Regulation of Interleukin-6 Production in Human Fetal Kupffer Cells

Inflammatory mediators such as interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) exhibit local autocrine and paracrine effects as well as distant systemic effects on target cells. Human Kupffer cells, the fixed tissue macrophages of the liver, may modulate immune and endocrine function in early fetal development. We purified and cultured human fetal Kupffer cells to investigate the production of the cytokine, IL-6. Fetal Kupffer cells treated with bacterial lipopolysaccharide (LPS) produced IL-6 in a dose-dependent fashion with maximal secretion (1000 pg per 10(6) cells) observed within 12 h using 10 micrograms of LPS/ml. Cortisol and dexamethasone, but not oestrogen, progesterone, or testosterone, dramatically suppressed the LPS-stimulated secretion of IL-6 by fetal Kupffer cells. None of the steroids tested altered basal production or enhanced the LPS-stimulated production of IL-6 by fetal Kupffer cells. The inhibition of glucocorticoids could be reversed by the addition of RU 486, indicating that this effect was mediated by the glucocorticoid receptor. These results demonstrate that the production of IL-6 by fetal hepatic macrophages can be activated by LPS and suppressed by glucocorticoids. These studies suggest that Kupffer cells express mature macrophage function in early gestation and would be capable of regulatory roles in the growth and development of the fetus.     

The Th1 and Th2 cytokines IFN-γ and IL-4 antagonize the inhibition of monocyte IL-1 receptor antagonist by glucocorticoids: involvement of IL-1

Monocytes express IL-1 and IL-1 receptor antagonist (IL-1Ra) in response to lipopolysaccharide (LPS). IL-1 self-induction contributes to the increase in IL-1 following LPS stimulation. LPS-stimulated IL-1 and IL-1Ra production are inhibited by glucocorticoids. In the present work we examined the regulation of IL-1Ra by Th1 cytokine IFN-γ, Th2 cytokine IL-4, glucocorticoids and IL-1 in human monocytes. We demonstrate that IL-1 contributes to LPS-induced IL-1Ra expression as shown by IL-1 blockade in LPS-stimulated monocytes using a specific anti-IL-1β antibody or recombinant IL-1Ra. Glucocorticoids inhibited IL-1β-stimulated IL-1Ra mRNA expression and protein production. Glucocorticoids inhibited both IL-1-mediated and non-mediated LPS stimulation of IL-1Ra expression. Both IFN-γ and IL-4 reversed the inhibitory effect of glucocorticoids on IL-1Ra expression and secretion. The effect of IFN-γ was blocked by pretreatment of monocytes with an anti-IL-1β blocking antibody, whereas the effect of IL-4 could not be blocked, demonstrating that IFN-γ acts through a mechanism dependent on endogenous IL-1 production, whereas IL-4 acts through an IL-1-independent one. Consistent with this finding, IFN-γ (but not IL-4) failed to reverse the inhibitory effect of glucocorticoids when stimulated by IL-1, and only IL-4 combined with IL-1 showed synergism resulting in an increase in IL-1Ra production. The differential regulation and involvement of IL-1 in the expression of IL-1Ra by IFN-γ, IL-4 and glucocorticoids sets the level of monocyte responsiveness during the Th1 or Th2 responses.     

3-Deazaadenosine--an inhibitor of interleukin 1 production by human peripheral blood monocytes

3-Deazaadenosine (c3Ado) has been reported to have properties of an immunosuppressive and anti-inflammatory agent. This study was designed to investigate whether c3Ado might exert anti-inflammatory activities through inhibiting Interleukin 1 (IL-1). c3Ado was found to be a potent inhibitor of IL-1 production by LPS stimulated human peripheral blood monocytes and acted at the level of synthesis rather than secretion. c3Ado also had direct effects on IL-1 biological activity in two separate assays; thymocyte proliferation and induction of prostaglandin release. Further experiments indicated that c3Ado also inhibited growth factor dependent proliferation driven by both Interleukin-2 and Interleukin-3 as well as the proliferation of a number of non-growth factor dependent cells. However, short term exposure to c3Ado resulted in no inhibition of 3H-thymidine incorporation by cells but a significant inhibition of 3H-uridine uptake into TCA precipitable material. These results suggest that c3Ado is a selective inhibitor of RNA synthesis and inhibits IL-1 production and activity by blocking new messenger RNA production induced by LPS or IL-1. General inhibition of RNA synthesis would also account for its anti-proliferative activity.     

