Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.     

Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-alpha: role of the p38 and p42/44 mitogen-activated protein kinases

IL-10 is an anti-inflammatory cytokine with potent immunomodulatory effects, including inhibition of cytokine production. However, regulation of monocyte IL-10 production is poorly understood. In this report we have investigated the mechanisms of LPS-induced IL-10 production by human peripheral blood monocytes and demonstrate that IL-10 synthesis is uniquely dependent on the endogenous proinflammatory cytokines IL-1 and/or TNF-alpha. LPS signal transduction in monocytes has been shown to involve activation of the p38 and p42 mitogen-activated protein kinase (MAPK) cascades. The results in this paper indicate that inhibition of p38 MAPK potently inhibited the production of IL-10, IL-1beta, and TNF-alpha, whereas blockade of the p42/44 MAPK pathway, while partially inhibiting TNF-alpha and IL-1beta production, had no effect on monocyte secretion of IL-10. Furthermore, neither the inhibition of monocyte TNF-alpha induced by IL-10 nor the stimulation of soluble TNF receptor production was affected by inhibition of the p42/44 MAPK pathway, suggesting that this signaling event is not involved in either monocyte production of or anti-inflammatory responses to IL-10. These data raise the interesting possibility that proinflammatory TNF-alpha-mediated effects may be selectively blocked without modulating the induction or the response to IL-10, whereas the signaling events associated with the anti-inflammatory events induced by IL-10 remain to be elucidated.     

Expression of type II nitric oxide synthase in primary human astrocytes and microglia: role of IL-1beta and IL-1 receptor antagonist

In this work, we studied the expression of type II nitric oxide synthase (NOS) in primary cultures of human astrocytes and microglia. Cytokine-activated human fetal astrocytes expressed a 4.5-kb type II NOS mRNA that was first evident at 8 h, steadily increased through 48 h, and persisted through 72 h. The inducing signals for astrocyte NOS II mRNA expression were in the order IL-1beta + IFN-gamma > IL-1beta + TNF-alpha > IL-1beta. SDS-PAGE analysis of cytokine-stimulated astrocyte cultures revealed an approximately 130-kDa single NOS II band that was expressed strongly at 48 and 72 h (72 h > 48 h). Specific NOS II immunoreactivity was detected in cytokine-treated astrocytes, both in the cytosol and in a discrete paranuclear region, which corresponded to Golgi-like membranes on immunoelectron microscopy. In human microglia, cytokines and LPS failed to induce NOS II expression, while the same stimuli readily induced TNF-alpha expression. In cytokine-treated human astrocytes, neither NOS II mRNA/protein expression nor nitrite production was inhibited by TGF-beta, IL-4, or IL-10. In contrast, IL-1 receptor antagonist exerted near complete inhibition of NOS II mRNA and nitrite induction. Monocyte chemoattractant peptide-1 mRNA was induced in TGF-beta-treated astrocytes, demonstrating the presence of receptors for TGF-beta in astrocytes. These results confirm that in humans, cytokines stimulate astrocytes, but not microglia, to express NOS II belonging to the high output nitric oxide system similar to that found in rodent macrophages. They also show that the regulation of type II NOS expression in human glia differs significantly from that in rodent glia. A crucial role for the IL-1 pathway in the regulation of human astrocyte NOS II is shown, suggesting a potential role for IL-1 as a regulator of astrocyte activation in vivo.     

TNF-alpha and IL-1beta selectively induce expression of 92-kDa gelatinase by human macrophages

Macrophages are present in inflammatory tissue sites where abnormal degradation of the extracellular matrix takes place. To evaluate the potential of macrophages to participate in such matrix destruction, we studied the effects of three cytokines present in inflammatory tissue sites, TNF-alpha, IL-1beta, and IFN-gamma, on the production of three matrix-degrading metalloproteinases, interstitial collagenase, stromelysin, and 92-kDa gelatinase, as well as their natural inhibitor, TIMP-1 (tissue inhibitor of metalloproteinases number 1), by human monocyte-derived macrophages differentiated in vitro. Spontaneous production of interstitial collagenase and stromelysin by these cells was minimal, and was not influenced by the cytokines. In contrast, the cells secreted substantial basal amounts of 92-kDa gelatinase, the secretion of which was stimulated (2- to 15-fold; on average 5-fold) by both TNF-alpha and IL-1beta, while the production of TIMP-1 was unaffected. IFN-gamma suppressed the production of the 92-kDa gelatinase induced by TNF-alpha- and IL-1beta. TNF-alpha and IL-1beta regulated the expression of 92-kDa gelatinase by monocyte-derived macrophages at the pretranslational level. The results show that expression of 92-kDa gelatinase, but not its natural inhibitor TIMP-1, by human tissue-type macrophages is selectively up-regulated by proinflammatory cytokines; which suggests that these cells, when actually present in an inflammatory environment, will actively participate in the destruction of the extracellular matrix.     

