Interleukin-10 differentially regulates cytokine inhibitor and chemokine release from blood mononuclear cells and fibroblasts

In this study we have examined the effects of interleukin-10 (IL-10) on blood mononuclear cells (MNC) and on skin as well as on synovial fibroblasts. In unstimulated MNC, we found that IL-10 is a potent stimulator of interleukin-1 receptor antagonist (IL-1ra) and monocyte chemoattractant protein-1 (MCP-1) production and an inhibitor of IL-8 release. In cells exposed to IL-1β, it also moderately stimulated IL-1ra production and release of soluble tumor necrosis factor receptor p75 (sTNF-R p75) and inhibited IL-8 and MCP-1 production. In addition, we have evidence that the biological effects of IL-10 are not restricted to hematopoietic cells. IL-10 stimulated sTNF-R p55 dose-dependently and inhibited MCP-1 release from IL-1β-activated fibroblasts, whereas IL-8 production was not affected. Taken together, these findings identify novel biological actions of IL-10 on blood mononuclear and connective tissue cells which support its regulatory functions as a suppressor of inflammatory processes.     

Divergent effects of interleukin-10 on cytokine production by mononuclear phagocytes and endothelial cells

Interleukin-10 (IL-10), a product of T helper type 2 (TH2) cells and monocytes, inhibits cytokine production in mononuclear phagocytes. Given the similarities and interrelationship between cells of the monocyte-macrophage lineage and endothelial cells, we examined the effects of IL-10 on vascular endothelium. Murine IL-10 induced low levels of IL-6 production and amplified induction of IL-6 by lipopolysaccharide (LPS) or IL-1 in the murine tEND.1 endothelioma line, used for these studies because it retains properties of normal endothelium. The effect was more evident after prolonged (48–72 h) exposure to IL-10. IL-10 had similar activity on other endothelioma lines, whereas it inhibited IL-6 production by peritoneal macrophages. Induction and amplification of cytokine production by IL-10 was associated with higher levels of mRNA, which were maintained longer (up to 48 h) than in controls. In addition to IL-6, murine IL-10 induced or amplified expression of the chemoattractant cytokines monocyte chemotactic protein-1 (MCP-1) and KG Human IL-10 inhibited IL-6 release by LPS-stimulated human peripheral blood mononuclear cells, whereas it did not interfere with cytokine production by LPS- or IL-1-stimulated human umbilical vein endothelial cells. The selective inhibitory action of IL-10 on mononuclear phagocytes versus endothelial cells may play a role in the pathophysiology of TH2-directed responses.     

Gene expression and secretion of cytokines and cytokine receptors from highly purified CD56+ natural killer cells stimulated with interleukin-2, interleukin-7 and interleukin-12

Interleukin (IL)-2 IL-7 and IL-12 stimulate the generation of lymphokine-activated killer activity and proliferation in natural killer (NK) cells by different mechanisms. In this study, we have compared the ability of IL-2, IL-7 and IL-12 to induce expression of cytokines and cytokine receptors both at the gene and protein level. IL-2 and IL-12 stimulated the CD56⁺ NK cells to release significant amounts of soluble p55 and p75 tumor necrosis factor receptor (TNFR), whereas less amounts of soluble TNFR were detected in IL-7-stimulated cultures. The p55 and p75 TNFR mRNA were expressed in resting NK cells, and no further induction was observed after cytokine-stimulation. Compared to the effects of IL-2, IL-7 induced lower, but substantial levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA, and IL-7 was a more potent GM-CSF-inducing stimulus than IL-12. IL-12 induced higher levels of interferon-γ (IFN-γ) mRNA than did IL-2, and IL-7 only weakly influenced the IFN-γ expression. In accordance with the mRNA studies, IL-7 induced the secretion of high amounts of GM-CSF and no or low levels of IFN-γ, whereas high amounts of IFN-γ and low levels of GM-CSF were detected in supernatants from IL-12-stimulated NK cells. In conclusion, IL-2, IL-7 and IL-12 differentially regulate expression of cytokines and cytokine receptors both at the gene and protein level.     

