Monocyte responses to sulfatide from Mycobacterium tuberculosis: inhibition of priming for enhanced release of superoxide, associated with increased secretion of interleukin-1 and tumor necrosis factor alpha, and altered protein phosphorylation.

In monocytes, sulfatide, a lipid from Mycobacterium tuberculosis, blocked priming for enhanced release of superoxide (O2-) by the macrophage activating factors lipopolysaccharide, gamma interferon, interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and muramyl dipeptide. Sulfatide, in the presence of lipopolysaccharide, also caused increased secretion of IL-1 beta and TNF-alpha into monocyte culture medium. Sulfatide altered the pattern of phosphorylation of monocyte proteins. Cell lysates prepared from monocytes treated with sulfatide showed decreased activity of protein kinase C, but sulfatide did not directly inhibit protein kinase C activity when added to lysates. A known inhibitor of protein kinase C, staurosporine, also inhibited O2- release and caused increased secretion of IL-1 beta. Thus, sulfatide appeared to indirectly affect protein kinase C, implicating protein kinase C as part of the mechanism of priming. Because sulfatide blocked priming for enhanced release of O2-, which could interfere with monocyte bactericidal activity, while causing enhanced secretion of IL-1 beta and TNF-alpha, which could promote formation of granulomata, sulfatide might be an important factor in the pathogenesis of M. tuberculosis.     

Staphylococcal toxic shock syndrome toxin 1-induced tumor necrosis factor alpha and interleukin-1 beta secretion by human peripheral blood monocytes and T lymphocytes is differentially suppressed by protein kinase inhibitors.

The signal transduction pathways by which staphylococcal toxic shock syndrome toxin 1 (TSST-1) induces tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion were examined with various protein kinase inhibitors. TNF-alpha secretion by normal human monocytes and T cells in response to TSST-1 was suppressed by inhibitors of protein kinase C (H7) and tyrosine kinases (genistein). In contrast, the secretion of IL-1 beta was blocked by a cyclic AMP- and cyclic GMP-dependent kinase inhibitor (HA1004) as well as by H7 and genistein. These results suggest that the secretion of TNF-alpha and IL-1 beta may be differentially regulated by TSST-1 and that protein kinases play an important role in mediating cytokine responses to the toxin.     

Ozone exposure of macrophages induces an alveolar epithelial chemokine response through IL-1alpha

Ozone is known to produce an acute influx of neutrophils, and alveolar epithelial cells can secrete chemokines and modulate inflammatory processes. However, direct exposure of alveolar epithelial cells and macrophages to ozone (O(3)) produces little chemokine response. To determine if cell-cell interactions might be responsible, we investigated the effect of alveolar macrophage-conditioned media after ozone exposure (MO(3)CM) on alveolar epithelial cell chemokine production. Serum-free media were conditioned by exposing a rat alveolar macrophage cell line NR8383 to ozone for 1 hour. Ozone stimulated secretion of IL-1alpha, IL-1beta, and IL-18 from NR8383 cells, but there was no secretion of chemokines or TNF-alpha. Freshly isolated type II cells were cultured, so as to express the biological markers of type I cells, and these cells are referred to as type I-like cells. Type I-like cells were exposed to diluted MO(3)CM for 24 hours, and this conditioned medium stimulated secretion of cytokine-induced neutrophil chemattractant-1 (CXCL1) and monocyte chemoattractant protein-1 (CCL2). Secretion of these chemokines was inhibited by the IL-1 receptor antagonist. Although both recombinant IL-1alpha and IL-1beta stimulated alveolar epithelial cells to secrete chemokines, recombinant IL-1alpha was 100-fold more potent than IL-1beta. Furthermore, neutralizing anti-rat IL-1alpha antibodies inhibited the secretion of chemokines by alveolar epithelial cells, whereas neutralizing anti-rat IL-1beta antibodies had no effect. These observations indicate that secretion of IL-1alpha from macrophages stimulates alveolar epithelial cells to secrete chemokines that can elicit an inflammatory response.     

