过氧化氢刺激RAW264.7巨噬细胞促炎细胞因子和趋化因子基因表达

免疫反应细胞经呼吸瀑布作用产生的活性氧是巨噬细胞促炎细胞因子和趋化因子表达的信号分子,但目前缺乏过氧化氢(H2O2)刺激巨噬细胞表达促炎细胞因子和趋化因子的直接证据．本研究以离体培养的小鼠RAW264.7巨噬细胞为研究体系,探讨外源H2O2对RAW264.7巨噬细胞促炎因子和趋化因子基因表达和生成的影响．MTT法结合实时荧光定量PCR(qRT-PCR)、酶联免疫吸附试验(ELISA)结果显示,RAW264.7细胞在H2O2浓度低于40 μmol/L时不影响RAW264.7细胞的增殖活力．20 μmol/L和40 μmol/L H2O2显著增强RAW264.7细胞TNF-α、IL-1β、MCP-1和MIP-2基因转录和蛋白质生成,并存在剂量依赖效应;而200 U/mL过氧化氢酶预处理则可减弱由H2O2刺激的TNF-α、IL-1β、MCP-1和MIP-2基因表达和蛋白生成．这些结果提示,H2O2是刺激巨噬细胞促炎因子和趋化因子表达或生成的重要因子,对机体炎症反应的发生具有重要作用．     

上皮源性细胞因子IL-33、IL-25和TSLP在哮喘发病机制中的作用

哮喘(asthma)是一种以气道高反应性、慢性气道炎症、气道重塑和可逆性的气流受阻为特征的常见慢性呼吸系统疾病。近年来,研究发现气道上皮细胞在霉菌、尘螨、花粉、病毒感染、空气污染物等各种损伤因素的作用下,可释放细胞因子白细胞介素-33(interleukin-33,IL-33)、白细胞介素-25(interleukin-25,IL-25)和胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin,TSLP),这些细胞因子不仅可作用于2型辅助性T细胞(type 2 helper T cells,Th2 cells),同时也可作用于固有淋巴样2型细胞(group 2 innate lymphoid cells,ILC2s),通过释放Th2型细胞因子,参与哮喘的发生与发展。尽管这3种细胞因子在哮喘的发生与发展中均起到重要作用,但其在哮喘病理、生理学效应及作用方式上并非完全相同。现就这3种上皮源性细胞因子IL-33、IL-25和TSLP在哮喘发病机制中的作用作一概述。     

人IL-37b转基因对小鼠哮喘模型作用的研究

目的通过研究人抑制性细胞因子白细胞介素37b(interleukin 37b, IL-37b)转基因(transgenic, TG)小鼠(Il37b TG)对卵清蛋白(ovalbumin, OVA)诱导的哮喘气道高反应性、气道炎症和气道重塑等指标的变化,以及IL-37b在体外对小鼠和人肺结构细胞的作用,评估IL-37b在治疗哮喘方面的潜在意义。方法采用野生型和Il37b TG的雌性C57BL/6小鼠,建立周期为24 d的OVA诱导的哮喘小鼠模型,检测其肺力学参数、炎性细胞分类和Th2细胞因子表达以及肺组织病理学改变。体外采用原核细胞表达系统,获得大量纯化的重组人IL-37b。继而分别预处理小鼠肺成纤维细胞系(MLg)和人肺泡上皮细胞系(A549细胞),随后加以脂多糖(lipopolysaccharide, LPS)刺激,收集培养上清,采用双抗体夹心ELISA检测炎性细胞因子IL-6含量。结果与野生型OVA模型组相比,Il37b TG OVA组的气道高反应性降低,差异有统计学意义(P=0.000 1,P0.05);支气管肺泡灌洗液和肺组织中嗜酸性粒细胞数量降低,差异有统计学意义(分别为P=0.000 1和P0.000 1,P0.05);肺组织炎症细胞浸润明显减少,气道炎症、杯状细胞增生和黏液分泌均明显减轻,苏木精-伊红染色和过碘酸-希夫染色评分也有所降低,差异有统计学意义(分别为P=0.000 1和P=0.000 2,P0.05),马松染色显示急性期胶原沉积的改变不明显;肺组织匀浆中Th2型细胞因子IL-5和IL-13的含量均有所降低。同时研究采用原核细胞表达系统得到的IL-37b预处理鼠肺成纤维细胞MLg和人肺上皮细胞A549,可以降低LPS所诱导IL-6的产生。结论 IL-37b可能通过对相关靶细胞的负向调控进而发挥对哮喘气道的高反应性及气道炎症的抑制作用,为IL-37b作为一种新的治疗方法在临床治疗哮喘的潜在应用提供了一定的实验依据。     

