Swine dust induces cytokine secretion from human epithelial cells and alveolar macrophages

Exposure to swine dust causes airway inflammation with increased levels of proinflammatory cytokines, and inflammatory cells in nasal and bronchoalveolar lavage fluid (BALF) in healthy subjects. Earlier studies have suggested that lipopolysaccharides (LPS) might be an important proinflammatory factor in swine dust. Since respiratory epithelial cells and alveolar macrophages are target cells for the inhaled dust, we therefore compared the release of proinflammatory cytokines from normal human bronchial epithelial cells (NHBE), an epithelial cell line (A549) and from human alveolar macrophages obtained from BALF from healthy subjects in vitro after incubation with dust collected in swine houses or LPS. Swine dust or LPS was added to the wells with A549 cells or macrophages and incubated for 8 h at concentrations of 12.5, 25, 50 and 100 μg/ml. NHBE cells were incubated with swine dust at a concentration of 25, 50 or 100 μg/ml or with LPS at a concentration of 50 or 100 μg/ml and incubated for 24 h. The supernatants were collected, centrifuged, and IL-6, IL-1β and tumour necrosis factor-alpha (TNF-α) production was measured using an ELISA method and expressed per 10⁶ cells. Swine dust and LPS caused a dose-dependant increase of IL-6 production in NHBE cells, swine dust being more potent than LPS. In A549 cells, only swine dust, but not LPS caused an increase of IL-6 production. Neither swine dust nor LPS induced IL-1β or TNF-α release from A549 cells. Both swine dust and LPS caused a dose-dependent increase of IL-1β, IL-6 and TNF-α in alveolar macrophages. Swine dust which contained 2.2 (0.2) ng endotoxin/100 μg swine dust (0.02‰) was almost as potent as LPS in inducing cytokine release from alveolar macrophages in vitro. We conclude that both epithelial cells and alveolar macrophages have the capability to contribute to the release of proinflammatory cytokines following exposure to swine dust. Some agent(s) other than LPS in the dust contribute to the marked airway inflammatory reaction.     

Quinacrine inhibits oxygen radicals release from human alveolar macrophages.

We examined the effect of quinacrine, an anti-malarial drug which inhibits phospholipase A2, on phorbol myristate acetate (PMA) stimulated alveolar macrophage oxygen radical secretion. This drug suppressed 30% of radicals release, which was 70% of the amount inhibited by superoxide dismutase, a specific inhibitor of oxygen radicals. In addition, this reduction was at the same magnitude as dexamethasone. This and previous results on other inflammatory cells support the assumption that quinacrine may have a beneficial effect on bronchial asthma.     

Changes in cytokine and nitric oxide secretion by rat alveolar macrophages after oral administration of bacterial extracts.

Oral administration of the bacterial immunomodulator Broncho-Vaxom (OM-85), a lysate of eight bacteria strains commonly causing respiratory disease, has been shown to enhance the host defence of the respiratory tract. In this study we examined the effect of orally administered (in vivo) OM-85 on stimulus-induced cytokine and nitric oxide secretion by rat alveolar macrophages in vitro. The results show that alveolar macrophages isolated from OM-85-treated rats secreted significantly more nitric oxide, tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta upon in vitro stimulation with lipopolysaccharide (LPS), whereas, in contrast, LPS-induced IL-6 secretion was significantly lower. The observed effects of in vivo OM-85 treatment on stimulus-induced cytokine secretion in vitro are not due to a direct effect of OM-85 on the cells, because in vitro incubation of alveolar macrophages with OM-85 did not result in altered activity, nor did direct intratracheal instillation of OM-85 in the lungs of rats result in altered alveolar macrophage activity in vitro. It is hypothesized that oral administration of OM-85 leads to priming of alveolar macrophages in such a way that immune responses are non-specifically enhanced upon stimulation. The therapeutic action of OM-85 may therefore result from an enhanced clearance of infectious bacteria from the respiratory tract due to increased alveolar macrophage activity.     