Cytokine regulation of human microglial cell IL-8 production

IL-8 involvement in neutrophil activation and chemotaxis may be important in inflammatory responses within the central nervous system, secondary to meningitis, encephalitis, and traumatic injury. The source of IL-8 within the brain during these inflammatory processes, however, is unknown. To explore the role of microglia in the production of IL-8, human fetal microglia, which are the resident macrophages of the brain, were treated with LPS and pro- and anti-inflammatory cytokines to determine their effects on IL-8 production. We found that IL-8 protein levels increased in response to LPS or IL-1 beta, or to TNF-alpha, which also corresponded to elevated IL-8 mRNA levels by RT-PCR. Pretreatment with IL-4, IL-10, or TGF-beta 1 potently inhibited the stimulatory effects of these proinflammatory agents. These findings indicate that human microglia synthesize IL-8 in response to proinflammatory stimuli, and that anti-inflammatory cytokines down-regulate the production of this chemokine. These results may have important therapeutic implications for certain central nervous system insults involving inflammation.     

Inhibition of IL-1 induced tissue factor (TF) synthesis and procoagulant activity (PCA) in purified human monocytes by IL-4, IL-10 and IL-13

Exposure of monocytes to pro-inflammatory cytokines or lipopolysaccharide (LPS) may induce synthesis and expression of tissue factor (TF). In this paper we have focused on the induction of TF-activity in human monocytes by the pro-inflammatory cytokines recombinant human interleukin 1 (rhIL-1 alpha) (rhIL-1 beta) (rhIL-6) and human tumour necrosis factor alpha (rhTNF-alpha), measured as procoagulant activity (PCA) in a microtitre plate-based clot assay. In addition we have studied the modulation of IL-1 alpha/beta induced TF-mRNA and PCA by rhIL-4, rhIL-10 and rhIL13. IL-1 alpha and IL-1 beta induced a concentration dependent increase in TF-activity. Neither IL-6 nor TNF-alpha gave rise to procoagulant activity at the concentrations tested (0.2-20 ng/ml). IL-4, IL-10 and IL-13, all effectively diminished IL-1 alpha/beta induced PCA, shown at the protein- and at the mRNA-level, while cell viability was unaffected. These results add to the previously demonstrated role of IL-4 and IL-10 as inhibitors of LPS-induced TF-activity, showing that these anti-inflammatory cytokines are not specific for LPS-activation but interfere with other stimulating substances such as IL-1, which may be involved in diseases where LPS is not present.     

In vitro IL-1 beta and TNF-alpha release from THP-1 monocytes in response to metal ions

OBJECTIVES: Previous studies have shown that biomaterials can activate macrophages to produce cytokines and promote an inflammatory response. Although the toxicity of many metal ions has been extensively investigated, little is known about the ability of these ions to alter cytokine release from macrophages. Yet the release of these ions from biomaterials has been well documented. Previous studies indicated that alterations in cytokine release might be expected because metal ions alter protein production in macrophages at sub-toxic concentrations. Thus, the hypothesis of this study was that metal ions can alter the secretion of cytokines from macrophages at sub-toxic concentrations. METHODS: The release of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) from macrophages was investigated when the macrophages were exposed to metal ions, with or without lipopolysaccharide (LPS), a component of dental plaque. Human THP-1 macrophages were exposed to ions of Ag, Au, Cu, Hg, and Ni for 24 h. In half of the cultures, LPS was added for the last 4 h. The release of IL-1 beta and TNF-alpha into the medium was measured using enzyme-linked immunosorbent assays. ANOVA and Tukey multiple comparison intervals were used to compare the various experimental conditions. RESULTS: None of the metal ions elevated the IL-1 beta or TNF-alpha levels after 24 h, but Ni ions significantly elevated the IL-1 beta and TNF-alpha levels after 72 h. With LPS added, Ag, Cu, and Ni significantly amplified the LPS-induced production of IL-1 beta but only Ni amplified the TNF-alpha response. These alterations in cytokine response occurred with metal ion concentrations which have been previously shown to be released from dental alloys in vitro and in vivo. SIGNIFICANCE: It appeared plausible that macrophage-cytokine mediated inflammatory responses may be altered by the presence of some metal ions in tissues, particularly Ni.     