Glucocorticoid inhibitory action on the response to PHA of residual circulatory lymphocytes in renal transplant patients following immunosuppressive therapy

The inhibitory effect of glucocorticoids on the in vitro response to phytohaemagglutinin of the residual circulating T lymphocytes in renal transplant patients during maintenance immunosuppressive therapy with glucocorticosteroids and azathioprine has been investigated. As in normal subjects, the steroid-induced inhibition of transplanted patients' lymphocyte response was inversely correlated with the mitogen concentration used. On the other hand, the response of the various lymphocyte preparations from transplanted patients appeared less inhibited by steroids than the corresponding preparations from normal subjects. The addition to the culture of the adherent cell product interleukin 1 was effective in removing to a similar extent the steroid inhibitory effect on lymphocytes from normal and transplanted subjects. Thus, the lesser inhibitory effect of glucocorticoids on transplanted patient lymphocytes could be explained by the higher percentages of monocytes present in all peripheral blood mononuclear cell preparations. These results suggest that during immunosuppressive therapy with glucocorticoids and azathioprine the residual circulating lymphocytes have a responsiveness to in vitro dexamethasone suppression similar to that of normal peripheral blood lymphocytes.     

Fluticasone and budesonide inhibit cytokine release in human lung epithelial cells and alveolar macrophages

Glucocorticoids are potent anti-inflammatory agents capable of influencing cytokine release in a number of cell types. The aim of the present study was to investigate whether glucocorticoids, frequently used in the treatment of asthma, interfere with cytokine secretion by lung epithelial cells and alveolar macrophages in vitro. Inhalation of swine dust induces airway inflammation with influx of inflammatory cells and release of proinflammatory cytokines in the lungs. Therefore, human lung epithelial cells (A549) and human alveolar macrophages were stimulated with swine dust or lipopolysaccharide (LPS), and the inhibitory effect of budesonide and fluticasone propionate on cytokine release was studied in a dose-response (10(-13)-10(-8) M) manner. The time course for the steroid effect was also investigated. Both steroids caused a dose-dependent, almost total, inhibition of swine dust-induced IL-6 and IL-8 release from epithelial cells and LPS-induced IL-6 and TNF-alpha from alveolar macrophages. The steroids only partially inhibited IL-8 release from alveolar macrophages. Budesonide was approximately 10 times less potent than fluticasone propionate. Preincubation with the steroids did not inhibit cytokine release more than simultaneous incubation with stimulus and steroid. In conclusion, budesonide and fluticasone propionate, in concentrations that probably occur in the airway lining fluid during inhalational therapy, inhibited cytokine release from human lung epithelial cells (IL-6, IL-8) and alveolar macrophages (TNF-alpha, IL-6, IL-8). In vitro, the onset of this effect was rapid.     

CD4-independent inhibition of lymphocyte proliferation mediated by HIV-1 envelope glycoproteins.