The effects of dexamethasone and chlorpromazine on tumour necrosis factor-alpha, interleukin-1 beta, interleukin-1 receptor antagonist and interleukin-10 in human volunteers.

Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are pro-inflammatory cytokines that play an important role in severe infections, whereas IL-1 receptor antagonist (IL-1ra) and IL-10 are anti-inflammatory cytokines that counteract their effects. Chlorpromazine and dexamethasone protect mice against lethal endotoxaemia by decreasing circulating concentrations of TNF-alpha and IL-1 beta. We investigated whether administration of chlorpromazine or dexamethasone to human volunteers is able to modulate the lipopolysaccharide (LPS)-stimulated cytokine production capacity in whole blood. Blood samples were taken before and several time-points after medication. Circulating cytokine concentrations were low in all samples. LPS-induced TNF-alpha and IL-1 beta production in whole blood was inhibited by dexamethasone treatment, while chlorpromazine had no effect. When peripheral blood mononuclear cells were stimulated in vitro with LPS, the addition of chlorpromazine (1-100 ng/ml) had no modulatory action on TNF-alpha, IL-1 beta, IL-1ra or IL-10 synthesis. The chlorpromazine concentrations measured in circulation of volunteers were eight to 40 times lower than the concentrations shown to be effective in mice. In conclusion, chlorpromazine inhibits TNF-alpha and IL-1 beta production in mice at concentrations that cannot be reached in humans, thus precluding its usage in clinical anti-cytokine strategies. In contrast, dexamethasone is an effective inhibitor of pro-inflammatory cytokine production.     

IL-13对LPS诱导人系膜细胞分泌IL-12的影响

目的: 探讨白细胞介素13（IL-13）对肾小球系膜细胞分泌白细胞介素12（IL-12）的影响作用。方法: 用酶联免疫吸附（ELISA）法和逆转录聚合酶链反应（RT-PCR）法检测系膜细胞IL-12蛋白和L-12p40 mRNA表达。结果: LPS诱导系膜细胞的IL-12p40 mRNA表达及其蛋白分泌（P<0.01）。IL-13在1-100 μg/L浓度范围内呈剂量依赖性抑制LPS诱导的系膜细胞IL-12分泌及其mRNA表达（P<0.05或P<0.01）。结论: IL-13可能通过抑制LPS诱导系膜细胞分泌IL-12，而调整了体内Th1/Th2细胞因子平衡。     

Effects of piroxicam on mononuclear cells

Piroxicam and other antiarthritic drugs were compared with respect to their effects on T-lymphocyte/monocyte/rheumatoid synovial cell interactions leading to inflammatory mediator production. Piroxicam inhibited PGE2 formation by blood mononuclear cells, but was less potent than indomethacin. Both drugs enhanced suboptimal phytohemagglutinin (PHA)-stimulated tritiated thymidine (³H-TdR) incorporation by mononuclear cells, although optimal responses were less affected. Exogenous interleukin-2 (IL-2) enhanced suboptimal but not optimal PHA responses, and the effects of the cyclo-oxygenase inhibitors were overcome by exogenous PGE₂, Thus piroxicam and indomethacin prevented the inhibition by endogenous monocyte-derived PGE₂ of IL-2 secretion and activity. Other antiarthritic drugs, including antimalarials, immunosuppressive agents and gold salts, inhibited PHA-induced lymphocyte proliferation regardless of the level of stimulation. Mepacrine and chioroquine were more effective in inhibiting the release of mononuclear cell factor (MCF) that stimulated PGE₂ synthesis by synovial cells. Cyclosporin-A, azathioprine and 6-mercaptopurine were more potent as antiproliferative agents than as inhibitors of mediator release. Sodium aurothiomalate and aurothioglucose selectively interfered with lymphocyte-mediated amplification of MCF release, whereas auranofin inhibited spontaneous production of monocytes and the action of MCF on synovial cells. In rheumatoid synovial cells, piroxicam and indomethacin inhibited PGE₂ production but not collagenase release. Suppression of MCF release could lead indirectly to reduction of IL-2 and collagenase as well as PGE₂ production and consequently to more profound inhibition of immunologically-mediated inflammation.     

