Differential regulation of interleukin-6, macrophage inflammatory protein-1, and JE/MCP-1 cytokine expression in macrophage cell lines.

Activated macrophages produce a number of proinflammatory cytokines including IL-6, JE, MIP-1 alpha and MIP-1 beta. The induction requirements for production of either IL-6 or the MIP-1 related inflammatory proteins (MIP-1 alpha, MIP-1 beta, and JE) have been analyzed independently using fibroblasts, monocytes, or endothelial cells. However, little is known about the regulation of these cytokines in macrophages. Since activated macrophages produce prostaglandins (PGE2) which may participate in the autoregulation of cytokine production by stimulation of adenylate cyclase and the induction of cAMP-dependent signal pathways, we determined the effects of PGE on the production of IL-6 and MIP-1-related proteins. Murine macrophage cell lines were incubated with PGE1, PGE2, cholera toxin, or dibutyryl cAMP in the presence of absence suboptimal doses of LPS. Pharmacologic agents alone did not induce IL-6 production but incubation of macrophages with combinations of adenylate cyclase stimulators and LPS or dcAMP and LPS led to the dose-dependent enhancement of IL-6 secretion and mRNA expression. In contrast, PGE1 inhibits LPS-induced JE, MIP-1 alpha, and MIP-1 beta mRNA expression and this inhibition is partially dependent on a cAMP-mediated pathway of signal transduction. In previous work we demonstrated that IFN-gamma and PMA do not stimulate the production of IL-6 by macrophages. Here we show that incubation of macrophages with either IFN-gamma or PMA induces the expression of JE, MIP-1 alpha and MIP-1 beta mRNA expression. JE mRNA expression is much more responsive to the stimulatory effects of IFN-gamma than are the MIP-1 genes. Finally, PGE inhibits PMA and IFN-gamma-induced JE and MIP-1-related mRNA expression.     

IFN-beta inhibits the ability of T lymphocytes to induce TNF-alpha and IL-1beta production in monocytes upon direct cell-cell contact.

Tumour necrosis factor (TNF)-alpha and interleukin (IL-)1beta, essential players in the pathogenesis of immuno-inflammatory diseases, are strongly induced in monocytes by direct contact with stimulated T lymphocytes. The present study shows that the latter mechanism is inhibited by interferon (IFN)-beta. In co-cultures of autologous T lymphocytes and monocytes stimulated by phytohaemagglutinin (PHA), IFN-beta inhibited the production of TNF-alpha and IL-1beta by 88 and 98%, respectively, whereas the simultaneous production of IL-1 receptor antagonist (IL-1Ra), was enhanced two-fold. The latter effects of IFN-beta were independent of modulations in IFN-gamma, IL-4 and IL-10 production. When monocytes were activated by plasma membranes of stimulated T cells, IFN-beta slightly inhibited the production of TNF-alpha and IL-1beta, while enhancing 1.5-fold that of IL-1Ra. The latter effect correlated with the persistence of high steady-state levels of IL-1Ra mRNA after 24 h of activation. Membranes isolated from T lymphocytes that had been stimulated in the presence of IFN-beta displayed a 80% decrease in their capacity to induce the production of IL-1beta and TNF-alpha in monocytes, whereas IL-1Ra induction was decreased by only 32%. These results demonstrate that IFN-beta modulates contact-mediated activation of monocytes by acting on both T lymphocytes and monocytes, decreasing the ability of T lymphocytes to induce TNF-alpha and IL-1beta production in monocytes and directly enhancing the production of IL-1Ra in the latter cells.     

Zinc-mediated inhibition of cyclic nucleotide phosphodiesterase activity and expression suppresses TNF-alpha and IL-1 beta production in monocytes by elevation of guanosine 3',5'-cyclic monophosphate

The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-alpha and IL-1beta release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-alpha and IL-1beta release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-alpha and IL-1beta by increasing intracellular cGMP levels.     

Inducible expression and regulation of the alpha 1-acid glycoprotein gene by alveolar macrophages: prostaglandin E2 and cyclic AMP act as new positive stimuli.