The cytopathic effects of HIV-1 produced by direct infection of human T cells do not account for the disproportionate loss of CD4-positive lymphocytes during the course of HIV infection. Previous studies have demonstrated the inhibition of uninfected human T cell activation and proliferation by the HIV-1 envelope glycoproteins, presumably due to gp120-CD4 interactions. To examine the ability of HIV-1 to inhibit T cell proliferation in the absence of both direct infection and gp120-CD4 interactions, we tested the effect of HIV-1 on mouse T cell proliferation. Culture media containing HIV-1 released from infected cells inhibited T lymphocyte proliferation in response to interleukin-2 (IL-2). Studies to explore the mechanism of this inhibition suggested that the decrease in proliferation resulted from interactions between HIV-1 and the mouse cells, but did not involve IL-2/IL-2 receptor interactions. We used monoclonal antibodies to demonstrate that the HIV-1 envelope glycoproteins were required for the inhibition of murine T cell proliferation. Anti-gp120 antibodies completely restored proliferation, indicating that the surface protein gp120 was primarily required for the inhibition of proliferation. However, antibodies directed against the transmembrane protein of HIV-1 (gp41) also partially restored lymphocyte proliferation. The functional significance of the HIV-1 envelope protein epitopes recognized by the monoclonal antibodies is discussed.     

Notch信号在脂多糖诱导的骨髓间充质干细胞中的作用

目的:探讨Notch信号对脂多糖(lipopolysaccharide,LPS)诱导的小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖及白细胞介素6(interleukin-6,IL-6)和趋化因子CXCL1分泌的影响。方法:全骨髓培养法制备BMSCs;采用q PCR与Western blot实验比较LPS对BMSCs中Notch信号通路配体、受体及靶基因表达的变化;MTT法和活细胞计数检测Notch信号对细胞增殖的影响;并以ELISA法检测Notch信号通路抑制剂DAPT对IL-6和CXCL1分泌的调节作用。结果:经10μg/L、100μg/L和1 mg/L的LPS刺激后,BMSCs增殖和IL-6分泌呈现上升趋势(P0.05或P0.01)。q PCR与Western blot结果显示Notch信号通路受体和配体在BMSCs中均有表达,但LPS对其mRNA与蛋白水平并无明显影响,然而LPS可显著诱导Notch信号靶基因Hes1与Hey1的蛋白表达。Notch信号通路抑制剂DAPT可以降低LPS诱导的BMSCs活力的上升(P0.01),同时对LPS诱导的BMSCs增殖有抑制作用。另外,LPS显著诱导BMSCs中IL-6与CXCL1的分泌,而抑制Notch信号通路可显著抑制LPS所诱导的IL-6与CXCL1的分泌(P0.05)。结论:抑制Notch信号可以抑制LPS诱导的BMSCs增殖,并抑制IL-6与CXCL1的分泌。     

Expanded adipose-derived stem cells suppress mixed lymphocyte reaction by secretion of prostaglandin E2

Multipotent mesenchymal stem cells (MSCs) in adult tissue are known to be less immunogenic and immunosuppressive. Previous study showed that primary cultures of human adipose-derived stem cells (ADSCs) shared their immunomodulatory properties with other MSCs. However, whether passaged human ADSCs can retain their immunomodulatory effect after in vitro expansion remains unknown. In addition, the mechanism of ADSC-mediated immunomodulatory effect remains to be elucidated. This study aimed to investigate these issues by using passaged human ADSCs as an in vitro study model. Flow cytometry showed that passaged ADSCs expressed human leukocyte antigen (HLA) class I but not class II molecules, which could be induced to express to a high level with interferon-gamma (IFN-gamma) treatment. The study found that passaged ADSCs could not elicit lymphocyte proliferation after co-culturing with them, even after IFN-gamma treatment. In addition, either IFN-gamma-treated or non-treated ADSCs could inhibit phytohemagglutinin (PHA)-stimulated lymphocyte proliferation. Moreover, passaged ADSCs could serve as the third-party cells to inhibited two-way mixed lymphocyte reaction (MLR). Further study using a transwell system also showed that this type of immunosuppressive effect was not cell-cell contact dependent. In defining possible soluble factors, we found that passaged ADSCs significantly increased their secretion of prostaglandin E2 (PGE2), but not transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF), when they were co-cultured with MLR. Furthermore, the result demonstrated that only PGE2 production inhibitor indomethacine, but not TGF-beta- and HGF-neutralizing antibodies, could significantly counteract ADSC-mediated suppression on allogeneic lymphocyte proliferation. These results indicated that in vitro expanded ADSCs retain low immunogenicity and immunosuppressive effect, and PGE2 might be the major soluble factor involved in the in vitro inhibition of allogeneic lymphocyte reaction.     