麻疹疫苗反复注射对哮喘患儿IL-12、IL-13水平的影响

目的与方法:通过观察轻-中度发作期哮喘患儿麻疹疫苗反复注射前后IL-12,IL-13及血清总IgE水平的变化,探讨麻苗治疗对哮喘患儿的免疫影响及其治疗小儿哮喘的作用机制。采用生物素-亲合素双抗夹心酶联免疫(ABC-ELISA)法测定麻苗治疗前后13例哮喘患儿(麻苗治疗组)血浆及外周血单个核细胞(PBMC)培养上清中的IL-12、IL-13与血清总IgE水平,并与12例单纯对症治疗组哮喘患儿及17例正常对照组小儿比较。结果:麻苗治疗后,IL-12水平与对症治疗组无显著差异(P＞0.05),IL-13与血清总IgE水平显著降低(P＜0.05)。血浆IL-12水平与血清总IgE水平呈负相关(r=-0.437,P＜0.05),PBMC培养上清中的IL-12水平与血清总IgE水平无相关性;血浆及PBMC培养上清中的IL-13水平与血清总IgE水平呈正相关(r=0.657及r=0.485,P＜0.01);血浆及PBMC培养上清中的IL-12水平与IL-13水平呈负相关(r=-0.460及r=-0.383,P＜0.05)。结论:麻疹疫苗反复注射是通过下调哮喘患儿IL-13水平,进而降低血清总IgE水平,而对IL-12水平无显著影响。     

Interleukin-17 augments tumor necrosis factor-alpha-induced elaboration of proangiogenic factors from fibroblasts

Interleukin-17 (IL-17) is a CD4 T cell cytokine. In this report, we investigated the effects of this cytokine on the elaboration of proangiogenic factors by lung fibroblasts. After stimulation with a wide range of doses of IL-17, fibroblasts produced more amount of various kinds of angiogenic factors including NO, HGF, MCP-1, KC, MIP-2, PGE1, PGE2 and VEGF in a dose-dependent manner. Treatment with a COX-1 and COX-2 inhibitor indomethacin did not impair IL-17-induced HGF and VEGF secretion in fibroblasts. In addition, TNF-alpha alone stimulated the elaboration of KC, MIP-2, PGE2 and VEGF in fibroblasts. IL-17 and TNF-alpha in combination up-regulated elaboration of these proangiogenic factors additively or synergistically. Moreover, conditioned media (CM) from IL-17-stimulated fibroblasts showed significantly higher activity on endothelial cell growth than those from non-treated control cells. These results indicate that IL-17 up-regulates elaboration of various proangiogenic factors, and modulates macrophage-derived TNF-alpha-induced production of KC, MIP-2, PGE2 and VEGF by fibroblasts. Our findings also demonstrate that IL-17 might be a potential contributor to the inflammatory angiogenesis via induction of proangiogenic factors by stromal fibroblasts.     

Regulation of Interleukin-2 Induced Soluble Fas Ligand Release from Human Peripheral Blood Mononuclear Cells

Adjuvant interleukin (IL)-2 immunotherapy has been used in the treatment of different malignant dieseases. However, clinical results have been rather disappointing. Therefore, further investigations on IL-2-induced mediators of cytotoxicity seem to be necessary in order to possibly create cytokine cocktails which could enhance the IL-2-induced cytotoxicity. We therefore investigated the regulation of IL-2-induced release of soluble Fas Ligand (sFasL), since this factor is known to possess anti-tumor activities. In CD3-stimulated peripheral blood mononuclear cells IL-2 induced sFasL in a dose-dependent fashion. Maximum sFasL concentrations were obtained after stimulation of MNC for 120 hrs. Inhibition of endogenous IL-12 production significantly reduced IL-2-mediated sFasL release by about 25%. In contrast, addition of IL-12 enhanced the IL-2-induced sFasL about 1,5-fold. IL-10 and IL-4 reduced the IL-2-stimulated sFasL by about 30%. Interestingly, these suppressive effects could be antagonized by the addition of IL-12. Not only exogenous IL-10 but also endogenously produced IL-10 decreased the sFasL release to that extent which had been stimulated by IL-12. Since IL-12 and IL-10 only marginally influenced the IL-2-mediated cell proliferation as well as the IL-2-induced cell death, the IL-12- and IL-10-controlled sFasL release seems to be based on an enhanced production per cell. However, the increase in cell numbers as well as the decrease of viability during cell culture might additionally contribute to the IL-2-induced increase of sFasL release. This secondary effect might explain why IL-2-mediated sFasL production is only partially controlled by regulatory cytokines such as IL-4, IL-10 or IL-12. In conclusion, addition of IL-12 might increase the efficacy of IL-2 immunotherapy by inhibition of the IL-10-mediated negative feed-back loop on IL-2-mediated sFasL release.     