内源性IL-17诱导MIP-2与IL-6表达在衣原体肺炎中促进中性粒细胞循环

目的 探讨衣原体肺炎中白细胞介素-17（interleukin -17,IL-17）对中性粒细胞（polymorphonuclear leucocyte,PMN）循环的调节作用及机制.方法 用40 μl含1×10^3包涵体形成单位（inclusion-forming units,IFU）的衣原体鼠肺炎株（Chlamydia muridarum,Cm）呼吸道感染BALB/c小鼠,诱导鼠衣原体肺炎.用抗鼠IL-17单克隆抗体吸入中和内源性IL-17,以相应独特型抗体（IgG2α）作为对照.用RT-PCR检测小鼠肺组织及肺上皮细胞系巨噬细胞炎性蛋白-2 （macrophage inflammatory protein-2,MIP-2）和IL-6 mRNA的表达.取小鼠支气管肺泡灌洗液细胞染色计数PMN,感染肺组织进行病理染色.结果 衣原体肺炎中,内源性IL-17中和小鼠肺组织PMN浸润显著降低,支气管肺泡灌洗液PMN数量显著低于对照组.IL-17与TNF-α协同可上调肺上皮细胞MIP-2和IL-6 mRNA表达,且内源性IL-17中和小鼠肺组织MIP-2和IL-6表达显著降低.结论 衣原体肺炎中IL-17通过促进肺组织细胞分泌趋化性细胞因子MIP-2和前症性细胞因子IL-6,诱导PMN循环,参与宿主抗衣原体炎性应答.     

EV-A71感染小鼠巨噬细胞诱导促炎细胞因子和趋化因子反应

为初步探索EV-A71在小鼠巨噬细胞中的复制情况和抗病毒的固有免疫应答,本文以小鼠巨噬细胞系RAW264.7为细胞模型,通过建立EV-A71绝对定量qPCR方法检测EV-A71病毒载量;EV-A71和紫外灭活的EV-A71感染RAW264.7,不同时间点提取总RNA,RT-qPCR检测促炎细胞因子、趋化因子和模式识别受体的mRNA表达变化水平。本研究成功建立了EV-A71的绝对定量qPCR检测方法,并发现EV-A71感染RAW264.7后随着感染时间的延长,EV-A71病毒载量呈递减趋势;EV-A71和紫外灭活的EV-A71可以引起IL-1β、IL-6、TNF-α促炎细胞因子和IP-10、MCP-1、MIP-1α趋化因子反应,上调TLR2、TLR1、TLR6、MDA5和RIG-I mRNA表达。研究结果显示,EV-A71在小鼠巨噬细胞中具有较低水平的复制,同时产生促炎细胞因子和趋化因子反应。     

Ghrelin抑制H2O2诱导的肺泡上皮A549细胞中IL-8的产生

目的:通过细胞培养的方法,初步研究了生长激素释放肽Ghrelin在氧化应激相关的肺泡上皮细胞炎症反应中的作用.方法:首先是用双氧水(H2O2)刺激A549细胞建立肺泡上皮细胞炎症反应模型,分别加入不同浓度的Ghrelin及普通培养基培养A549细胞,用酶联免疫吸附技术(ELISA)从蛋白水平检测上清液中IL-8的含量,采用逆转录聚合酶链反应技术(RT-PCR)检测炎性细胞因子IL-8mRNA的表达.结果:H2O2可以使A549细胞IL-8蛋白的释放及IL-8mRNA的表达明显升高(P＜0.05),而用Ghrelin干预后IL-8的释放及IL-8mRNA的表达被抑制,明显低于单纯H2O2刺激的模型组(P＜0.05),且随着浓度的增加Ghrelin的这种抑制作用逐渐增强.结论:分别从蛋白水平和基因水平证明了Ghrelin能够抑制H2O2诱导的肺泡上皮A549细胞中IL-8的产生,由此推测Ghrelin可能能够抑制以COPD、支气管哮喘为代表的氧化应激相关的肺部炎症反应.     

