Prostaglandins regulate melanoma-induced cytokine production in macrophages

Tumor-secreted products can affect macrophage cytokine expression and in that way alter the immune response. Prostaglandins (PGs) are found in the tumor microenvironment and have been associated with local and regional immunosuppression. We investigated whether tumor-secreted factors could induce PG synthesis in macrophages and whether these PGs could alter macrophage production of immunoregulatory cytokines. In both murine and human models, melanoma conditioned medium (MCM) induced macrophage production of PGE(2), IL-6, and TNF-alpha. PGE(2) production increased over 24 h and was accompanied by an increase in cyclooxygenase-2 (COX-2) expression, while COX-1 expression remained unchanged. In the presence of 10 microM NS398, a selective COX-2 inhibitor, MCM-stimulated PGE(2) synthesis was almost completely suppressed, while production of IL-6 and TNF-alpha proteins and mRNA also was partially abrogated. In the murine model, 200 microM NS398 resulted in more significant inhibition of cytokine protein and mRNA production. Although MCM induced NFkappaB and NF-IL-6 activation, neither dose of NS398 altered this effect. We conclude that melanoma-secreted products stimulate COX-2 expression and PGE(2) synthesis in macrophages and that inhibition of COX-2-derived PG synthesis results in partial abrogation of macrophage cytokine production.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells

Murine IL-10 (cytokine synthesis inhibitory factor) inhibits cytokine production by Th1 cell clones when they are activated under conditions requiring the presence of APC. By preincubating APC with IL-10, we demonstrate that IL-10 acts principally on APC to inhibit IFN-gamma production by Th1 clones. Moreover, IL-10 is not active when Th1 cells are stimulated with glutaraldehyde-fixed APC, which also indicates that its action involves regulation of APC function. Furthermore, IL-10 inhibits cytokine synthesis by Th1 cells stimulated with the super-antigen Staphylococcus enterotoxin B, which does not appear to require processing. Flow microfluorimetry purified splenic or peritoneal B cells and macrophages, and B cell and macrophage cell lines can present Ag to Th1 clones. However, IL-10 acts only on sorted macrophages and the macrophage cell line to suppress IFN-gamma production by Th1 clones. IL-10 does not show this effect when B cells are used as APC. In contrast, IL-10 does not impair the ability of APC to stimulate cytokine production by Th2 cells. IL-10 does not decrease IFN-gamma-induced I-Ad levels on a macrophage cell line. Inasmuch as IL-10 also inhibits IL-2-induced IFN-gamma production by Th1 cells in an Ag-free system requiring only the presence of accessory cells, these data suggest that IL-10 may inhibit macrophage accessory cell function which is independent of TCR-class II MHC interactions.     

Regulation of lipopolysaccharide-induced tumor necrosis factor production by cyclosporin A in mice primed with muramyl dipeptide

Abstract The effect of cyclosporin A (CsA) on tumor necrosis factor (TNF) or interleukin-6 (IL-6) production was evaluated in vivo in primed or unprimed mice challenged with lipopolysaccharide (LPS). Both pretreatment with BCG infection or with muramyl dipeptide (MDP) prior to LPS challenge resulted in an increase in the cytokine bioactivity level in the blood. CsA administration inhibited the TNF production. In unprimed mice, either normal or sensitized to LPS lethality by galactosamine treatment, a marked decrease in the cytokine level was observed after injection of CsA. After adrenalectomy, the yield of both TNF and IL-6 following LPS injection was markedly elevated but decreased by CsA administration. Ex vivo experiments have shown that the inhibitory effect of CsA could be demonstrated at the level of macrophages from mice previously given the drug. If mice had received MDP, in vitro responses of cells to LPS were enhanced but again CsA decreased the mRNA expression and protein secretion.     

Nitric Oxide Inhibits Rhinovirus-Induced Cytokine Production and Viral Replication in a Human Respiratory Epithelial Cell Line

To better understand the early biochemical events that occur in human rhinovirus (HRV) infections, we examined the kinetics and mechanisms of interleukin-8 (IL-8) and IL-6 production from infected epithelial cells. Several HRV strains caused IL-8 and IL-6 production, but HRV-16 induced maximal IL-8 and IL-6 mRNA expression and protein production more rapidly than did HRV-14, despite similar rates of replication of the two viral strains. Viral induction of cytokine mRNA does not require new protein synthesis, since it was unaffected by cycloheximide treatment. The potent glucocorticoid budesonide did not affect viral replication or cytokine mRNA induction but modestly inhibited cytokine protein production. Interestingly, the nitric oxide donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (NONOate) inhibited both rhinovirus replication and cytokine production in a dose-dependent fashion without reducing levels of cytokine mRNA. The NONOate effects were due to release of nitric oxide, because NONOate that had been depleted of its nitric oxide content had no effect. Thus, nitric oxide may play an important anti-inflammatory and antiviral role in colds and nitric oxide donors may represent a novel therapeutic approach.     