Synergistic effect of coumarin (1,2 benzopyrone) and endotoxin in the induction of human interleukin-1.

Coumarin as well as its derivatives 7-OH coumarin and 4-OH coumarin were found to stimulate interleukin-1 beta (IL-1 beta) release from freshly isolated human mononuclear cells (MNC) if the culture medium contained fetal calf serum. Under serum-free conditions, almost no induction of IL-1 beta release was observed and the former effect could be completely eliminated by polymyxin B. Therefore, the combined action of endotoxin and coumarin was tested on MNC IL-1 beta production. The coumarins were able to potentiate human MNC IL-1 beta production by lipopolysaccharide (LPS) in a dose-dependent manner. That the effect was due to the presence of coumarins and not endotoxin contamination was shown by negative Limulus amebocyte lysate tests and pre-incubation of the coumarins with polymyxin B-agarose. The latter procedure was able to block endotoxin induced IL-1 beta production but the synergism between coumarin and endotoxin was not influenced by pre-incubating the coumarins with polymyxin B-agarose. Cycloheximide as well as actinomycin D eliminated the induction of IL-1 release by coumarin and LPS demonstrating that the cytokine was newly synthesized after MNC stimulation. In addition, both the total amount of MNC IL-1 beta (cell-associated + extracellular) and the extracellular portion of the cytokine were synergistically decreased if coumarin or its derivatives were added to endotoxin-stimulated cultures. Synergism of coumarin and endotoxin in the induction of interleukin-6 or tumour necrosis factor-alpha could be observed in a smaller percentage of donors. These findings demonstrate an immunomodulatory effect of coumarin on cytokine production by monocytes in vitro which might help to explain some of the biological activities attributed to the drug upon its application in tumour patients.     

Regulation of interleukin-2 induced soluble Fas ligand release from human peripheral blood mononuclear cells

Adjuvant interleukin (IL)-2 immunotherapy has been used in the treatment of different malignant dieseases. However, clinical results have been rather disappointing. Therefore, further investigations on IL-2-induced mediators of cytotoxicity seem to be necessary in order to possibly create cytokine cocktails which could enhance the IL-2-induced cytotoxicity. We therefore investigated the regulation of IL-2-induced release of soluble Fas Ligand (sFasL), since this factor is known to possess anti-tumor activities. In CD3-stimulated peripheral blood mononuclear cells IL-2 induced sFasL in a dose-dependent fashion. Maximum sFasL concentrations were obtained after stimulation of MNC for 120 hrs. Inhibition of endogenous IL-12 production significantly reduced IL-2-mediated sFasL release by about 25%. In contrast, addition of IL-12 enhanced the IL-2-induced sFasL about 1,5-fold. IL-10 and IL-4 reduced the IL-2-stimulated sFasL by about 30%. Interestingly, these suppressive effects could be antagonized by the addition of IL-12. Not only exogenous IL-10 but also endogenously produced IL-10 decreased the sFasL release to that extent which had been stimulated by IL-12. Since IL-12 and IL-10 only marginally influenced the IL-2-mediated cell proliferation as well as the IL-2-induced cell death, the IL-12- and IL-10-controlled sFasL release seems to be based on an enhanced production per cell. However, the increase in cell numbers as well as the decrease of viability during cell culture might additionally contribute to the IL-2-induced increase of sFasL release. This secondary effect might explain why IL-2-mediated sFasL production is only partially controlled by regulatory cytokines such as IL-4, IL-10 or IL-12. In conclusion, addition of IL-12 might increase the efficacy of IL-2 immunotherapy by inhibition of the IL-10-mediated negative feed-back loop on IL-2-mediated sFasL release.     

