Cloning and characterization of recombinant rhesus macaque IL-10/Fc(ala-ala) fusion protein: a potential adjunct for tolerance induction strategies

The powerful anti-inflammatory and immunosuppressive activities of IL-10 make it attractive for supplemental therapy in translational tolerance induction protocols. This is bolstered by reports of IL-10-mediated inhibition of innate immunity, association of human stem cell and nonhuman primate (NHP) islet allograft tolerance with elevated serum IL-10, and evidence that systemic IL-10 therapy enhanced pig islets survival in mice. IL-10 has not been examined as adjunctive immunosuppression in NHP. To enable such studies, we cloned and expressed rhesus macaque (RM) IL-10 fused to a mutated hinge region of human IgG1 Fc to generate IL-10/Fc^ala-ala. RM IL-10/Fc^ala-ala was purified to 98% homogeneity by affinity chromatography and shown to be endotoxin-free (<0.008 EU/μg protein). The biological activity of IL-10/Fc^ala-ala was demonstrated by (1) costimulation of the mouse mast cell line, MC/9 proliferation in a dose-dependent fashion, (2) suppression of LPS-induced septic shock in mice and (3) abrogation of LPS-induced secretion of proinflammatory cytokines/chemokines in vitro and in vivo in NHP. Notably, RM IL-10/Fc^ala-ala had significantly greater potency than human IL-10/Fc^ala-ala and exhibited a circulating half-life of 14 days. The availability of this reagent will facilitate definitive studies to determine whether supplemental therapy with RM IL-10/Fc^ala-ala can influence tolerance outcomes in NHP.     

An unusual pro-inflammatory role of interleukin-10 induced by arabinosylated lipoarabinomannan in murine peritoneal macrophages

Various species of Mycobacteria produce a major cell wall-associated lipoglycan, called Lipoarabinomannan (LAM), which is involved in the virulence of Mycobacterial species. In this study, we tried to establish the role of the increased IL-10 secretion under Arabinosylated-LAM (Ara-LAM) treatment, the LAM that induces apoptosis in host macrophages or PBMC. We have studied the survival and apoptotic factors by western blotting, and estimated nitrite generation by Griess reaction, quantified iNOS mRNA by semi-quantitative RT-PCR, and ultimately the fate of the cells were studied by Flow Cytometric Analysis of AnnexinV-FITC binding. As per our observations, neutralization of released IL-10 in C57BL/6 peritoneal macrophages prior to Ara-LAM treatment, as well as macrophages from IL-10 knockout (KO) mice treated with Ara-LAM, showed significant down regulation of pro-apoptotic factors and up regulation of survival factors. These effects were strikingly similar to those when peritoneal macrophages were subjected to TNF-α and IL-12 neutralization followed by Ara-LAM-treatment. However, under similar conditions virulent Mannosylated-LAM (from Mycobacterium tuberculosis) treatment of macrophages clearly depicts the importance of IL-10 in the maintenance of pathogenesis, proving its usual immunosuppressive role. Thus, from our detailed investigations we point out an unusual pro-inflammatory action of IL-10 in Ara-LAM treated macrophages, where it behaves in a similar manner as the known Th1 cytokines TNF- α and IL-12. This work is financed by the Council of Scientific and Industrial Research (CSIR), Govt. of India.     

Immune activation in advanced cancer patients treated with recombinant IL-21: multianalyte profiling of serum proteins

Purpose  Recombinant interleukin-21 (rIL-21) is an immune stimulating cytokine recently tested in two Phase 1 trials for immune responsive cancers. A secondary objective of these trials was to characterize pharmacodynamic responses to rIL-21 in patients. Here, we report the effects of systemic rIL-21 on serum markers of immune stimulation. Experimental design  Recombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 μg/kg using two distinct treatment regimens: thrice weekly (‘3/w’) for 6 weeks; or once daily for five consecutive days followed by nine dose-free days (‘5 + 9’). In the absence of dose limiting toxicity, additional cycles of dosing were initiated immediately following the nine dose-free days. An array of 70 different proteins was profiled in subject serum samples from several time points during the course of the study. Hierarchical clustering analysis was performed on a normalized subset of these data. Results  Systemic administration of rIL-21 affected the serum levels of several cytokines, chemokines, acute-phase proteins and cell adhesion proteins. The magnitude and duration of response were dose dependent for a subset of these biomarkers. The 5 + 9 dosing regimen generally produced cyclic changes that were of greater magnitude, as compared to a more chronic stimulation with the 3/w dosing regimen. Despite these differences, rIL-21 effects on many analytes were similar between regimens when averaged over the time of treatment. Based on similar temporal, between-subject and dose response changes, groups of analytes were identified that exhibited distinct components of the rIL-21-mediated immune activation. Biomarkers indicative of lymphocyte activation (increased IL-16, decreased RANTES), acute phase response (increased CRP, ferritin), myeloid activation (increased MDC, MIP-1 alpha), and leukocyte chemotaxis/trafficking (increased sCAMs, MCP-1) were strongly modulated in subjects treated with rIL-21. Conclusions  Administration of rIL-21 resulted in activation of multiple cell types and immune response pathways. The changes observed in serum proteins were consistent with coincident processes of lymphoid and myeloid cell activation and trafficking, and acute phase response. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