IL-10 synergizes with IL-4 and transforming growth factor-beta to inhibit macrophage cytotoxic activity.

After activation with IFN-gamma, thioglycollate-elicited murine peritoneal macrophages kill schistosomula of Schistosoma mansoni in vitro by an L-arginine-dependent mechanism which involves the production of reactive nitrogen oxides (NO). In the present study we demonstrate that the regulatory cytokines IL-10, IL-4, and transforming growth factor-beta (TGF-beta) are potent inhibitors of this extracellular killing function of activated macrophages. Each cytokine was found to suppress killing of schistosomula in a dose-dependent fashion. The activity of IL-10 was not permanent, because subsequent treatment with additional IFN-gamma 2 to 6 h later reversed the inhibition of macrophage larval killing. More importantly, the combination of suboptimal levels of any two of these three cytokines was found to give a potent synergistic suppression of schistosomulum killing by IFN-gamma-treated macrophages. Similarly, IL-10, IL-4, or TGF-beta alone blocked the production of NO, and when used in combination these cytokines exhibited an enhanced inhibitory effect on nitrite production. Macrophage-mediated killing of schistosomula through the generation of NO has been shown previously to be a major effector mechanism of schistosome immunity. The results presented here suggest that the suppression of this mechanism by induction of the regulatory cytokines IL-10, IL-4, and TGF-beta, which are known to be produced during schistosome infection, may be an important strategy used by the parasite to evade macrophage-mediated immune destruction.     

CYCLOSPORIN A INHIBITS THE IN VIVO PRODUCTION OF INTERLEUKIN-1β AND TUMOUR NECROSIS FACTOR α, BUT NOT INTERLEUKIN-6, BY A T-CELL-INDEPENDENT MECHANISM

The effect of cyclosporin A (CsA) on cytokine production in the tissue chamber model of acute inflammation was investigated. CsA caused a dose-related inhibition of interleukin 1β (IL-1β) production in both normal and athymic mice, confirming earlier conclusions that this effect is not T cell dependent (ED₅₀s 40 and 53 mg/kg p.o., respectively).Tumour necrosis factor alpha (TNF-α) levels were similarly affected with ED₅₀s of 40 and 58 mg/kg p.o. for normal and athymic mice, respectively. By contrast, CsA inhibited interleukin 6 (IL-6) production only in normal mice (ED₅₀27 mg/kg p.o.)Differences in the absolute production of the three cytokines in normal and athymic mice were also noted. IL-1β and IL-6 levels were two-fold higher in athymic mice, while for TNF-α, there was no difference between the two groups.The present findings support the authors' original hypothesis, that the inhibitory mechanism of CsA on IL-1β is not mediated via T cells. The same mechanism also seems responsible for the inhibition of TNF-α production, but not for IL-6, where inhibition by CsA appears to require the presence of T cells.     

Macrophage cell line RAW264.7 but not P-388D1 is an appropriate in vitro-model for studying oxidative burst as well as cytokine production in context of fatty acid enrichment

Oxidative burst and cytokines synthesis by macrophages is a crucial point for successful pathogen defense. However, macrophage cell lines commonly used in inflammatory research differ in their responses to external stimuli. Thus, there is the necessity to carefully characterize the cells before experimental usage. In this study we investigated the applicability of two widely-used macrophage cell lines, RAW264.7 and P-388D1, for studying oxidative burst and cytokine synthesis. Cells were tested for NADPH oxidase activity, iNOS-mRNA levels, and the release of NO, TNF-α, IL-6 and IL-10. Stimulation of RAW264.7 triggered oxidative burst as well as synthesis of TNF-α, IL-6 and IL-10. In contrast, following stimulation P-388D1 produced TNF-α and IL-6 only. Our findings confirm the relevance of cell line selection for reliability of in vitro-experiments. Moreover, the results approve RAW264.7 cells to be a suitable model to investigate the modulation capability of macrophages e.g. in context of fatty acid supplementation.