Inhibitory effects of interleukin-10 on synovial cells of rheumatoid arthritis.

This paper describes the immunoregulatory effects of interleukin-10 (IL-10) on synovial cells in vitro. Synovial cells were cultured with IL-10 in the presence or absence of various cytokines. Following incubation, the costimulatory molecule expression on synovial cells and cytokine production in culture supernatants were analysed by an indirect immunofluorescence method and enzyme-linked immunosorbent assay, respectively. We also examined the effect of IL-10 on the function of synovial cells as antigen-presenting cells (APC). Synovial cells spontaneously express several kinds of costimulatory molecule and produce various kinds of cytokines. Stimulation of synovial cells with interferon-gamma (IFN-gamma), IL-1 beta, or 12-O-tetradecanoyl phorbol 13-acetate (TPA) markedly enhanced the expression of costimulatory molecules and cytokine production of these cells. Both spontaneous and up-regulated costimulatory molecule expression and cytokine production were significantly suppressed by the addition of IL-10. Autologous T-cell proliferation was stimulated by purified protein derivative (PPD) in IFN-gamma-treated synovial cells and treatment of these synovial cells with IL-10 also suppressed T-cell proliferation. Our results suggest that IL-10 has an inhibitory effect on synovial cells and is an important immunoregulatory component of the cytokine network in rheumatoid arthritis.     

Effect of the late complement components C5b-9 and of platelet-derived growth factor on the prostaglandin release of human synovial fibroblast-like cells

We examined the prostaglandin E (PGE) synthesis of cultured adherent synovial fibroblast-like cells (SFC) from patients with osteoarthritis (OA) in the noninflammatory state as well as with rheumatoid arthritis (RA). In cells from RA patients the spontaneous PGE release was generally higher compared to that of OA patients, but decreased fast with time in culture. After cell passage, similar PGE baseline levels were seen in cells of the two patient groups. The cells could then be stimulated by the terminal complement components C5b-9 or C5b-8. PGE synthesis was also stimulated by the platelet-derived growth factor (PDGF), interleukin-1 (IL-1), or lipopolysaccharide (LPS). The amount of PGE synthesis after incubation with PDGF, LPS and IL-1 was comparable to that released after C5b-9. Thus, like other inflammatory mediators C5b-9 and PDGF trigger the increased PGE production by SFC and thus may participate in the development of synovial inflammation and contribute to the pathogenesis of RA.     

Cytokine regulation of the interleukin-1 receptor antagonist protein in U937 cells

A naturally occurring receptor-level antagonist of interleukin-1 (IRAPorIL-1 ra) has recently been cloned. To determine what stimuli might regulate this inhibitor, cytokines were tested for their effects on the steady-state level of IRAP mRNA in phorbol ester-differentiated U937 cells. The cytokines tested fell into one of three groups: (a) inducers: granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, (b) weak inducers (<2-fold stimulation): [IL-lα, IL-Iγ, and transforming growth factor-β (TGF-γ)] and (c) cytokines with no effect: (IL-2, platelet-derived growth factor, acidic fibroblast grouth factor, basic fibroblast growth factor, epidermal growth factor, granulocyte colony-stimulating factor, IL-3, IL-5, IL-6, interferon-y, multi-colony stimulating factor, tumor necrosis factor -a and IRAP itself. One hundred U/ml of either GM-CSF or IL-4 was the dose inducing peak IRAP mRNA expression; that peak expression occurred 12 h after addition of cytokine. GM-CSF induced a 34 ±15-fold increase in IRAP mRNA, and IL-4 induced a 15± 6-fold increase. In the same RNA samples, GM-CSF increased IL-ip mRNA 5.9 ± 1.7-fold, but IL-4 decreased IL-Iγ mRNA to half that of control levels (0.45 ± 0.17). Thus, a single stimulus (IL-4) decreased the expression of an agonist (IL-1) while it increased the expression of an antagonist (IRAP). When U937 cells were treated with both IL-4 and GM-CSF, the level of IRAP mRNA induced was additive, suggesting that the cytokines acted differently to increase IRAP mRNA levels. The level of IL-1 mRNA in cells treated with both IL-4 and GM-CSF was intermediate. Dexamethasone and cycloheximide inhibited all mRNA increases and did not reverse IL-4-induced decreases in IL-1 mRNA. These studies have identified two cytokines which induce IRAP in the monocytic cells studied, and have partially characterized the differential regulation of IL-1 and its antagonist, IRAP.     