Enhanced release of IL-1beta and TNF-alpha following endotoxin challenge from rat alveolar macrophages cultured in low-mg(2+) medium.

Our previous data have demonstrated that LPS-stimulated alveolar macrophages produce higher levels of IL-1beta and TNF-alpha mRNA in low-Mg(2+) medium than in normal-Mg(2+) medium. In this study, we examined whether the increased mRNA levels are correlated with the release of both cytokines. LPS-stimulated alveolar macrophages released higher amounts of IL-1beta and TNF-alpha in low-Mg(2+) medium than in normal-Mg(2+) medium. The enhanced release of IL-1beta was completely suppressed by pretreatment with verapamil (a calcium entry blocker), U73122 (a phospholipase C inhibitor), W-7 (a calmodulin inhibitor), and curcumin (an activator-protein [AP]-1 inhibitor), and weakly suppressed by dexamethasone (which inhibits nuclear factor [NF]-kappaB and AP-1). On the other hand, the enhanced release of TNF-alpha was completely suppressed by U73122, and strongly suppressed by TMB-8 (which inhibits calcium release from the endoplasmic reticulum) and W-7, and weakly suppressed by pyrrolidine dithiocarbamate (a NF-kappaB inhibitor). From these results, we conclude that the enhanced release of IL-1beta and TNF-alpha from LPS-stimulated alveolar macrophages in low-Mg(2+) medium depends partly on the enhanced synthesis of both cytokines, and occurs partly via identical, and partly via different, signaling pathways.     

Interferon-gamma, tumor necrosis factor-alpha, and interleukin 1-beta suppress the replication of hepatitis B virus through oxidative stress

The effect of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and H2O2, on hepatitis B virus (HBV) replication was analyzed in the HBV DNA transfected human hepatoblastoma-derived cell line HB 611. Secretion of HBV DNA from HB611 cells was inhibited by IFN-gamma, TNF-alpha, IL-1beta, and H2O2 in a dose-dependent manner. These cytokines and H2O2 also decreased HBV mRNA expression in HB611 cells, meaning that these reagents decreased the synthesis of all virally encoded components of the HBV virion. The level of manganese-SOD mRNA, indicative of occurrence of oxidative stress, increased immediately after treatment with IFN-gamma, TNF-alpha, IL-1beta, and H2O2. Moreover, the antioxidant N-acetyl-L-cysteine substantially inhibited the antiviral effect. Our findings suggest that oxidative stress may be a common factor in the antiviral effects of IFN-gamma, TNF-alpha, and IL-1beta on HBV.     

Differential expression and oxidation of MKP-1 modulates TNF-alpha gene expression

Monocytic cells are integral in the pathogenesis of inflammatory disorders. We have shown previously that asbestos-induced p38 mitogen-activated protein (MAP) kinase activation and TNF-alpha expression are mediated by H(2)O(2) in blood monocytes. Due to the high expression and activity of catalase and glutathione peroxidase, normal alveolar macrophages do not respond in a manner similar to that of blood monocytes. Since kinase activity is tightly regulated by phosphatases, we hypothesized that the dual specificity phosphatase MAP kinase phosphatase (MKP)-1 regulates p38 activity and TNF-alpha production in alveolar macrophages due to insufficient H(2)O(2) generation in response to asbestos. We found that MKP-1 was highly expressed in alveolar macrophages, while blood monocytes had minimal expression. Inhibition of expression and activity of MKP-1 or overexpression of a catalytic mutant MKP-1 recovered p38 activity in alveolar macrophages. We questioned whether MKP-1 oxidation played a role dictating the contrasting responses of these cells to asbestos exposure, and found that overexpressed wild-type MKP-1 in monocytes was oxidized, while the mutant MKP-1 remained in the reduced form. Monocytes overexpressing either catalase or wild-type MKP-1 had decreased p38 activation and TNF-alpha production, respectively. In addition, TNF-alpha gene expression was regained in alveolar macrophages overexpressing the catalytic mutant MKP-1. These data suggest that MKP-1, through increased expression and lack of oxidation, modulates the inflammatory response in alveolar macrophages exposed to asbestos.     