IL-12-and IL-23 in health and disease

Interleukin (IL)-12 and IL-23 play important roles in the development of experimental autoimmune disease models and numerous afflictions affecting humans. Preclinical data over the last 20 years combined with successful clinical trials has identified a clear relationship between IL-12, IL-23 and the generation of pathogenic T helper cells capable of orchestrating tissue inflammation. Observations made in the clinic have shown that IL-12p40, a common subunit shared by IL-12 and IL-23, is critical to pathologies associated with psoriasis, inflammatory bowel disease (IBD) and tumor growth. These advancements have set in motion the development of a number of potential therapeutics aimed at manipulating IL-12/23 signaling pathways in both mice and humans. This review will discuss a brief history of the understanding and expansion of the IL-12 cytokine family, some difficulties associated with preclinical data interpretation and finally the medicinal interventions that have been developed to combat IL-12/23-driven autoimmune disorders.     

胆囊切除术后患者IL-6、IL-10 及TNF-alpha水平的变化及意义

目的：探讨胆囊摘除术后患者IL-6,IL-10 以及TNF-alpha水平变化。方法：选取我院收治的行胆囊切除术患者120 例,根据手术 方式的不同随机分为实验组60 例,为腹腔镜胆囊切除术患者,对照组60 例,为常规开腹切除术患者。观察并比较术前、术后第1 天、第2 天、第3 天患者的IL-6,IL-10、TNF-alpha水平以及术后患者的疲劳情况。结果：①手术后,两组患者较手术前IL-6 均有所升 高,且对照组较实验组升高明显,差异有统计学意义(P＜0.05);两组患者较手术前IL-10 均有所降低,且对照组较实验组降低明 显,差异有统计学意义(P＜0.05)。②手术后,两组与手术前相比较TNF-alpha水平明显升高,与实验组相比较,对照组升高明显,差异 有统计学意义(P＜0.05)。③手术后,两组的疲劳情况相比较,实验组明显低于对照组,差异有统计学意义(P＜0.05)。结论：胆囊切除 术后患者IL-6,IL-10,以及TNF-alpha均有所升高,说明应激反应正在发生,进而出现术后疲劳。     

CD38 ligation plays a direct role in the induction of IL-1beta,IL-6, and IL-10 secretion in resting human monocytes

CD38 signaling, either induced by ligation with specific agonistic monoclonal antibody (mAb) or after interaction with CD31, its cognate counter-receptor, is involved in release of IL-1beta, IL-6, and IL-10 cytokines in resting human monocytes. CD38 ligation by the F(ab')(2) IB4 mAb did not induce signals relevant for cytokine secretion and the block of the Fcgamma receptor I (FcgammaRI) by anti-CD64 or FcgammaRII by anti-CD32 mAb did not inhibit CD38-mediated IL-1beta release. Dimerization or multimerization of the CD38 molecule by: (i) cross-linking of the receptor ligated by F(ab')(2) or by (ii) increasing CD38 expression by treating monocytes with IFNgamma were able to restore the truncated CD38-mediated signals involved in cytokine secretion. These data indicate that CD38 receptor-mediated signals operate directly suggesting a Fcgamma receptorial surface molecule independent activation pathway. The key element for the receptor mediated signaling is represented by surface density of CD38 on resting monocytes.     