Subpopulation of alveolar macrophages inhibits superoxide anion production by macrophages

Pig alveolar macrophages are a heterogeneous population of cells. Three subpopulations or bands exist when the whole population is separated according to density. Band 1 cells are the least dense cells and constitute 9% of the total population. Bands 2 and 3 represent 44 and 47% of the total population. The three subpopulations generate superoxide anions, although to varying degrees. Band 3 cells are the most active, while band 1 cells are the least active. The amount of superoxide anions released in a mixed population of bands 1, 2, and 3 cells was less than the sum of that produced from each band assayed separately. Band 1 cells were found to inhibit by 47% the production of superoxide anions by band 3 cells. Conditioned medium from band 1 cells contains a heat-sensitive, nondialyzable, soluble factor responsible for this inhibition.     

Lipocortin I production by human alveolar macrophages.

Lipocortin I, in some cells, may be a potent inhibitor of phospholipase A2 activity. These studies evaluated the relative amounts of lipocortin I in human alveolar macrophages compared with blood monocytes, using a specific polyclonal antibody and the technique of Western analysis. Lipocortin I was detected in all isolates of human alveolar macrophages and had molecular masses of 37,000 and 33,000 D. Corticosteroids increased amounts of lipocortin I in these cells in a dose-dependent manner. This effect was specific for corticosteroids as related steroids had no effect. Blood monocytes, when compared with alveolar macrophages, contained relatively small amounts of lipocortin I. We conclude that lipocortin I is present in relatively large amounts in human alveolar macrophages and that amounts of the protein can be induced by corticosteroids. We further speculate that the relative amounts of lipocortin I within monocytes/macrophages may be a marker of differentiation.     

Differences in cytokine secretion by intestinal mononuclear cells, peripheral blood monocytes and alveolar macrophages from HIV-infected patients.

Mononuclear cells of the lamina propria (LpMNC), isolated from endoscopically taken biopsies of the large bowel from AIDS patients, were analysed for their ability to secrete tumour necrosis factor-alpha (TNF-alpha), IL-1 beta and IL-6. Stimulation of LpMNC from normal controls with pokeweed mitogen (PWM) led to a time- and dose-dependent enhancement of TNF-alpha, IL-1 beta and IL-6 secretion. In contrast, PWM stimulation of LpMNC from AIDS patients resulted in only a small increase in TNF-alpha release. Constitutive secretion of IL-1 beta and IL-6 in these patients was already increased to the concentration range of stimulated cells from normal controls and could not be further increased, probably due to maximal in vivo stimulation. Secretion of TNF-alpha, IL-1 beta and IL-6 by peripheral blood monocytes (PBM) and alveolar macrophages from AIDS patients was elevated with or without stimulation compared with normal controls. Obviously, the regulation of TNF-alpha secretion is dependent on the microenvironment. Since it is known that interferon-gamma (IFN-gamma) may induce the production of TNF-alpha, the secretion of this cytokine was examined. Release of IFN-gamma was constitutively and under stimulation lowered in LpMNC from AIDS patients compared with normal controls. Addition of IFN-gamma to LpMNC did not result in enhanced TNF-alpha secretion. Our data indicate a defective function of intestinal mononuclear cells in AIDS patients as shown by the diminished TNF-alpha secretion.     

Interleukin 1 secretion from human alveolar macrophages in lung disease

Interleukin 1 secretion from human alveolar macrophages was studied in patients with interstitial pulmonary fibrosis, sarcoidosis, and the acquired immunodeficiency syndrome with pneumonitis and compared to secretion from alveolar macrophages of normal volunteers. Macrophages lavaged from the lungs were stimulated with 10 µg/ml of lipopolysaccharide and cultured for 24 hr. In some cases macrophages were also stimulated with 1 µg/ml lipopolysaccharide. After dialysis of the culture supernatants, interleukin 1 secretion was quantified by the thymocyte proliferation assay and probit analysis and expressed in terms of secretion from 1 million macrophages. Results showed that, on average, macrophages derived from patients secreted more interleukin 1 after stimulation with lipopolysaccharide compared to normal subjects. Mean secretion was significantly greater from macrophages of patients with acquired immunodeficiency syndrome and interstitial pulmonary fibrosis when stimulated with 10 µg/ml lipopolysaccharide. Of the 24 individuals studied, spontaneous interleukin 1 secretion was detected from unstimulated macrophages in only 1 patient and 1 normal volunteer. We conclude that alveolar macrophages lavaged from the lungs of patients with inflammatory lung disease have an increased capacity to secrete interleukin 1 onin vitro stimulation with lipopolysaccharide. Possible mechanisms for this increase are discussed.     