We have reported that alpha 1-acid glycoprotein (AGP) gene expression was induced in lung tissue and in alveolar type II cells during pulmonary inflammatory processes, suggesting that local production of this immunomodulatory protein might contribute to the modulation of inflammation within the alveolar space. Because AGP may also be secreted by other cell types in the alveolus, we have investigated the expression and the regulation of the AGP gene in human and rat alveolar macrophages. Spontaneous AGP secretion by alveolar macrophages was increased 4-fold in patients with interstitial lung involvement compared with that in controls. In the rat, immunoprecipitation of [35S]methionine-labeled cell lysates showed that alveolar macrophages synthesize and secrete AGP. IL-1 beta had no effect by itself, but potentiated the dexamethasone-induced increase in AGP production. RNase protection assay demonstrated that AGP mRNA, undetectable in unstimulated cells, was induced by dexamethasone. Conditioned medium from LPS-stimulated macrophages as well as IL-1 beta had no effect by themselves, but potentiated the dexamethasone-induced increase in AGP mRNA levels. In addition to cytokines, PGE2 as well as dibutyryl cAMP increased AGP mRNA levels in the presence of dexamethasone. When AGP expression in other cells of the monocyte/macrophage lineage was examined, weak and no AGP production by human blood monocytes and by rat peritoneal macrophages, respectively, were observed. Our data showed that 1) AGP expression is inducible specifically in alveolar macrophages in vivo and in vitro; and 2) PGE2 and cAMP act as new positive stimuli for AGP gene expression.     

Induction of human monocyte IL-1 mRNA and secretion during anti-CD3 mitogenesis requires two distinct T cell-derived signals.

The T cell signals that regulate the induction of human monocyte IL-1 during primary immune activation were investigated by using anti-CD3 mitogenesis. The induction of monocyte IL-1 alpha and beta mRNA during anti-CD3 mitogenesis was rapid (less than or equal to 1 h) and required the presence of both T cells and anti-CD3. The addition of T cells plus a nonmitogenic anti-CD5 antibody failed to induce IL-1 alpha or beta mRNA, indicating that IL-1 mRNA induction by anti-CD3 required T cell activation. Experiments using double chamber culture wells revealed that the major initial phase of IL-1 alpha and beta mRNA induction (1 to 12 h) required direct cell contact between monocytes and T cells. A subsequent minor late phase (greater than or equal to 12 h) of IL-1 mRNA was induced independently of cell contact in monocytes that received only soluble factors generated during anti-CD3 mitogenesis and was temporally associated with the appearance in culture supernatants of the late phase IL-1-inducing cytokines, IL-2, IFN-gamma, and TNF-alpha. Metabolic inactivation of T cells using paraformaldehyde demonstrated that the ability of T cells to induce IL-1 mRNA via cell contact was acquired only after activation of T cells via solid phase anti-CD3. Furthermore, pretreatment of T cells with the protein synthesis inhibitor emetine had no effect on T cell-mediated induction of monocyte IL-1 mRNA or cell-associated IL-1 alpha and beta, indicating that the expression of the IL-1 inductive signal did not require protein synthesis. Despite their ability to induce monocyte IL-1 alpha and beta mRNA, activated T cells treated with paraformaldehyde or emetine were no longer able to induce monocytes to secrete IL-1 beta into culture supernatants. However, supernatants from purified T cells that were activated with solid-phase anti-CD3 restored the ability of paraformaldehyde or emetine-treated T cells to induce IL-1 secretion. These studies provide evidence that supports a two-signal model of monocyte IL-1 production during primary immune activation. The first signal leads to the induction of monocyte IL-1 mRNA and is mediated by direct contact with activated T cells, and the second signal is provided by soluble T cell factors and results in IL-1 secretion.     

Interleukin 4 inhibition of prostaglandin E2 synthesis blocks interstitial collagenase and 92-kDa type IV collagenase/gelatinase production by human monocytes.