Pulmonary surfactant inhibits activation of human monocytes by recombinant interferon-gamma.

During an inflammatory reaction in the alveoli, the functional activities of monocytes, macrophages and granulocytes are regulated by a complex network of inflammatory mediators. The primary cytokine involved in activation of these phagocytes is interferon-gamma (IFN-gamma). The possible influence of local factors, such as pulmonary surfactant, on the activation process has not been studied until now. The aim of the present study was to investigate the effects of surfactant on the activation of monocytes by recombinant (r)IFN-gamma. The results revealed that human surfactant significantly inhibited both the increase in the expression of the high-affinity receptor for IgG, i.e. Fc gamma RI, and the production of H2O2 by rIFN-gamma-activated monocytes. Since our surfactant preparation stimulated the basal production of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) by monocytes, the effect of Survanta, a surfactant extract, on the rIFN-gamma-induced production of these cytokines by monocytes was studied. The results revealed that Survanta caused 80-90% inhibition of the rIFN-gamma-induced production of TNF-alpha and IL-1 beta by these cells. Together, these results could mean that surfactant is involved in the protection of the alveolar epithelium against injury caused by reactive oxygen intermediates (ROI) and TNF-alpha, and in the down-regulation of the production of inflammatory mediators. In view of these considerations, surfactant therapy may not only improve lung compliance and gas exchange but may also be beneficial in reducing the inflammatory reaction in the lungs.     

Mechanisms underlying the enhanced elevation of IL-1beta and TNF-alpha mRNA levels following endotoxin challenge in rat alveolar macrophages cultured with low-Mg2+ medium.

We have previously shown that interleukin (IL)-1beta and tumour necrosis factor (TNF)-alpha mRNA levels in rat alveolar macrophages are increased in by endotoxin (lipopolysaccharide; LPS)- stimulation and further enhanced by culturing with low-Mg2+ medium. We have now investigated the mechanisms of underlying this enhancement by using some specific signal transduction inhibitors. The enhanced elevation of both mRNAs levels was suppressed by pretreatment with TMB-8 (which inhibits calcium release from the endoplasmic reticulum) or dexamethasone (which inhibits nuclear factor [NF]-kappaB and activator protein [AP]-1), but not with verapamil or nifedipine (which inhibits calcium channels). The enhancment of IL-1beta, but not TNF-alpha mRNA levels, was suppressed by pretreatment with W-7 (which inhibits calmodulin), whereas the enhancement of TNF-alpha mRNA levels was suppressed by pretreatment with U73122 (which inhibits phospholipase C). Curcumin (an inhibitor of AP-1), suppressed the increases in both mRNAs induced by low-Mg2+ medium alone, but had no suppressive effect on the levels of either mRNA after LPS-stimulation in low-Mg2+ medium. Pyrrolidine dithiocarbamate (an inhibitor of NF-kappaB) prevented the elevation of TNF-alpha mRNA levels induced by low-Mg2+ medium without LPS-stimulation, but had no suppressive effect on IL-1beta mRNA levels. From these results, we conclude that the enhanced elevation of IL-1beta and TNF-alpha mRNA levels seen in LPS-stimulated alveolar macrophages in low-Mg2+ medium occurs partly via the same, and partly via different, signaling pathways.     

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.     