Production of hydroxyl radical by human alveolar macrophages.

Stimulated human alveolar macrophages were demonstrated to oxidize B-methyl proprionaldehyde (methional) or 2-keto-4-thiomethylbutyric acid to ethylene (C2H4). Agents which are believed to scavenge the hydroxyl radical (.OH), sodium benzoate, and mannitol, as well as scavengers of superoxide anion (O2-) or hydrogen peroxide, decreased C2H4 production, implicaing .OH as the oxidizing radical. Differences in C2H4 rpoduction, as well as oxygen uptake and O2- release between human alveolar macrophages and polymorphonuclear leukocytes, were also documented.     

Production of C2 by human alveolar macrophages.

Human and rat alveolar macrophages produce haemolytic C2 during in vitro culture. We conclude that C2 synthetic ability is maintained during in vivo maturation of human monocytes to macrophages and that production of complement components by mature tissue macrophages may be important for optimal generation of inflammatory responses.     

Cigarette smoke exposure attenuates cytokine production by mouse alveolar macrophages

HIV-1 infection impairs alveolar macrophage immune function and renders patients susceptible to pneumonia by poorly understood mechanisms. Alveolar macrophage maturation and function depends on granulocyte-macrophage colony-stimulating factor (GM-CSF), which is produced and secreted by the alveolar epithelium. Macrophages respond to GM-CSF through the GM-CSF receptor (GM-CSFR), which has a binding subunit (GM-CSFRalpha) and a signaling subunit (GM-CSFRbeta). In this study, we measured GM-CSFR expression and alveolar macrophage function in a transgene HIV-1 rat model (NL4-3Delta gag/pol); this construct bears a pro-virus with gag and pol deleted, but other HIV-1-related proteins, such as gp120 and Tat, are expressed, and the rats develop an AIDS-like phenotype as they age. We first determined that HIV-1-transgenic expression selectively decreased alveolar macrophage expression of GM-CSFRbeta and impaired bacterial phagocytosis in vitro. Next, we examined the role of zinc (Zn) deficiency as a potential mechanism underlying these effects, and determined that HIV-1-transgenic rats have significantly lower levels of Zn in the alveolar space and macrophages. To test the direct effect of Zn deficiency on macrophage dysfunction, we treated rat alveolar macrophage cell line with a Zn chelator, N,N,N',N'-tetrakis-(2-pyridyl-methyl) ethylenediamine, and this decreased GM-CSFRbeta expression and phagocytosis. In parallel, treatment with Zn acetate in vitro for 48 hours restored intracellular Zn levels and phagocytic function in alveolar macrophages from HIV-1-transgenic rats. Taken together, these data suggest that pulmonary Zn deficiency could be one of the mechanisms by which chronic HIV-1 infection impairs alveolar macrophage immune function and renders these individuals susceptible to serious lung infections.     

Expression of Ia like (HLA-DR) antigens on human alveolar macrophages.

The distribution of Ia like (HLA-DR) antigens on human alveolar macrophages (HAM phi) has been investigated by indirect immunofluorescence staining of viable macrophages with a panel of monoclonal antibodies (MoAb) to common determinants of these antigens. HAM phi were characterized by non-specific esterase stain, plastic adherence, phagocytosis and IgG-Fc receptor expression. Ia like antigens were expressed in approximately 45-80% of HAM phi, being localized as patchy and lineal fluorescence along the membrane. Ia like expression was higher in macrophages from non-smoker subjects (P less than 0.025). No difference in Ia like antigen expression was found between adherent and non-adherent HAM phi subsets. Ia like positive HAM phi from both smoker and non-smoker subjects consisted of a large subpopulation of phagocytic cells (60-70%) and a smaller non-phagocytic subpopulation (20-25%). These subpopulations were also present in the Ia like negative HAM phi. The percentage of Ia like positive macrophages showed variable results depending on the MoAb used, suggesting that not all anti-Ia like antibodies recognize the same antigenic determinants. Moreover, lack of staining of one macrophage subset occurred with all MoAb tested, over a large range of concentrations.     

Tumor necrosis factor production by human sarcoid alveolar macrophages.

