/- 3.3 h) compared with unstimulated human AMs (104.9 +/- 2.4 h; P < 0.01; n = 5 rabbits). The supernatants from rabbit AMs incubated with 0.1 mg/ml of PM(10) (n = 4 rabbits) caused a similar shortening in the PMN transit time through the bone marrow (91.5 +/- 1.6 h) compared with human AMs. We conclude that mediators released from AMs after phagocytosis of PM(10) induce a systemic inflammatory response that includes stimulation of the bone marrow.

Time-dependent inhibition of immune complex-induced lung injury by catalase: relationship to alterations in macrophage and neutrophil matrix metalloproteinase elaboration

Rats were subjected to acute lung injury by the intra-alveolar formation of IgG immune complexes of bovine serum albumin (BSA) and anti-BSA. In this model of injury, complement activation occurs and large numbers of neutrophils invade the interstitium and alveolar space. In the present study, animals were treated with intratracheal catalase concomitantly with anti-BSA or after a lag period of 5–120 min. Catalase treatment at time-zero or at 5 min post injury failed to prevent lung injury as indicated by permeability change, histological features, and neutrophil influx. However, treatment after a delay of 15–30 min (but not 120 min) afforded substantial protection. Consistent with past findings [19], lung injury was accompanied by an accumulation of matrix metalloproteinase 9 (MMP-9) in bronchoalveolar lavage (BAL) fluid. There was a strong correlation between inhibition of injury and reduction in MMP-9 levels. In vitro studies conducted in parallel revealed that unstimulated alveolar macrophages did not produce measurable MMP-9, while there was a large induction following exposure to the same immune complexes that initiated injury in vivo. MMP-2 was also slightly upregulated under the same conditions. Concomitant treatment with catalase greatly inhibited MMP-9 production by macrophages in response to immune complexes, but this treatment had little effect on basal production of either MMP-9 or MMP-2 by macrophage. The same concentration of catalase that suppressed MMP-9 elaboration also inhibited the production of tumor necrosis factor α. In contrast, when neutrophils were treated with catalase and then exposed to immune complexes, the antioxidant failed to prevent the release of either MMP-2 or MMP-9. Taken together, these findings demonstrate that antioxidant treatment interferes with elaboration of MMPs by alveolar macrophages. Protection against lung injury is correlated with reduction in MMP levels in the BAL fluid.

Phenotypic features of alveolar monocytes/macrophages and IL-8 gene activation by IL-1 and TNF-alpha in asthmatic patients.

The alveolar macrophage (AM), a major defense cell in the lung, participates in immune and inflammatory reactions through the release of several regulatory and chemotactic cytokines. In particular, macrophages are considered to play a pivotal proinflammatory role in the production and maintenance of airway inflammation and bronchial hyperreactivity. To assess the phenotypic pattern of AM from asthmatic subjects, we performed the following experiments: 1) cytofluorometric analysis of specific phenotypic features (CD11b, CD14, CD16, CD45, HLA-DR, CD71, CD95, and CD44) 2) assessment of the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and the chemotactic regulatory cytokine IL-8 by unstimulated and lipopolysaccharide-stimulated AM. In these patients, we phenotypically characterized the AM, showing their strong proinflammatory activity also in patients with mild asthma. Their activity has been clarified by our biomolecular data that showed a constitutive basal IL-8 production by AM, and also indicated that IL-1 and TNF-alpha were able to upregulate the ability of activated human AM to produce IL-8 at the protein and messenger ribonucleic acid (mRNA) levels.