PGE2对肝病大鼠创伤弧菌攻击后血清TNF-α、IL-10水平的影响及其肾组织的保护作用

目的探讨前列腺素E2（prostaglandin E2,PGE2）免疫干预对肝病大鼠创伤弧菌（Vibrio vulnifcus,Vv）攻击后TNF-α、IL-10的影响以及与肾组织超微结构改变的相关性。方法正常大鼠9只和肝病大鼠36只,分别为正常大鼠Vv攻击组、肝病大鼠Vv攻击组、肝病Vv攻击后氧氟沙星药物治疗组、肝病Vv攻击PGE2氧氟沙星联合保护组和肝病大鼠NS组（每组n=9）。ELISA法测定血清TNF-α和IL-10含量。取各组大鼠肾标本电镜下观察超微结构病理改变。结果肝病大鼠较正常大鼠Vv攻击后IL-10低而TNF-α高（P〈0.05）;PGE2免疫干预组肾组织超微结构明显改善。结论PGE2免疫干预能上调肝病大鼠血清IL-10,抑制TNF-α的分泌,对肝病大鼠Vv攻击后肾组织具有免疫保护作用。     

氧化苦参碱对哮喘大鼠肺组织IL-10 表达的影响

目的:观察不同浓度氧化苦参碱(Oxymatrinem,Oxy)对哮喘大鼠肺组织IL~(-1)0表达的影响,并探讨其作用机制。方法:构建哮喘大鼠模型,将40只清洁级健康雌性SD大鼠随机分成5组,每组8只:A:哮喘组(仅卵蛋白(Ovalbumin,OVA)致敏)、B:低浓度组(Oxy 50 mg/kg)、C:中浓度组(Oxy 100 mg/kg)、D:高浓度组(Oxy 150 mg/kg)、E:对照组(生理盐水),末次激发24 h后处死全部大鼠,取大鼠肺脏,HE染色观察肺组织病理改变,采用RT-PCR、Western Blot测定各组肺组织中IL~(-1)0基因及蛋白水平的表达。结果:HE结果显示,哮喘组可见大量炎症细胞浸润,气管平滑肌明显增厚。对照组肺泡壁薄且光滑,未见明显炎性细胞的浸润,不同浓度氧化苦参碱药物干预组其肺组织炎症细胞浸润及气管平滑肌病变程度随着用药浓度的增高呈逐渐减轻趋势。RT-PCR以及Western blot检测IL~(-1)0发现,哮喘组、氧化苦参碱低浓度组、氧化苦参碱中浓度组与对照组相比IL~(-1)0的表达均有所减低(P0.05),而氧化苦参碱高浓度组与对照组比较,IL~(-1)0的表达无统计学意义(P0.05);氧化苦参碱中浓度组、氧化苦参碱高浓度组与哮喘组相比IL~(-1)0的表达均有所增高(P0.05),氧化苦参碱低浓度组与哮喘组相比IL~(-1)0的表达无统计学意义(P0.05)。结论:氧化苦参碱抑制、控制哮喘发作可能与促进肺组织中IL~(-1)0基因、蛋白的表达相关,且促进程度在一定范围内与浓度呈正比。     

Dithiocarbamates and viral IL-10 collaborate in the immortalization and evasion of immune response in EBV-infected human B lymphocytes

Epstein-Barr virus (EBV) is implicated in the development of a number of human malignancies including several subtypes of non-Hodgkin lymphoma (NHL) [G. Pallesen, S.J. Hamilton-Dutoit, X. Zhou, The association of Epstein-Barr virus (EBV) with T cell lymphoproliferations and Hodgkin's disease: two new developments in the EBV Field, Adv. Cancer Res. 62 (1993) 179-239]. Lymphoproliferative disease and NHL occurring in severely immunosuppressed individuals almost always involve EBV and have been extensively studied and modeled in vitro. EBV has also been causally associated with some cases of NHL occurring in otherwise immunocompetent individuals. However, a direct role for EBV in the pathogenesis of neoplasms developing in the presence of an otherwise competent immune system has not been established. We investigated potential interactions between dithiocarbamates (DTC), an important class of thiono-sulfur compounds, and EBV leading to immortalization of human B lymphocytes and evasion of cell-mediated immune response in culture. Primary lymphocyte cultures employing wild-type and recombinant EBV mutants were used to assess the respective roles of DTC and viral genes in lymphocyte transformation and survival. Pretreatment of EBV-infected human B lymphocytes with DTC directly enhanced transformation in the absence of T cells (5 nM) and independently increased survival of transformed cells in the presence of competent autologous T cells (10 nM). Both DTC-induced transformation and immortalization of EBV-infected B lymphocytes were dependent on the expression of viral IL-10. These results provide a biological basis for studying collaborations between chemical and virus that alter lymphocyte biology, and provide a rationale for further molecular epidemiology studies to better understand the potential influence of these interactions on the development of NHL and perhaps other viral-associated malignancies.     

IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells

Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL-10 had no effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 or IL-15.     

Inhibition of interleukin-10 (IL-10) production from MOPC 315 tumor cells by IL-10 antisense oligodeoxynucleotides enhances cell-mediated immune responses

 Interleukin-10 (IL-10) has both inhibitory and stimulatory effects on diverse cell types of the immune system. It inhibits the antigen-presenting capacity of monocytes/macrophages and stimulates T cell proliferation. Although many tumors spontaneously release IL-10, the physiological relevance of this phenomenon to the in vivo antitumor immune response is not known. To elucidate the physiological role of tumor-released IL-10, we used IL-10-specific antisense oligodeoxynucleotides (AS-ODN) for the inhibition of IL-10 production from the tumor cells. Incubation of MOPC 315 plasmacytoma with IL-10 AS-ODN in vitro resulted in inhibition of IL-10 production and also in enhancement of the expression of major histocompatibility complex (MHC) class I, MHC class II, and B7-1 molecules. MOPC 315 cells incubated with IL-10 AS-ODN (MOPC-IL10AS) for 16 h in vitro showed reduced tumorigenicity in Balb/c mice. The mice implanted with MOPC-IL10AS effectively rejected the tumor graft, and showed strong cytotoxic T lymphocyte (CTL) activity against the parental MOPC 315 cells. In addition, MOPC-IL10AS were more effective as stimulator cells in mixed lymphocyte/tumor cell culture, and as target cells in a CTL assay. These results imply that IL-10 spontaneously released from MOPC 315 cells inhibits their immunogenicity and that the inhibition of IL-10 production by IL-10 AS-ODN may be a way to enhance the host cellular antitumor immune response. Received: 11 November 1999 / Accepted: 6 April 2000     

各个肠段IL-10 水平与气候变化的关系

目的：季节气候的变化能够影响人体肠道的生理功能和病理变化,这在中医学的长期临床实践和现代流行病学调查中都有 记载和证实,但是相关的机制并不清晰。因此本研究通过观察不同气候下大鼠空肠、回肠、结肠的IL-10 水平变化,从而从免疫功 能的角度探讨季节气候变化对肠道功能的影响机制。方法：根据北京地区气象资料,大致可将一年之中气候划分为五种季节气 候,分别由春、夏、长夏、秋和冬来代表;实验分为正常组和松果腺摘除模型组;分别把两组大鼠饲养五种正常自然季节气候下一 个月,然后用ELISA 方法检测大鼠空肠、回肠和结肠的IL-10 水平。结果：①正常组空肠IL-10水平在长夏季节明显升高（P<0. 001）;② 回肠的IL-10 表达水平则是在长夏与冬季这两个季节含量最多（P<0.01, P<0.001）;③ 结肠IL-10 含量则在冬季呈现出高 峰（P<0.01）。结论：季节气候变化对肠道功能的影响的机制与肠道免疫功能的季节性变化有关。具体表现在：① 肠道IL-10 水平 是有季节气候变化规律的,而且空肠、回肠和结肠表现出来的季节气候规律是不相同的。②松果腺参与了肠道免疫功能气候调 节过程。     

Inhibition of IL-6, TNF-alpha, and cyclooxygenase-2 protein expression by prostaglandin E2-induced IL-10 in bone marrow-derived dendritic cells

Several endogenously produced mediators, including cytokines such as IL-6, IL-10, and TNF-alpha and prostanoids such as prostaglandin E(2) (PGE(2)), regulate dendritic cell (DC) function and contribute to immune homeostasis. In this study, we report that exogenous PGE(2) enhances the production of IL-10 from bone marrow-derived DC (BM-DC). IL-6, but not TNF-alpha, release is enhanced by PGE(2) in the presence of anti-IL-10, suggesting that endogenous IL-10 masks PGE(2)-induced IL-6. Furthermore, both exogenous IL-10 and PGE(2) inhibit LPS-induced IL-6 and TNF-alpha, whereas selective inhibition of cyclooxygenase-2 (COX-2) or addition of anti-IL-10 causes the reverse effects. Exogenous IL-10, but not IL-6, dose-dependently suppresses COX-2 protein expression and PGE(2) production, and TNF-alpha does not reverse this effect. In contrast, anti-IL-10 up-regulates prostanoid production by LPS-stimulated BM-DC. Taken together, our results show that in response to PGE(2), BM-DC produce IL-10, which in turn down-regulates their own production of IL-6-, TNF-alpha-, and COX-2-derived prostanoids, and plays crucial roles in determining the BM-DC pro-inflammatory phenotype.     