Comparison of the immunosuppressive activities of the antimycotic agents itraconazole, fluconazole, ketoconazole and miconazole on human T-cells.

Four antifungal agents have been screened in vitro for their immunosuppressive effects on proliferative responses in human mixed lymphocyte cultures (MLC). A hierarchy of inhibitory activity was observed, where itraconazole was greater than ketoconazole greater than miconazole greater than fluconazole, with itraconazole as suppressive as cyclosporin A, and fluconazole completely without suppressive activity. The mechanism of inhibition did not involve blockade of T-cell growth factor production and, consistent with this, interleukin-2-dependent T-cell clone proliferation was blocked by these agents in the same order of decreasing activity as in MLC. The secretion of cytokines without known T-cell growth factor activity (interferon-gamma, tumour necrosis factor-alpha) was also not significantly blocked by these agents. These results therefore demonstrate that antifungal azole drugs may be variably strongly immunosuppressive for human T-lymphocyte proliferation in vitro, but none appear to be so via a mechanism involving inhibition of cytokine secretion.     

S-adenosil-l-methionine is able to reverse the immunosuppressive effects of chenodeoxycholic acid in vitro

The study was conceived to evaluate if S-adenosil-L-methionine, a substance commonly used in the treatment of cholestasis in patients with cirrhosis and chronic hepatitis, exerts any immunological effect and if it is able to counterbalance bile acid-mediated immunosuppression. Proliferation and interleukin 2 and interferon-gamma secretion of human lymphocytes, collected from healthy subjects and exposed to mitogenic stimuli (phytohemagglutinin, pokeweed and anti-CD3 monoclonal antibodies), were analysed in the basal condition or after exposure to S-adenosil-L-methionine and/or chenodeoxycholic acid. Chenodeoxycholic acid inhibited phytohemagglutinin-induced lymphocyte proliferation and interferon-gamma secretion, and phytohemagglutinin and pokeweed-mediated interleukin 2 secretion. S-adenosil-L-methionine did not affect lymphocyte proliferation while it reduced interleukin 2 secretion upon phytohemagglutinin and pokeweed stimulation and interferon-gamma secretion upon all stimuli tested. Moreover, S-adenosil-L-methionine counteracted chenodeoxycholic acid-mediated inhibition of lymphocyte proliferation and interleukin 2 secretion. The results of our study confirm the immunosuppressive role of chenodeoxycholic acid on both secretive and proliferative lymphocyte functions and provide evidence of immunomodulatory activities of S-adenosil-L-methionine and its capacity to antagonize chenodeoxycholic acid-mediated inhibition of lymphocyte proliferation and interleukin 2 secretion.     

Taurine chloramine inhibits lymphocyte proliferation and decreases cytokine production in activated human leukocytes

Previously, we described the inhibition of proinflammatory mediators such as nitric oxide, tumor necrosis factor-alpha (TNF-alpha), and prostaglandin E2 by taurine chloramine (Tau-Cl) in activated rodent macrophages. We also demonstrated that Tau-Cl suppressed superoxide anion, IL-6, and IL-8 production in activated human polymorphonuclear leukocytes separated from peripheral blood. In these studies, we report the effect of Tau-Cl on lymphocyte proliferation and the production of cytokines by activated human peripheral blood mononuclear leukocytes. Adherent and nonadherent leukocytes were activated using lipopolysaccharide (LPS) and phytohemagglutinin (PHA), respectively, in the presence or absence of Tau-Cl. Tau-Cl significantly suppressed lymphocyte proliferation as measured by tritiated (3H) thymidine. Production of IL-6, IL-8, and IL-2 in PHA-activated nonadherent leukocytes was inhibited by Tau-Cl. The production of IL-1beta, IL-6, and IL-8 was also decreased in LPS-activated adherent monocytes by Tau-Cl. These data demonstrate that the ability of Tau-Cl to modulate the immune response is not species specific and extends to human leukocytes.     