Tumor necrosis factor (TNF) is an oncolytic peptide that may also exert many other biologic effects. Experimentally, immunologically activated mononuclear phagocytes stimulated with endotoxin (LPS) produce TNF, while resting mononuclear phagocytes stimulated with LPS produce little TNF. To date, the ability of human alveolar macrophages (AMs) to produce TNF has not been clearly delineated. As pulmonary sarcoidosis is a granulomatous inflammatory disorder characterized by immunologically activated AMs, we investigated the production of TNF by AMs obtained by bronchoalveolar lavage from 7 normal volunteers and 13 patients with pulmonary sarcoidosis. The AMs were cultured with and without LPS, and TNF production was assessed by an in vitro cytotoxicity assay. Unstimulated sarcoid and normal AMs produced little TNF, but LPS stimulation enhanced TNF production by both normal and sarcoid AMs. Furthermore, LPS-stimulated sarcoid AMs produced more TNF than normal AMs (84.9 +/- 16.7 versus 32.5 +/- 10.2 units/million cells, P less than 0.05). It is concluded that human AMs can produce TNF and that sarcoid AMs are primed and can produce significantly more TNF, compared with normal AMs.     

Chlamydicidal activity of human alveolar macrophages.

Pneumonia due to Chlamydia trachomatis is a disease limited mainly to infants under 6 months of age. Rare cases have been reported in immunocompromised adults. One possible reason for the propensity of the pneumonia to occur in the very young may be related to differences in the phagocytic and bactericidal capacity of alveolar macrophages (AMs) in young infants and adults. At birth a function of AMs is clearance of surfactant-related material from the alveolar surface. Studies in animals have suggested that engorgement of AMs with surfactant-related lipids may reduce the microbicidal capacity of these cells. In the present study we determined that AMs obtained from healthy, nonsmoking adults were capable of killing both human biovars of C. trachomatis, with complete killing observed by 48 h after inoculation. Preincubation of AMs from adults with surfactant did not reduce the capacity of the cells to kill C. trachomatis.     

Interleukin-4 production by human alveolar macrophages

BACKGROUND: IL-4 is a key factor for T helper type 2 (Th2) differentiation and Ig class switching to IgE and IgG(4) during the development of immune responses. IL-4 is produced by T cells, mast cells, basophils, and eosinophils. However, there is also evidence suggesting that rat alveolar macrophages (AMs) produce IL-4. OBJECTIVE: Given the importance of AMs and Th2-related diseases in the lung, we investigated the production of IL-4 by human AMs. METHODS: Human AMs were isolated from bronchoalveolar lavage, purified, and IL-4 production was investigated at mRNA and protein levels using real-time PCR, flow cytometry, immunocytochemistry, and ELISA. The presence of IL-4 was investigated in subjects with asthma or asymptomatic airway hyper-responsiveness, and in normal non-smokers. RESULTS: IL-4 and IL-4delta2 (a splice variant found in other IL-4 producing cells) mRNAs were found in all these subjects, but IL-4 expression could not be correlated with a particular disease. Protein production was verified by immunocytochemistry and flow cytometry analysis demonstrating, respectively, up to 69% and 59% positive AMs, regardless of the subject condition. Furthermore, phorbol-12-myristate-13-acetate and calcium ionophore stimulated the release of IL-4 after 48 h treatment in the presence of anti-IL-4 receptor antibody. CONCLUSION: Our results show for the first time that IL-4 and IL-4delta2 mRNA are expressed and IL-4 protein produced and released by human AMs, suggesting a contribution of these cells in the modulation of Th2 immune response.     

Mycobacterium tuberculosis 19-kilodalton lipoprotein inhibits Mycobacterium smegmatis-induced cytokine production by human macrophages in vitro