The roles of IL-10 and TGF-beta in controlling IL-4 and IFN-gamma production during experimental Fasciola hepatica infection

Hosts infected with Fasciola hepatica experience immunosuppression during the acute and chronic phases of the disease. This immunosuppression may allow parasite survival in the face of an ongoing immune response. In bovine hosts early IL-4 and continued IgG1 production is one of the few remaining features of the characteristic type 0/2 helper (Th0/2) response present in the chronic stage of disease. Here we demonstrate elevated levels of parasite-specific, in vitro peripheral blood mononuclear cell (PBMC)-derived transforming growth factor (TGF)-β1 from the early phases of infection and increasing levels of IL-10 as the infection becomes chronic. In vitro neutralisation of these cytokines during culture of PBMCs from experimentally-infected cattle increased IL-4 and IFN-γ production in response to parasite-specific and non-specific stimulation. At 4 weeks p.i. neutralisation of TGF-β results in an increase in parasite driven IL-4, while also having a greater role, compared with IL-10, in influencing specific and non-specific IFN-γ. At 12 weeks p.i. ex vivo parasite driven IL-4 was not restored by inhibiting either IL-10 or TGF-β. However IL-10 influenced both parasite-specific and non-specific IFN-γ production at this time. This highlights the roles of IL-10 and TGF-β in fasciolosis, however the cellular sources of these have yet to be defined. This suggests that suppression of IFN-γ production by parasite molecules occurs during infection and it is possible that the suppression of IFN-γ production may mediate parasite survival in this disease.     

中国白兔白介素-10基因的克隆、表达及其抗体的制备

目的克隆并表达中国白兔IL-10基因,制备抗IL-10多克隆抗体。方法运用RT-PCR对从ConA诱导后的中国白兔外周血单核细胞(PMBCr)总RNA中扩增IL-10基因,克隆后进行测序和遗传进化分析,同时将其亚克隆pET28a中,在大肠杆菌中诱导表达,并用纯化的表达产物制备多克隆抗体。结果该基因全长537 bp,编码178个氨基酸。与欧洲兔IL-10基因同源性很高,但与鼠、鸡、河豚和斑马鱼等不同物种IL-10基因差异较大。经IPTG诱导后,重组菌体裂解物经SDS-PAGE电泳可检测到相对分子质量为23.4×103的重组蛋白。Western blot分析表明,中国白兔IL-10基因已经表达。重组表达的蛋白量可占菌体蛋白的15.2%。结论成功实现了中国白兔IL-10的原核表达,制备了小鼠抗兔IL-10的多克隆抗体。     

The IL-12p70/IL-10 interplay is differentially regulated by free heme and hemozoin in murine bone-marrow-derived macrophages

The outcome of malarial anemia is determined by a complex interplay between pro-inflammatory and anti-inflammatory cytokines, its severity associated with accumulation of hemozoin (Hz) in macrophages, elevated IL-10 responses and impaired IL-12 production. Although free heme contributes to malarial anemia by inducing oxidative damage of red blood cells (RBCs) and enhancing their clearance by phagocytes, its impact on IL-12/IL-10 interactions has not been fully characterized. Herein, the effect of hemin (HE) on IL-12 and IL-10 responses was studied in murine bone marrow-derived macrophages (BMDM) and compared with synthetic Hz. Our data reveal that HE induces modest inhibition of IL-12p70 responses to lipopolysaccharide (LPS) whereas Hz significantly impairs IL-12p70 responses to IFNγ/LPS through down-regulation of IL-12p35 and p40 gene expression. Although reactive oxygen species (ROS) are generated after short-term exposure to HE and Hz, prolonged exposure to these iron protoporphyrins has opposite effects on the cellular redox status, HE being the only compound able to promote persistent ROS production. Accordingly, the inhibitory effect of HE on IL-12p70 seems sustained by redox-dependent induction of IL-10 and is partially controlled by the p38 mitogen-activated protein kinase (MAPK) signalling pathway. Indeed, treatment with n-acetylcysteine (NAC) or with the p38 MAPK inhibitor SB203580 inhibits IL-10 responses and significantly restores IL-12p70 responses to IFNγ/LPS in HE-conditioned BMDM. Our results suggest that oxidant stress induced by free heme may potentially contribute to sustained production of IL-10 and down-regulation of IL-12 responses in malaria.