The in vivo effects of PIXY321 therapy on human monocyte activity

In this report we describe the time-dependent effects of PIXY321 (a synthetic hybrid cytokine) treatment (500 and 750 micrograms/m2/day for 14 days) on six sarcoma patients. Blood was taken prior to PIXY321 injection (day 0), on days 1, 7, and 14 of treatment, and 7 days posttreatment (day 21). The number of isolated monocytes quadrupuled by day 7 and sustained a significant increase through day 14. There were significant increases in the percentage of circulating monocytes relative to total mononuclear cells on days 1 and 7 of therapy. There were no significant changes in monocyte cell surface antigens (15 checked), suggesting that the increase in monocyte numbers was not due to increased numbers of immature monocytes. The basal activity of the monocytes was not markedly altered during treatment; however, they were primed for significantly increased phorbol 12,13-dibutyrate-stimulated superoxide anion production and endotoxin-stimulated release of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) on days of 1 and 7 of therapy. There was a significant increase of IL-1 beta mRNA levels (unstimulated cells) on days 1 and 7, but TNF-alpha mRNA levels increased significantly on day 1 only. Consistent with the increase in superoxide anion production, there were increases in monocyte protein kinase C (PKC) levels on all days of therapy. There was a significant increase in PKCII beta mRNA only on the first day of treatment. All significant changes in monocyte number and function produced by PIXY321 infusion were reversible, as there were no sustained effects on day 21 (7 days after therapy). These results indicate that the effects of PIXY321 may be mediated through up-regulation of PKC resulting in monocytes primed for increased functional activity in response to an appropriate second stimulus.     

Enhanced IL-1 beta and tumor necrosis factor-alpha release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure

Idiopathic pulmonary fibrosis (IPF) and asbestosis are fibrotic interstitial lung diseases characterized by alveolar wall fibrosis with accumulation of extracellular matrix, interstitial remodeling, and increased numbers of activated alveolar macrophages. Animal models and in vitro studies have shown that macrophage cytokines, namely IL-1 beta and TNF-alpha, play significant roles in the development of fibrosis. We found significant increases for TNF-alpha release in both diseases (p < 0.01) and a significant increase for IL-1 beta release in asbestosis compared to normal controls (p < 0.01). Also, the mRNA expression of these cytokines was increased in alveolar macrophages from patients with IPF or asbestosis compared with normals. The level of TNF-alpha release in macrophage supernatants correlated with the number of neutrophils per milliliter bronchoalveolar lavage fluid returned. Chrysotile, crocidolite, amosite asbestos, and silica stimulated IL-1 beta and TNF-alpha release and up-regulated their respective mRNA in macrophages or monocytes. To evaluate the role of IL-1 beta and TNF-alpha in the accumulation of extracellular matrix, we studied collagen types I and III and fibronectin gene expression in human diploid lung fibroblasts after short term (2 h) serum-free exposure to recombinant cytokines. Both cytokines up-regulated these genes 1.5- to 3.6-fold. These cytokines have the potential to influence the remodeling and fibrosis observed in the lower respiratory tract in IPF and asbestosis.     

Human macrophages induced in vitro by macrophage colony-stimulating factor are deficient in IL-12 production

IL-12 is important for Th1 differentiation. Myeloid-derived antigen-presenting cells (APC) such as monocytes, macrophages (Mphi) and dendritic cells (DC) are believed to be major sources of IL-12 in vivo. We have compared IL-12 production of fresh monocytes with Mphi differentiated in vitro using macrophage colony-stimulating factor (M-CSF) or human plasma, and in vitro generated dendritic cells, since these differentiated cell types represent APC at sites of antigen challenge. Macrophages stimulated with lipopolysaccharide (LPS) or heat-killed Listeria monocytogenes in the presence or absence of IFN-gamma produced minimal IL-12 p70 by comparison with DC or monocytes, despite comparable production of TNF-alpha. M-CSF-induced Mphi produced low levels of IL-10 constitutively and high levels after stimulation with LPS, but neutralization of IL-10 did not augment Mphi IL-12 production. Exposure of Mphi to TNF-alpha, granulocyte-macrophage CSF or IFN-gamma did not substantially up-regulate IL-12. Therefore M-CSF induces a differentiated Mphi phenotype in which IL-12 production is down-regulated, perhaps irreversibly. This may be the default pathway for monocyte-Mphi development in the absence of inflammation.     