二氢青蒿素对超高分子量聚乙烯颗粒诱导的巨噬细胞源性炎性因子释放的影响

目的:研究二氢青蒿素(Dihydroartemisinin,DHA)对超高分子量聚乙烯(Ultra highmolecularweightpolyethylene,UHMWPE)颗粒诱导的小鼠巨噬细胞系RAW264.7细胞源性炎性因子释放的影响。方法:建立UHMWPE颗粒诱导的小鼠巨噬细胞系RAW264.7细胞源性炎性因子释放模型;施加不同浓度的二氢青蒿素观察药物对细胞的影响,酶联免疫分析法(Enzyme-linked immuno sorbent assay,ELISA)检测细胞培养液上清中TNF-α,IL-1β,IL-6和IL-10含量,MTT法检测细胞毒性反应。结果:酶联免疫分析方法结果表明,二氢青蒿素可以显著抑制由UHMWPE颗粒诱导的小鼠RAW264.7细胞促炎细胞因子TNF-α,IL-1和IL-6的表达,并显著促进抗炎因子IL-10的释放,其效应具有剂量依赖性。结论:二氢青蒿素具有显著的抗炎作用,可以抑制UHMWPE颗粒诱导的巨噬细胞炎症反应,其在预防人工关节置换术后假体无菌性松动的药物治疗方面具有潜在的作用。     

IL-33通过活化巨噬细胞NF-κB信号通路参与低氧性肺动脉高压的发生发展

目的探讨IL-33/ST2应答轴是否通过激活巨噬细胞NF-κB通路促进低氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)的发生、发展。方法采用野生型(WT)、IL-33转基因(Il33 Tg)小鼠和St2基因敲除(St2-/-)小鼠制备HPH小鼠模型,采集小鼠支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)和肺组织标本;进行小鼠BALF总细胞和分类计数;用免疫荧光和免疫组化法检测IL-33、ST2在小鼠肺组织的表达水平和MAC-2+巨噬细胞聚集;用ELISA检测肺组织匀浆中的细胞因子含量;用免疫印迹(Western blot)检测转录因子NF-κB在模型鼠肺组织及体外IL-33刺激的小鼠巨噬细胞系RAW264.7的表达水平。结果 IL-33、ST2在HPH模型小鼠肺组织中表达上调并伴有MAC-2+巨噬细胞增加;Il33 Tg模型鼠肺部以巨噬细胞为主的炎性细胞浸润;低氧可诱导WT小鼠肺组织表达促炎因子IL-1β和巨噬细胞趋化蛋白(monocyte chemotactic protein 1,MCP-1),而St2基因缺失则下调上述细胞因子表达;低氧亦可诱导上调NF-κB在WT小鼠肺组织表达,而St2基因缺失则可抑制低氧诱导NF-κB的表达。体外实验显示IL-33能上调巨噬细胞NF-κB的表达。结论低氧可促进IL-33/ST2表达增强,进而诱导巨噬细胞内NF-κB通路的激活,导致促炎细胞因子MCP-1、IL-1β的产生,加重炎症反应并间接引起肺动脉血管重塑参与HPH的发生、发展。     