Trypanosoma cruzi induces regulatory dendritic cells in vitro

A main feature of acute infection with Trypanosoma cruzi is the presence of immunological disorders. A previous study demonstrated that acute infection with the virulent RA strain downregulates the expression of major histocompatibility complex class II (MHC-II) on antigen-presenting cells and impairs the T-cell stimulatory capacity of splenic dendritic cells (DC). In the present work, we assessed the ability of trypomastigotes (Tp) to modulate the differentiation stage and functionality of bone marrow-derived DC in vitro. We observed that the Tp stage of T. cruzi failed to activate DC, which preserved their low expression of MHC-II and costimulatory molecules, as well as their endocytic activity. We also show that Tp induced transforming growth factor beta (TGF-beta) secretion by DC and enhanced the gap between interleukin-10 (IL-10) and IL-12p70 production, showing a higher IL-10/IL-12p70 ratio upon lipopolysaccharide (LPS) treatment. In addition, we observed that Tp prevented DC full activation induced by LPS, thereby downregulating their MHC-II surface expression and inhibiting their capacity to stimulate lymphocyte proliferation. In vitro IL-10 neutralization during the differentiation process of DC with Tp+LPS showed a reversion of their inhibitory effect during mixed lymphocyte reaction. In contrast, only simultaneous neutralization of IL-10 and TGF-beta, after DC differentiation, was involved in the partial restitution of lymphocyte proliferation. Since both TGF-beta and IL-10 are immunosuppressive cytokines essential in the modulation of the immune response and important in the induction of tolerance, our results suggest for the first time that Tp are responsible for the generation of regulatory DC in vitro.     

The Combined Effects of Zafirlukast,Prednisone, and Inhaled Budesonide on IL-13 and IFN-γ Secretion

Therapy for asthma often includes the combined use of glucocorticoids and leukotriene receptor antagonists. The short-term, combined effects of these drugs on cytokine secretion and lymphocyte proliferation are ill-defined. The aim of this study was to analyze allergen and mitogen-induced cytokine secretion and lymphocyte proliferation in asthmatics and to determine the effect of combined therapy on these immune responses. Peripheral blood mononuclear cells were isolated from mild, persistent adult asthmatics (n = 28) and analyzed for cat allergen (Fel d 1) and mitogen (phytohemagglutinin) induced IL-13 and IFN-γ secretion and lymphocyte proliferation. Samples were analyzed before and after 10 days of therapy with oral zafirlukast, prednisone (0.5 mg/kg/day), and inhaled budesonide (1600 mcg/day). Both Fel d 1 and mitogen stimulation resulted in IL-13 and IFN-γ secretion. Combination drug therapy resulted in a significant decrease in allergen-induced IFN-γ secretion (p = 0.018) and allergen-specific lymphocyte proliferation (p = 0.02), while IL-13 secretion was unchanged (p = 0.109). This study indicates a role for Th1 cytokines as well as Th2 cytokines in the allergic response.     

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)     

LPS刺激诱导人卵巢癌细胞株Toll样受体4表达及细胞免疫调节

目的 探讨脂多糖(LPS)刺激对体外培养的人卵巢癌细胞株SKOV3的生长、Toll样受体4(TLR4)的表达、细胞活性氧(ROS)表达及6种炎性细胞因子分泌水平变化的影响.方法 用流式细胞仪测定不同浓度LPS刺激SKOV3 4 h后TLR4的表达水平;用LPS分别刺激SKOV3细胞不同时间后,MTT法检测细胞增殖情况,流式细胞仪分析TLR4表达、细胞周期分布、ROS表达水平以及细胞因子分泌水平.结果 TLR4表达与LPS作用浓度之间存在浓度依赖性和最大量效曲线关系;LPS刺激组与正常组的细胞增殖、细胞周期PrI值(细胞增殖指数,S+G_2/M)、ROS表达水平、细胞因子分泌水平均有显著性差异.结论 LPS具有诱导卵巢癌细胞TLR4表达、活性氧表达、炎症因子分泌以及细胞增殖和抑制的作用.     