Different G(i)-coupled chemoattractant receptors signal qualitatively different functions in human neutrophils

fMLP- or TNF-alpha-stimulated neutrophils produced H(2)O(2) when they adhered to fibrinogen-coated surfaces but not when they adhered to collagen I-, collagen IV-, or Matrigel-coated surfaces. In contrast, LTB4- or IL-8-stimulated neutrophils did not produce H(2)O(2) when they adhered to any of these surfaces. fMLP and TNF-alpha were much more potent than LTB4 and IL-8 in stimulating neutrophils to up-regulate and to activate their alpha(M)beta(2) integrins, as measured by the binding of specific monoclonal antibodies. Pretreatment of neutrophils with pertussis toxin completely blocked their production of H(2)O(2) on fibrinogen-coated surfaces in response to fMLP and their migration through Matrigel in response to fMLP, LTB4, and IL-8. These data show that although the fMLP, LTB4, and IL-8 receptors are coupled to pertussis toxin-sensitive Galpha proteins, they signal neutrophils to initiate qualitatively different effector functions. We propose that the qualitative differences in effector functions signaled by different chemoattractants reflect qualitative differences in using G-protein beta and/or gamma subunits or other factors by their cognate receptors.     

Macrophages from C3H/HeJ mice require an additional step to produce monokines: synergistic effects of silica and poly(I:C) in the release of interleukin 1.

Resident macrophages from C3H/HeJ mice, in contrast with those of other strains of mice such as BDF1 mice, did not release a 35 kD m.w. factor having macrophage replacing activity (FRM) or interleukin 1 (IL-1) when, respectively, cultured alone or in the presence of silica. C3H/HeJ macrophages were nevertheless capable of producing an intracellular IL-1-like activity. In addition, after a two-step activation process, macrophages from BDF1 mice spontaneously released IL-1, whereas silica was required to induce the release of IL-1 from similarly treated C3H/HeJ macrophages. Such in vivo primed and in vitro stimulated macrophages failed to release FRM. In contrast, poly(I:C) was able to induce the release of FRM by C3H/HeJ macrophages but not that of IL-1; moreover, the addition of silica to poly(I:C)-stimulated cells led to an IL-1 release similar to that obtained with normal mice treated with silica alone. Since poly(I:C) is able to elicit the production of interferons (IFN), the involvement of IFNs was investigated in poly(I:C) activity. Neither IFN-alpha/beta nor IFN-gamma, when used alone or in the presence of silica, could induce the release of IL-1 by C3H/HeJ macrophages. In addition, antibodies to IFN-alpha/beta and IFN-gamma were unable to affect the poly(I:C) and silica induced release of IL-1. Thus, the signal provided by poly(I:C) does not appear to be mediated by IFN(s).     

Role of IL-12 in macrophage activation during intracellular infection: IL-12 and mycobacteria synergistically release TNF-alpha and nitric oxide from macrophages via IFN-gamma induction

IL-12 is believed to play an important role in cell-mediated immunity against intracellular infection primarily by acting on T and NK cells. Recent evidence has suggested, however, that IL-12 has broader cellular targets than previously thought. In this study, we examined the role of IL-12 in macrophage TNF-alpha and nitric oxide (NO) release by using an in vitro model of intracellular infection. IL-12 alone released relatively little TNF-alpha and NO, whereas live mycobacteria alone released TNF-alpha markedly but little NO from murine alveolar macrophages. However, IL-12 and mycobacteria together enhanced TNF-alpha and NO release synergistically. Because IL-12 and mycobacteria also released IFN-gamma from macrophages synergistically, and exogenous IFN-gamma with mycobacteria enhanced TNF-alpha and NO release synergistically, we examined the role of endogenous IFN-gamma in IL-12/mycobacteria-stimulated macrophage activation. Using macrophages from mice deficient in IFN-gamma, we found that IL-12/mycobacteria-enhanced macrophage TNF-alpha and NO release was mediated through endogenous IFN-gamma. We further demonstrated that IFN-gamma and mycobacteria together had a selective effect on macrophage cytokine release because they released TNF-alpha synergistically but not macrophage chemotactic protein-1 (MCP-1). These findings reveal that IL-12 can activate macrophages potently during intracellular infection, and this activating effect is mediated primarily through its effect on macrophage IFN-gamma release.