Activation of human monocytes results in the production of interstitial collagenase through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Inasmuch as interleukin 4 (IL-4) has been shown to inhibit PGE2 synthesis by monocytes, we examined the effect of IL-4 on the production of human monocyte interstitial collagenase. Additionally, we also assessed the effect of IL-4 on the production of 92-kDa type IV collagenase/gelatinase and tissue inhibitor of metalloproteinase-1 (TIMP-1) by monocytes. The inhibition of PGE2 synthesis by IL-4 resulted in decreased interstitial collagenase protein and activity that could be restored by exogenous PGE2 or dibutyryl cyclic AMP (Bt2cAMP). IL-4 also suppressed ConA-stimulated 92-kDa type IV collagenase/gelatinase protein and zymogram enzyme activity that could be reversed by exogenous PGE2 or Bt2cAMP. Moreover, indomethacin suppressed the ConA-induced production of 92-kDa type IV collagenase/gelatinase. These data demonstrate that, like monocyte interstitial collagenase, the conA-inducible monocyte 92-kDa type IV collagenase/gelatinase is regulated through a PGE2-mediated cAMP-dependent pathway. In contrast to ConA stimulation, unstimulated monocytes released low levels of 92-kDa type IV collagenase/gelatinase that were not affected by IL-4, PGE2, or Bt2cAMP, indicating that basal production of this enzyme is PGE2-cAMP independent. IL-4 inhibition of both collagenases was not a result of increased TIMP expression since Western analysis of 28.5-kDa TIMP-1 revealed that IL-4 did not alter the increased TIMP-1 protein in response to ConA. These data indicate that IL-4 may function in natural host regulation of connective tissue damage by monocytes.     

Adherence induces selective mRNA expression of monocyte mediators and proto-oncogenes

Adherence is an important regulatory signal for several monokines and the proto-oncogenes c-fms and c-fos in human peripheral blood monocytes. Although there is little if any constitutive expression of the IL-1 beta, TNF-alpha and CSF-1 genes in freshly isolated monocytes, adherence is sufficient to induce high steady-state levels of mRNA for TNF and c-fos and more slowly that of CSF-1. Expression of mRNA for the CSF-1R gene, c-fms, was transiently down-regulated by 4 h. In contrast, the induction of high levels of IL-1 beta mRNA were achieved independent of culture conditions. Although all of these genes could be induced by adherence, actual secretion of the mediators required the exposure to a second signal derived from LPS. Thus adherence rapidly primes monocytes for a variety of inflammatory responses, the magnitude of which depends on the nature of a second "activating" signal. It is likely that some of these products act locally as paracrine or autocrine factors to further regulate the phenotype of the differentiating macrophage.     

Cyclic AMP modulates interleukin-1 action in a cytotoxic T-cell hybridoma

The induction of cytolytic activity in PC60, a murine T-cell hybridoma, is paralleled by a rise in the level of BLT-esterase (N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase), a serine esterase specific for activated T-cells. Both interleukin-1 (IL-1) and dibutyryl cAMP were albe to increase the esterase activity in a dose-dependent and saturable manner. When added in combination the two activators showed a strong synergism: BLT-esterase levels were up to three times higher than the sum of the levels due to dibutyryl cAMP and IL-1 added separately. Stimulators of the adenylate cyclase, such as forskolin and cholera toxin, induced a similar enhancement of the BLT-esterase response to IL-1. PC60 cells did not produce any cAMP in response to IL-1. When the two stimuli were added sequentially a second effect for cAMP emerged: preincubation with dibutyryl cAMP or activators of the adenylate cyclase for 4 h or longer completely blocked the action of subsequently added IL-1. Taken together, the data demonstrate a dual modulatory role for cAMP in T-lymphocytes activated by IL-1.     

Cyclic AMP stimulates platelet-derived growth factor B chain mRNA expression in murine macrophage cell lines

Prostaglandin E(2) plays a role in cytokine production presumably by altering intracellular levels of cAMP. In this paper, we report on the differential expression of cytokine genes in murine macrophages in response to stimulation with activators of cAMP. Macrophages were cultured with or without cAMP activators in the presence or absence of LPS. Prior to treatment, macrophages do not express interleukin-1beta, but do express low levels of tumour necrosis factor alpha and platelet-derived growth factor B chain mRNAs. After culture with cAMP-inducers, including PGE(2), dibutyryl cAMP and forskolin, PDGF B chain mRNA is induced. Forskolin, for example, induced expression PDGF B chain mRNA to a level ranging from 25% to 200% of the level induced by LPS in 6 h. In contrast, cAMP-inducers enhance the expression of IL-1beta and TNF-alpha mRNAs, but only in the presence of LPS. The combination of forskolin and LPS does not appear to act synergistically on PDGF B chain mRNA levels, suggesting that LPS-stimulated effects are not mediated through a cAMP-dependent pathway. Furthermore, macrophages differentially express cytokine genes in response to treatment with inducers of intracellular cAMP.     