Vaccination of mice with Mycobacterium vaccae or M. smegmatis induces some protection against M. tuberculosis challenge. The 19-kDa lipoprotein of M. tuberculosis, expressed in M. vaccae or M. smegmatis (M. smeg19kDa), abrogates this protective immunity. To investigate the mechanism of this suppression of immunity, human monocyte-derived macrophages (MDM) were infected with M. smeg19kDa. Infection resulted in reduced production of tumor necrosis factor alpha (TNF-alpha) (P < 0.01), interleukin-12 (IL-12) (P < 0.05), IL-6 (P < 0.05), and IL-10 (P < 0.05), compared to infection with M. smegmatis vector (M. smegV). Infection with M. smeg19kDa and with M. smegV had no differential effect on expression of costimulatory molecules on MDM, nor did it affect the proliferation of presensitized T cells cocultured with infected MDM. When MDM were infected with M. smegmatis expressing mutated forms of the 19-kDa lipoprotein, including non-O-glycosylated (M. smeg19NOG), nonsecreted (M. smeg19NS), and nonacylated (M. smeg19NA) variants, the reduced production of TNF-alpha or IL-12 was not observed. When the purified 19-kDa lipoprotein was added directly to cultures of infected monocytes, there was little effect on either induction of cytokine production or its inhibition. Thus, the immunosuppressive effect is dependent on glycosylated and acylated 19-kDa lipoprotein present in the phagosome containing the mycobacterium. These results suggest that the diminished protection against challenge with M. tuberculosis seen in mice vaccinated with M. smegmatis expressing the 19-kDa lipoprotein is the result of reduced TNF-alpha and IL-12 production, possibly leading to reduced induction of T-cell activation.     

Regulatory effects of miR-155 and miR-146a on repolarization and inflammatory cytokine secretion in human alveolar macrophages in vitro

Macrophages play an important role in inflammatory responses; however, miRNA-mediated repolarization of macrophages is essential for fulfilling this function. To clarify a series of changes at the RNA level in alveolar macrophages under normal and inflammatory conditions, bronchial alveolar lavage liquid (BALF) was collected from healthy volunteers or patients with pneumonia. This approach, which differs from that used in previously, provides more accurate information about the states of macrophages in different lung microenvironments. In this study, the density plots of macrophage subtypes (M1 and M2) in the BALF of healthy volunteers differed from that of the patients with pneumonia. The M2 subtype dominated in healthy volunteers and was rapidly repolarized to M1 in response to miRNA-mediated gene regulation. Differential miRNA expression in the two macrophage subtypes revealed lower expression of miR-155 and MIR-146a in patients with pneumonia compared with healthy volunteers; this may be related to inflammation and the use of anti-inflammatory drugs. We also found increased TNF-α and IL-6 expression at the RNA level, while macrophage galactose-type C-type lectin 1 (MGL-1) expression decreased with downregulation of miR-155 and miR-146a expression. These results indicate that the gene regulation mediated by miR-155 and miR-146a contributes to human alveolar macrophage phenotype repolarization, thus leading to an early switch from pro-inflammatory to anti-inflammatory cytokine production.     

Extrinsic allergic alveolitis: inhibitory effects of pentoxifylline on cytokine production by alveolar macrophages.

BACKGROUND: Pentoxifylline is a well-established drug with hemorheologic properties. Various evidence suggests an additional therapeutic potential in regard to inflammation and immunomodulation. Extrinsic allergic alveolitis (EAA) is a granulomatous disease that is driven by T-cell and alveolar macrophage (AM)-derived cytokines. OBJECTIVE: To investigate the effects of pentoxifylline on the production of tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta, IL-6, IL-8, IL-10, and the soluble TNF receptors (sTNFR1 and sTNFR2) from AMs in EAA compared with dexamethasone. METHODS: The AMs from 9 patients with EAA were cultured for 24 hours with RPMI medium alone or lipopolysaccharide (LPS) (100 ng/mL) and with pentoxifylline at concentrations of 0.01, 0.1, and 1 mmol/L or 0.1-mmol/L dexamethasone. Cytokines in the culture supernatants were analyzed by enzyme-linked immunosorbent assay. RESULTS: Pentoxifylline induced a dose-dependent suppression of spontaneous TNF-alpha and IL-10 release from AMs in EAA. The spontaneous production of other cytokines was unaffected by pentoxifylline at all tested concentrations. Dexamethasone inhibited significantly only the spontaneous release of TNF-alpha. Pentoxifylline and dexamethasone also inhibited the LPS-stimulated production of all cytokines except IL-1beta and sTNFR1. CONCLUSION: Our results may be the basis for clinical trials to evaluate the role of pentoxifylline as an immunotherapeutic agent in the treatment of EAA.