Ghrelin抑制H202诱导的肺泡上皮A549细胞中IL-8的产生

目的：通过细胞培养的方法,初步研究了生长激素释放肽Ghrelin在氧化应激相关的肺泡上皮细胞炎症反应中的作用。方法：首先是用双氧水（H202）刺激A549细胞建立肺泡上皮细胞炎症反应模型,分别加入不同浓度的Ghrelin及普通培养基培养A549细胞。用酶联免疫吸附技术（ELISA）从蛋白水平检测上清液中IL-8的含量,采用逆转录聚合酶链反应技术（RT—PCR）检测炎性细胞因子IL．8mRNA的表达。结果：H2O2可以使A549细胞IL-8蛋白的释放及IL-8mRNA的表达明显升高（P〈0．05）,而用Gllrelin干预后IL．8的释放及IL-8mRNA的表达被抑制,明显低于单纯H2O2刺激的模型组（P〈0．05）,且随着浓度的增加Ghrelin的这种抑制作用逐渐增强。结论：分别从蛋白水平和基因水平证明了Ghrelin能够抑制H202诱导的肺泡上皮A549细胞中IL-8的产生,由此推测Ghrelin可能能够抑制以COPD、支气管哮喘为代表的氧化应激相关的肺部炎症反应。     

RAW264.7小鼠巨噬细胞与阿萨希毛孢子菌相互作用后TNF-α、IL-1β和CCL3的表达变化

目的研究RAW264.7小鼠巨噬细胞与T.asahii相互作用后TNF-α,IL-1β和CCL3的表达水平变化。方法RAW264.7细胞与T.asahii菌液共孵育3 h、6 h、9 h、12 h,对照组不加菌液。在各孵育时间点取上清液,采用双抗体夹心酶联免疫吸附试验(ELISA)试剂盒检测上清液中TNF-α、IL-1β和CCL3的含量。台盼蓝拒染法计各孵育时间点RAW264.7细胞的活细胞数。结果 RAW264.7细胞与T.asahii共孵育3 h、6 h、9 h、12 h后,TNF-α、IL-1β、CCL3的表达水平均明显高于对照组,有显著性差异(P0.05)。3种细胞因子的表达水平随共孵育时间延长而增高,共孵育9 h的表达水平最高。RAW264.7细胞的活细胞数随共孵育时间延长而逐渐下降,共孵育12 h后的活细胞数量与其他各时间点有统计学差异(P0.05)。结论 RAW264.7小鼠巨噬细胞面临T.asahii感染时可分泌TNF-α、IL-1β、CCL3等细胞因子,这些炎性细胞因子可能参与了抗T.asahii感染的免疫反应。     

Production of IL-13 in spleen cells by IL-18 and IL-12 through generation of NK-like cells

Treatment of Nylon wool-passed cells (NWC) prepared from the spleen of C57BL/6 mice with IL-18 and IL-12, but not with IL-18 alone, resulted in induction of IFN-gamma, a Th1 cytokine, and GM-CSF at 24 h, and IL-13, a Th2 cytokine at 72 h. The induction of IL-13 was suppressed by anti-GM-CSF antibody, indicating involvement of GM-CSF in IL-13 production. When NWC incubated with IL-18 and IL-12 for 72 h ("primary treatment") were treated again with the same cytokines ("secondary treatment"), IL-13 was induced much more quickly than observed in the primary treatment. Flow cytometric analysis of NWC after the primary treatment showed marked increases in the CD4(-)CD8(-) non-T cell population bearing CD25(+), CD45RB(super high) and CD122(+). These cells were positive for CD49b but negative for NK1.1, indicating that they were not typical but NK-like cells. The NK-like cells produced IL-13 in response to the treatment with IL-18 alone, indicating that the generation of these cells in the primary treatment likely accounts for the quick production of IL-13 in the secondary treatment. These results show that IL-18 and IL-12 generates the NK-like cells in NWC by a process mediated by GM-CSF that are ready for producing IL-13.     