Macrophage inflammatory protein-3 beta enhances IL-10 production by activated human peripheral blood monocytes and T cells.

We report that the addition of human macrophage inflammatory protein-3 beta (MIP-3 beta) to cultures of human PBMCs that have been activated with LPS or PHA results in a significant enhancement of IL-10 production. This effect was concentration-dependent, with optimal MIP-3 beta concentrations inducing more than a 5-fold induction of IL-10 from LPS-stimulated PBMCs and a 2- to 3-fold induction of IL-10 from PHA-stimulated PBMCs. In contrast, no significant effect on IL-10 production was observed when 6Ckine, the other reported ligand for human CCR7, or other CC chemokines such as monocyte chemoattractant protein-1, RANTES, MIP-1 alpha, and MIP-1 beta were added to LPS- or PHA-stimulated PBMCs. Similar results were observed using activated purified human peripheral blood monocytes or T cells. Addition of MIP-3 beta to nonactivated PBMCs had no effect on cytokine production. Enhancement of IL-10 production by MIP-3beta correlated with the inhibition of IL-12 p40 and TNF-alpha production by monocytes and with the impairment of IFN-gamma production by T cells, which was reversed by addition of anti-IL-10 Abs to the cultures. The ability of MIP-3 beta to augment IL-10 production correlated with CCR7 mRNA expression and stimulation of intracellular calcium mobilization in both monocytes and T cells. These data indicate that MIP-3 beta acts directly on human monocytes and T cells and suggest that this chemokine is unique among ligands binding to CC receptors due to its ability to modulate inflammatory activity via the enhanced production of the anti-inflammatory cytokine IL-10.     

Production of IL-1 receptor antagonist by human in vitro-derived macrophages. Effects of lipopolysaccharide and granulocyte-macrophage colony-stimulating factor.

The objective of these experiments was to evaluate the production of IL-1ra, a specific receptor antagonist of IL-1, by human in vitro-derived macrophages, a model for differentiated macrophages. IL-1ra protein levels in supernatants and lysates of cultured cells were determined by a specific ELISA. Relative steady-state IL-1ra mRNA levels were measured using a specific cDNA probe. Human monocytes were differentiated by 6 days culture in either medium or granulocyte-macrophage colony-stimulating factor (GM-CSF), after which the effects of subsequent LPS and/or GM-CSF on the production of IL-1ra were evaluated. In vitro-derived macrophages cultured in medium for 6 days constitutively produced IL-1ra protein during the 24-h period of the 7th day in culture. The constitutive production of IL-1ra by medium-aged cells correlated with low steady-state IL-1ra mRNA levels determined over this same time period. In contrast, cells cultured for 6 days in GM-CSF synthesized significantly increased levels of IL-1ra protein during the 7th day in culture but the secreted levels remained unchanged. Cells differentiated in GM-CSF displayed enhanced steady-state levels of IL-1ra mRNA in comparison with cells aged in medium. Stimulation of in vitro-derived macrophages aged for 6 days in medium or in GM-CSF, with LPS or adherent IgG, did not result in increased levels of IL-1ra protein production in comparison with non-LPS stimulated cells. The IL-1ra protein detected in the supernatants of cells differentiated in GM-CSF was biologically active in the IL-1-augmented murine thymocyte proliferation assay. By Western blot analysis, the IL-1ra protein in the in vitro-derived macrophage supernatants was predominantly the 22- to 24-kDa glycosylated species, whereas the lysates contained additional lower molecular weight forms. These results suggest that as monocytes differentiate in vitro into macrophages, they constitutively produce IL-1ra protein and that this production is enhanced by the continuous presence of GM-CSF.