Effect of schistosome-derived inhibitory factor on the cell cycle of T lymphocytes

A schistosome-derived inhibitory factor (SDIF) with immunosuppressive properties has been investigated for its effect on human T cell proliferation. We show here that SDIF has no effect on the process of lymphocyte activation because peripheral blood leukocytes (PBL) stimulated with lectin in the presence of SDIF increased normally their RNA content and showed normal acquisition of interleukin 2 (IL-2) and transferrin receptors. IL-2 production was not altered by SDIF but utilization of IL-2 was decreased, suggesting that SDIF blocked cells before or in the early s phase. Jurkat T cell line cells physically enriched for G1 cells were also more susceptible to SDIF inhibition. On the contrary, normal PBL or Jurkat cells which were already in the s phase were no more inhibited by SDIF. While SDIF has no effect on T lymphocyte activation and on production of regulatory lymphokines it selectively blocks T cell proliferation at G1 transition of the cell cycle.     

Immunological studies in uremic and kidney transplanted patients

Some aspects of the cellular in vitro immune response were studied in uremic and kidney transplanted patients. In particular, the immunosuppressive effects of glucocorticoids were examined in uremic patients and in cadaver kidney graft recipients with special reference to clinical kidney transplantation. 1) In vitro, lymphocytes from patients on hemodialysis had impaired responses to stimulation with mitogen. Variations in the culture conditions including changes in; (i) mitogen concentrations, (ii) culture periods, (iii) aerobic growth conditions, (iiii) and cellular synthesis of prostaglandins did not normalize the uremic lymphocyte response. 2) A possible effect of hemodialysis per se could not be excluded. However, in short term experiments accumulation in uremic plasma of inhibitory factors and/or deprivation of supportive factors could only partly explain the decreased cell responses. 3) In vitro, glucocorticoids inhibit the proliferation of mitogen stimulated normal lymphocytes in a dose-dependent way. Moreover, the in vitro immunosuppressive effects of some glucocorticoids did not correlate with their anti-inflammatory potencies. In uremic cell cultures the in vitro lymphocyte sensitivity to glucocorticoids was 5-8 fold increased in comparison to the control cultures. 4) Lymphocytes from patients on peritoneal dialysis (CAPD) and non-dialyzed uremic patients had normal transformation responses but were also very sensitive in vitro to glucocorticoids. 5) Cytotoxic effector cell functions (NK and K) remained normal in hemodialyzed patients which is in contrast to the decreased transformation responses presupposing interleukin-2 (IL-2) dependent cell proliferation (DNA synthesis). Furthermore, both control and uremic NK and K cell functions were resistant in vitro to the suppressive effects of glucocorticoids. 6) The decreased mitogen response of patients on hemodialysis was improved by addition of IL-2 to the cell cultures. Moreover, IL-2 normalized the increased uremic sensitivity in vitro to glucocortiooids. In accordance, the production of IL-2 was decreased in mitogen stimulated cell cultures from patients on hemodialysis. There was no evidence that the impaired transformation responses of lymphocytes from patients on hemodialysis was due to a lack of cells positive for IL-2 receptors. These results suggested that a deficient production of IL-2 may be part of; (i) the decreased transformation response (ii) and the increased sensitivity in vitro to glucocorticoids of lymphocytes from hemodialyzed patients.(ABSTRACT TRUNCATED AT 400 WORDS)