Localization of mRNA for inflammatory cytokines in radicular cyst tissue by in situ hybridization, and induction of inflammatory cytokines by human gingival fibroblasts in response to radicular cyst contents

The expression of mRNA encoding the inflammatory cytokines interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha(TNF-alpha) have been examined in radicular cysts by in situ hybridization. Furthermore, the biological activity of the contents of radicular cysts (RCC) has been assayed by adding extracts of RCC to cultured human gingival fibroblasts (HGFs) and analyzing the culture medium for the release of inflammatory cytokines. In the epithelial layer, keratinocytes expressed all cytokine mRNAs examined at various levels. Basal layer cells expressed mRNA for each cytokine. In the subepithelial granulation tissue of the cysts, fibroblasts and macrophages expressed mRNA for IL-6, IL-8, IL-1beta and TNF-alpha mRNA at varying levels; especially clear expression of TNF-alpha and IL-1beta mRNA was detected on macrophages. The infiltrating lymphoid cells, largely composed of T cells and plasma cells, expressed these cytokine mRNAs, especially those encoding IL-6 and IL-8, at various levels. In vitro analysis indicated dose-dependent release of both IL-6 and IL-8 by HGFs in response to RCC. After heating to 100 degrees C for 10 min, RCC almost completely failed to stimulate IL-6 release from HGFs. Furthermore, anti-IL-1beta antibody (neutralization test) did not prevent the stimulation of IL-6 release by RCC. Significant amounts of IL-6 and IL-8 were detected in RCC in two cases, and a trace amount of IL-1beta was detected in one case. This study demonstrated the wide expression of mRNA encoding inflammatory cytokines in radicular cyst tissues, and RCC itself was capable of stimulating IL-6 and IL-8 production from HGFs.     

In vivo expression of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumour necrosis factor-alpha and interferon-gamma in the fetal murine thymus

Cytokines are known to play a role in T-cell lymphopoiesis as potent growth or differentiation factors, but many experiments focusing on their role in the thymus have been conducted only in vitro. We have thus used frozen sections obtained from fetal thymuses of normal C57BL 6 mice to investigate by immunohistochemistry the presence of interleukin-1 beta (I4-1 beta), IL-2. IL-4. IL-6. interferon-7 (IFN-7) and tumour necrosis facor-alpha (TNF-alpha). The results reveal that apart from IL-2, which was not detected, all these cytokines display a time-dependent expression pattern in the normal fetal thymus. First, production of IL-4, IL-6 and TNF-alpha is detected around days 13 14; this is followed by a second wave on days 16 17, with a production of IL-1 beta, IL-4 and IL-6, and finally, just before birth (day 19), by a third wave of IL-1 beta, IL-4, IL-6, IFN-7 and TNF-alpha production. This supports the hypothesis that cytokines play a rote in T-cell lymphopoiesis.     

Divergent effects of LPS on expression of IL-1 receptor family members in mononuclear phagocytes in vitro and in vivo

Three molecules, interleukin 1 (IL-1) receptor I (IL-1RI), IL-1 receptor II (IL-1RII or decoy) and IL-1 receptor accessory protein (IL-1R AcP or IL-1RIII), are involved in IL-1 binding and signal transduction. In addition, three homologous genes (T1/ST2, MyD88 and rsc786) have been identified. Expression of the signal transducing type I R and of the decoy type II R in human monocytes is regulated by pro- and anti-inflammatory signals. The present study was designed to evaluate comprehensively how a prototypic pro-inflammatory signal, bacterial lipopolysaccharide (LPS), affects expression of IL-1R family members in mononuclear phagocytes in vitro and in vivo. Resting human monocytes expressed high levels of IL-1RII, IL-1R AcP, MyD88 and rsc786, whereas low levels of IL-1RI and T1/ST2 were present. In vitro exposure to LPS augmented expression of IL-1RI, T1/ST2 and MyD88, whereas it inhibited that of IL-1RII and rsc786. Expression of IL-1R AcP in monocytes was less substantially affected by LPS. The expression of IL-1R family members was also studied in organs of mice given LPS. As expected on the basis of in vitro results, organs (e.g. spleen, lungs and peritoneal exudate cells) from LPS-treated mice showed increased levels of IL-1RI, T1/ST2 and MyD88. Intriguingly, while expression of IL-1RII was inhibited in peritoneal macrophages after LPS, in accordance with in vitro results, increased IL-1RII mRNA was observed in organs such as liver, lungs and spleen. This unexpected effect of LPS was drastically reduced in mice rendered neutropenic by 5-fluorouracil. Therefore, we conclude that the apparent induction of IL-1RII in certain organs of LPS-treated mice is due to recruitment of myeloid cells which express high levels of decoy RII. Therefore, members of IL-1R family are independently and divergently regulated in mononuclear phagocytes exposed to the prototypic pro-inflammatory signal LPS in vitro and in vivo.