Detection of granulocyte-macrophage colony-stimulating factor in patients with pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is an idiopathic lung disease in which the alveolar spaces are filled with surfactant. Recently, it has been proposed that PAP is caused by deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) because GM-CSF-knockout mice develop the disease. To examine this possibility, we tested the two hypotheses that lung GM-CSF levels are low and that alveolar macrophages (AM) do not respond to GM-CSF in patients with PAP. Data from 10 adult patients with PAP who underwent therapeutic whole-lung lavage were compared with those of 10 healthy volunteers who underwent bronchoalveolar lavage (BAL) by fiberoptic bronchoscopy. Bronchoalveolar lavage fluid (BALF) and plasma were collected and analyzed for total protein and levels of GM-CSF, interleukin-3, and tumor necrosis factor (TNF)-alpha. Isolated AM were cultured with or without lipopolysaccharide (LPS) or GM-CSF, and production of GM-CSF and TNF-alpha was measured after 24 h. GM-CSF in BALF and plasma was higher in PAP than in control subjects (p </= 0.05), and was detectable under both reducing and nonreducing conditions as a 28-kD protein in BALF from the PAP patients. GM-CSF release by unstimulated AM from PAP patients was higher than in cells from control subjects, but the responses to LPS were similar. Mean TNF-alpha release by AM in response to GM-CSF was higher in control subjects than in PAP patients due to a low response in three patients. In conclusion, unbound immunoreactive GM-CSF is detectable in BALF and plasma of PAP patients. Most PAP patients also had intact AM responses to GM-CSF, although some may have had defects in GM-CSF receptor or signal-transduction mechanisms. Although these data exclude lack of GM-CSF production as a common etiology of human PAP, defects in GM-CSF function in PAP are under investigation.

Importance of histamine in the cytokine network in the lung through H2 and H3 receptors: stimulation of IL-10 production.

Histamine, a well-known inflammatory mediator, has been implicated in various immunoregulatory effects that are poorly understood. Thus, we tested the hypothesis that histamine inhibits the release of a proinflammatory cytokine, namely TNF, by stimulating the release of an anti-inflammatory cytokine, IL-10. Alveolar macrophages (AMs) from humans, Sprague Dawley rats, and the AM cell line, NR8383, were treated with different concentrations of histamine (10-5-10-7 M) for 2 h prior to their stimulation with suboptimal concentration of LPS (1 ng/ml) for 4 h. Histamine inhibited TNF release in a dose-dependent manner. This inhibition was mimicked by H2 and H3 receptor agonists, but not by H1 receptor agonist. Furthermore, we demonstrated the expression of H3 receptor mRNA in human AMs. Interestingly, treatment of AMs with anti-IL-10, anti-PGE2, or a NO synthase inhibitor (Nomega-nitro-l -arginine methyl ester) before the addition of histamine abrogated the inhibitory effect of the latter on TNF release. Histamine treatment (10-5 M) increased the release of IL-10 from unstimulated (2.2-fold) and LPS-stimulated (1. 7-fold) AMs. Unstimulated AMs, NR8383, express few copies of IL-10 mRNA, as tested by quantitative PCR, but expression of IL-10 was increased by 1.5-fold with histamine treatment. Moreover, the stimulation of IL-10 release by histamine was abrogated by pretreatment with anti-PGE2 or the NO synthase inhibitor, Nomega-nitro-l -arginine methyl ester. Thus, histamine increases the synthesis and release of IL-10 from AMs through PGE2 and NO production. These results suggest that histamine may play an important role in the modulation of the cytokine network.

Heterogeneity of procoagulant activity and cytokine release in subpopulations of alveolar macrophages and monocytes.

We have studied the expression of tissue factor (TF) and fibrinopeptide A (FPA) generation as well as the release capacity of TNF-alpha, IL-1beta, and IL-6 in density-defined subpopulations of alveolar macrophages (AM) and monocytes (Mo). TF was equally expressed on all AM subpopulations and Mo, while the FPA-forming capacity was at the same level in low density AM as in Mo and was significantly (P < 0.05) higher in low density AM than in high density AM. The lipopolysaccharide (LPS)-induced release of TNF-alpha was higher (P < 0.05) in high density AM than in low density AM and in Mo. IL-1beta release was undetectable in unstimulated AM and in LPS-stimulated low density AM, while the LPS-induced IL-1beta release in high density AM was low compared to the levels demonstrated in Mo. LPS-stimulated IL-6 release was not distinctively different in the AM subpopulations and Mo. The presented study showed that FPA generation and LPS-stimulated TNF-alpha release were dependent on the density (i.e., maturity) of AM. This implies that a skewed distribution of AM subpopulations induced by disease processes may profoundly influence the inflammatory reactions, including extravascular activation of coagulation.