IL-1 stimulates IL-6 production in endothelial cells

Leukocytes and vascular cells interact closely in inflammation and immunity and lymphokines are important mediators of this interaction. The present study was designed to define the possible role of IL-6 as a communication signal between vascular and immunocompetent cells. IL-6 was measured as hybridoma growth factor (HGF) on the 7TD1 cell line in the supernatants of human endothelial cells (HEC). HEC released appreciable levels of HGF activity in the absence of deliberate stimulation. In vitro exposure to recombinant IL-1 beta markedly increased (usually 10 to 15-fold) HGF production by HEC. Optimal stimulation was observed with 0.1 to 50 U/ml for 4 to 20 h of incubation. Human and murine rIL-1 alpha stimulated HGF production in HEC. Anti-IL-6 antibodies inhibited the HGF activity of the HEC supernatants, thus confirming, together with the cytokine specificity of the assay, the nature of HEC-produced cytokine. IL-1-treated HEC expressed high levels of IL-6 mRNA as detected by Northern blot analysis. Inasmuch as IL-1 elicits a complex series of changes in HEC, it was important to assess whether IL-6, produced after exposure to IL-1, modified HEC function. Natural or rIL-6 did not affect the functional status of HEC as assessed by proliferative capacity, production of procoagulant activity and prostacyclin, ability to induce adhesion of polymorphonuclear leukocytes. The capacity to produce IL-6 may represent an important mechanism by which endothelial cells participate in inflammatory and immune reactions.     

Activation of IL-4 and IL-6 secreting cells by antigen

The number, antigenic specificity and phenotype of cells secreting IL-4 and IL-6 in mice immunized with ovalbumin or keyhole limpet haemocyanin (KLH) in Freund's adjuvant (FA) was studied. The frequency of cells producing either of these cytokines began to rise 6 days post immunization, peaked at 11–14 days post-immunization, and fell to background by 21 days. The number of spleen cells secreting IL-6 was higher than the number producing IL-4 at all time points. Boosting elicited an anamnestic response characterized by a significant increase in the number of cytokine secreting cells within 4 days. Cytokine production was induced in multiple strains of normal mice, and was critically dependent on the use of Complete FA in addition to antigen. Immunization induced IL-4 and IL-6 production in vivo while ‘priming’ additional cells to release these cytokines when reexposed to soluble antigen in vitro. The latter response was antigen specific and was dominated by non-B/non-T cells. Those cells may serve to boost the immune response in cases of persistent or repeated antigenic challenge.     

IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells

Recent studies have shown that IL-4 can affect lymphocyte responses to IL-2. To evaluate the effects of IL-4 on T cell responses to physiologically relevant stimuli, we studied normal human T cells cultured with a low concentration of anti-CD3 mAb and IL-2 in the presence and absence of added IL-4. The addition of IL-4 to cultures of T cells stimulated with anti-CD3 mAb and IL-2 reduced the proliferative response by 49 to 59%. The inhibitory effect was observed in 3-, 5-, and 7-day cultures. Inhibition was dose-dependent with maximal inhibition at concentrations greater than or equal to 5 to 10 U/ml IL-4. IL-4-mediated inhibition occurred early during the T cell response, inasmuch as addition of IL-4 after stimulation for 24 h did not result in significant inhibition. Phenotypic analyses of cells cultured in the presence of anti-CD3 mAb, IL-2, and IL-4 suggested that the mechanism of regulation by IL-4 involves the inhibition of IL-2R expression. The proportion of both CD4+ and CD8+ cells that expressed IL-2R in response to IL-2 was diminished in the presence of IL-4, although HLA-DR levels were unaffected. Soluble IL-2R was also reduced in supernatants of cultures stimulated with anti-CD3 mAb, IL-2, and IL-4 as compared to cultures stimulated with anti-CD3 mAb and IL-2. These findings indicate that when normal human T cells are stimulated in vitro in a manner that approximates a physiologic interaction with Ag in vivo, rIL-4 provides a potent inhibitory signal to IL-2 responsive cells that is likely mediated by IL-4-induced inhibition of IL-2R expression.     