Reduced mitogen stimulation of DNA synthesis in human lymphocytes by a human recombinant interleukin-1 receptor antagonist.

The monokine interleukin-1 is produced by monocytes/macrophages after antigen/LPS stimulation and is an important early signal for the activation of resting T cells to become antigen specific T cells. However, little is known about the regulation and inhibition of IL-1. Recently, a new monokine has been described, generated by human macrophages, called interleukin-1 receptor antagonist (IL-1ra). This new monokine adheres to IL-1 in solution and blocks IL-1 receptor binding. IL-1ra is a glycoprotein structurally similar to IL-1 beta but having no interleukin-1-like activity. Using as a model mitogen (PHA 20 micrograms/ml)-stimulated lymphocyte DNA synthesis, we found that hrIL-1ra (30 min lymphocyte pretreatment) inhibits [3H]thymidine incorporation in a dose-dependent manner. This effect is most probably due to the inhibition of endogenous IL-1, which is a very important signal for T cell activation. The inhibition was maximum at the highest hrIL-1ra concentration used (250 ng/ml). However, when hrIL-1ra was added 2 h after PHA (20 micrograms/ml), a little, if any, inhibition of lymphocyte proliferation was found. The addition of hrIL-1ra simultaneously to the cell cultures with [3H]thymidine [( 3H]TdR) 6 h before the end of culture incubation did not significantly modify the results compared to the cells treated with PHA alone, indicating no interference of hrIL-1ra on [3H]TdR lymphocyte incorporation. We also found that the antibody anti-IL-1 beta inhibits mitogen stimulated lymphocyte DNA synthesis in dose-dependent concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effect of triptolide on interleukin-18 and its receptor in rheumatoid arthritis synovial fibroblasts

OBJECTIVE: To determine the effects of triptolide (TP) on the expression of interleukin-18 (IL-18) and its receptor in phorbol 12-myristate 13-acetate (PMA)-stimulated rheumatoid arthritis synovial fibroblasts (RASF). MATERIALS AND METHODS: RASF were obtained from the synovial tissue of patients with RA. RASF were pretreated with TP (0~100 ng/ml) for 2 h before stimulation with PMA (50 ng/ml). The bioactivity of IL-18 in the supernatant was detected based on IFN-gamma secretion from IL-18-responding human myelomonocytic KG-1 cells. IL-18 level was analyzed by ELISA. In situ expression of IL-18Ralpha was determined by immunofluorescence assay. To estimate the protein and mRNA expression of IL-18 and IL-18Ralpha in RASF, western blot and quantitative RT-PCR were performed. Nuclear factor-kappaB (NF-kappaB) activity in the whole-cell extract of treated RASF was also measured using an ELISA-based method. RESULTS: TP effectively inhibited the bioactivity of IL-18 in PMA-stimulated RASF. The expression of IL-18 and IL-18R at protein and gene levels was reduced by TP. NF-kappaB activity in PMA-stimulated RASF was profoundly suppressed by TP. These effects showed a high correlation with TP concentration (0~100 ng/ml). CONCLUSION: TP effectively inhibited the expression of IL-18 and its receptor in PMA-stimulated RASF. These results suggest a mechanism of TP in RA therapy.