Identification of IFN-gamma-producing cells in IL-12/IL-18-treated mice

Both IL-12 and IL-18 have been characterized as effective IFN-gamma-inducing cytokines. Concomitant treatment with IL-12 and IL-18 has been shown to synergistically induce IFN-gamma and may be an effective therapy for treating cancer, allergy, and infectious diseases. To understand the mechanisms underlying the strong induction of IFN-gamma by IL-12/IL-18 in mice, we focused our studies on the IFN-gamma-producing cells in various lymphoid organs and tissues and utilized the intracellular cytokine staining method to detect such cells in situ. After combined treatment with IL-12 and IL-18, IFN-gamma-positive cells in C57BL/6 mice were detected in the liver (12.18%), spleen (0.68%), bone marrow (1.80%), and peritoneum (2.12%), but not in the thymus or lymph nodes (<0.05 and <0.08%, respectively). A two-color staining method revealed that the majority of IFN-gamma-producing cells in the liver were NK1.1(+) cells, while those in the spleen were mostly CD3(+) cells, and to a lesser degree NK1.1(+) cells. Both CD4(+) and CD8(+) cells in the liver and in the spleen produced IFN-gamma. The CD19(+) B cell population was not definitely shown to produce IFN-gamma in our induction experiments. NKT cells, which are a subpopulation of NK1. 1(+) CD3(+) cells, were diminished in the liver and did not seem to contribute to IFN-gamma production arising from IL-12/IL-18 treatment. Further in vitro experiments confirmed the responsiveness of hepatic mononuclear cells to IL-12/IL-18 stimulation. This study is the first to show the IFN-gamma-producing mechanisms of IL-12/IL-18 treatment at the phenotypic level.     

Up-regulation of IL-33 expression in various types of murine cells by IL-3 and IL-4

The crucial roles of the novel cytokine IL-33 in allergic, inflammatory, infectious and autoimmune diseases are becoming characterized. However, the cytokines which regulate IL-33 expression and secretion are still largely unknown. In this study, IL-3 and IL-4 were found to up-regulate IL-33 mRNA expression in mouse peritoneal exudate cells by a two-color DNA microarray and further confirmed by real time PCR and ELISA. IL-3 and IL-4 synergistically promote IL-33 mRNA expression and IL-33 intracrine in the heterogeneous cell populations as peritoneal exudates cells, bone marrow cells and splenic cells. IL-3 and IL-4 also induced IL-33 introcrine in the peritoneal exudate cells from the macrophage-deficient op/op mice, suggesting that macrophage is not the only target of IL-3 and IL-4 in the heterogeneous peritoneal exudate cells. Furthermore, IL-3 and IL-4 were verified to promote the IL-33 intracrine in the homogeneous cell population as fibroblasts and mast cells. These results indicate that up-regulation of IL-33 expression by IL-3 and IL-4 is not a feature particular to a specific type of cells. Up to 100 cytokines were screened, but none of them stimulated the secretion or release of IL-33 in the culture system. In summary, we confirm for the first time that IL-3 and IL-4 are critical for IL-33 intracrine in murine cells of various types, indicating that IL-3 and IL-4 may play an important role in the constitutive expression of IL-33 in vivo.     

Astrocytes as antigen-presenting cells: expression of IL-12/IL-23

Interleukin-12 (IL-12, p70) a heterodimeric cytokine of p40 and p35 subunits, important for Th1-type immune responses, has been attributed a prominent role in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Recently, the related heterodimeric cytokine, IL-23, composed of the same p40 subunit as IL-12 and a unique p19 subunit, was shown to be involved in Th1 responses and EAE. We investigated whether astrocytes and microglia, CNS cells with antigen-presenting cell (APC) function can present antigen to myelin basic protein (MBP)-reactive T cells, and whether this presentation is blocked with antibodies against IL-12/IL-23p40. Interferon (IFN)-gamma-treated APC induced proliferation of MBP-reactive T cells. Anti-IL-12/IL-23p40 antibodies blocked this proliferation. These results support and extend our previous observation that astrocytes and microglia produce IL-12/IL-23p40. Moreover, we show that stimulated astrocytes and microglia produce biologically active IL-12p70. Because IL-12 and IL-23 share p40, we wanted to determine whether astrocytes also express IL-12p35 and IL-23p19, as microglia were already shown to express them. Astrocytes expressed IL-12p35 mRNA constitutively, and IL-23 p19 after stimulation. Thus, astrocytes, under inflammatory conditions, express all subunits of IL-12/IL-23. Their ability to present antigen to encephalitogenic T cells can be blocked by neutralizing anti-IL-12/IL-23p40 antibodies.     

B cells as antigen-presenting cells: antigen-specific IL-2 production by cloned T cells without expression of IL-2 receptors

A murine T cell clone, 24-2C, responds specifically to human IgG (HGG) in the context of I-Ab. B cells purified from mouse spleen cells were examined for their function as antigen-presenting cells (APC) in the response of 24-2C cells to HGG. B cells functioned as APC for IL-2 production but not for proliferation, whereas spleen cells or spleen-adherent cells functioned as APC for both IL-2 production and proliferation. LPS-activated B cells also failed to induce the proliferative response. The addition of the culture supernatant of 24-2C cells stimulated with HGG presented by irradiated spleen cells to the culture of 24-2C cells, irradiated B cells, and HGG induced the proliferative response of 24-2C cells, whereas IL-1, IL-3, and/or interferon-gamma did not reconstitute the proliferation. The expression of IL-2 receptors (IL-2R) on 24-2C cells was examined using a monoclonal anti-mouse IL-2R antibody AMT 13 or 7D4. 24-2C cells cultured with spleen cells as APC expressed IL-2R. Those cultured alone or with B cells as APC did not express IL-2R. Enlargement of 24-2C cells in response to HGG was also examined, and the relative cell size of those cultured with B cells or spleen cells as APC was larger than that of those cultured alone. These results demonstrate that B cells as APC induce IL-2 production and cell size enlargement in the response of 24-2C cloned T cells to HGG, but not IL-2R expression nor proliferation.     

IL-27 Regulates IL-18 binding protein in skin resident cells

IL-18 is an important mediator involved in chronic inflammatory conditions such as cutaneous lupus erythematosus, psoriasis and chronic eczema. An imbalance between IL-18 and its endogenous antagonist IL-18 binding protein (BP) may account for increased IL-18 activity. IL-27 is a cytokine with dual function displaying pro- and anti-inflammatory properties. Here we provide evidence for a yet not described anti-inflammatory mode of action on skin resident cells. Human keratinocytes and surprisingly also fibroblasts (which do not produce any IL-18) show a robust, dose-dependent and highly inducible mRNA expression and secretion of IL-18BP upon IL-27 stimulation. Other IL-12 family members failed to induce IL-18BP. The production of IL-18BP peaked between 48-72 h after stimulation and was sustained for up to 96 h. Investigation of the signalling pathway showed that IL-27 activates STAT1 in human keratinocytes and that a proximal GAS site at the IL-18BP promoter is of importance for the functional activity of IL-27. The data are in support of a significant anti-inflammatory effect of IL-27 on skin resident cells. An important novel property of IL-27 in skin pathobiology may be to counter-regulate IL-18 activities by acting on keratinocytes and importantly also on dermal fibroblasts.     

IL-6 and IL-1 synergize to stimulate IL-2 production and proliferation of peripheral T cells

Purified T cells can be induced to proliferate and to produce the autocrine growth factor IL-2 with mAb to the TCR and costimulatory cytokines. In a previous report we demonstrated that human IL-6 stimulates IL-2 production and proliferation of purified T cells, in conjunction with the insolubilized anti-TCR V beta 8 mAb, F23.1. Here we show that when CD4+ T cells are rigorously purified to greater than 99% CD4+CD8-, they respond only weakly to F23.1 and IL-6. Instead, there is an additional requirement for IL-1, which dramatically synergizes with IL-6 to induce prolonged (greater than 7 days) proliferative responses and IL-2 production. Similar results were observed when the highly mitogenic anti-CD3 mAb 145-2C11 was substituted for F23.1. The proliferation induced by F23.1, IL-1, and IL-6 was substantially (greater than 80%) inhibited by a mAb to mouse IL-2, and was not inhibited by an anti-IL-4-mAb. In accordance with this finding, medium conditioned by the activated CD4+ cells contained large amounts of IL-2, which increased over a 7-day culture period. These results demonstrate that IL-6 and IL-1 stimulate T cell proliferation by inducing production of the autocrine growth factor IL-2. In addition, the two lymphokines must be present simultaneously for activation to occur. The possible roles of IL-6 and IL-1 in IL-2 gene regulation and in Ag-induced T cell activation are discussed.