An update on leptin as immunomodulator

Until the discovery of leptin 20 years ago, adipose tissue was considered only as a fat storage organ, involved in the regulation of energy homeostasis. At present, it is well known that adipokines, being leptin the forerunner of this superfamily, may act in different biological processes, including inflammation and immunity. In this review, we have explored the recent evidence about the relationship between leptin and immune system, summarizing the most important findings related to the involvement of leptin in both innate and adaptive immune response.     

Role of leptin in immunity

Leptin, a protein hormone produced by the adipocytes, has long been recognized to regulate metabolism, neuroendorine and other physiological functions. Early findings of increased leptin production during infection and inflammation and dysregulated immune response in leptin signaling-deficient mice provide strong evidence for the involvement of leptin in the immune responses. Recent data have established the regulatory function for leptin in immunity similar to the function of a pro-inflammatory cytokine, while gene-targeting studies also demonstrated an essential role of leptin in regulating hematopoiesis and lymphopoiesis. Moreover, there has been increasing evidence that leptin is involved in the pathogenesis of various autoimmune diseases. This review discusses recent advances in understanding the role of leptin in immunity and leptin-signaling pathways involved in modulating immune homeostasis and autoimmune pathogenesis. Cellular ＆ Molecular Immunology. 2007;4（1）：1-13.     

Lactobacillus isolates from healthy volunteers exert immunomodulatory effects on activated peripheral blood mononuclear cells

As probiotics in the gut, Lactobacilli are believed to play important roles in the development and maintenance of both the mucosal and systemic immune system of the host. This study was aimed to investigate the immuno-modulatory function of candiate lactobacilli on T cells. Lactobacilli were isolated from healthy human feces and the microbiological characteristics were identified by API 50 CHL and randomly amplified polymorphic DNA (RAPD) assays. Anti-CD3 antibody activated peripheral blood mononuclear cells (PBMCs) were treated by viable, heat-killed lactobacilli and genomic DNA of lactobacilli, and cytokine profiles were tested by ELISA. Isolated lactobacilli C44 and C48 were identified as L. acidophilus and L. paracacei, which have properties of acid and bile tolerance and inhibitor effects on pathogens. Viable and heat-killed C44 and C48 induced low levels of proinflammatory cytokines (TNF-α, IL-6 and IL-8) and high levels of IFN-γ and IL-12p70 in PBMCs. In anti-CD3 antibody activated PBMCs, viable and heat-killed C44 increased Th2 cytokine levels (IL-5, IL-6 and IL-10), and simultaneously enhanced Th1 responses by inducing IFN-γ and IL-12p70 production. Different from that of lactabacillus strains, their genomic DNA induced low levels of IL-12p70, IFN-γ and proinflammatory cytokines in PBMCs with or without anti-CD3 antibody activation. These results provided in vitro evidence that the genomic DNA of strains of C44 and C48, especially C44, induced weaker inflammation, and may be potentially applied for treating allergic diseases.     

慢性乙型肝炎患者外周血单个核细胞中IL—18表达水平

目的探讨白细胞介素 18(IL- 18)在乙型肝炎病毒感染中的作用。方法应用流式细胞免疫学方法 ,对 30例慢性乙型肝炎活动期、缓解期 ,15例 HBS Ag阳性无症状携带者 ,10例正常对照外周血单个核细胞 (PBMC) IL- 18的表达进行检测。结果无症状携带者表达最低 ,慢性乙型肝炎缓解期低于正常对照 (P<0 .0 1) ,活动期与正常对照无显著差异 (P=0 .2 5 )。活动期 IL- 18的表达与肝组织炎症活动度有关 (P<0 .0 1) ,与血清 AL T水平呈正相关 (r=0 .6 3,P<0 .0 1)。结论 IL- 18与慢性乙型肝炎病情的活动相关 ,与肝组织炎症程度相关     

人脐血单个核细胞向神经细胞分化

目的：观察培养前后人脐血单个核细胞(MNCs)的形态学及免疫反应性变化,探讨其能否向神经细胞的分化及机制。方法：密度梯度离心脐血中单个核细胞,接种并用。EGF和bFGF刺激细胞生长,倒置显微镜下观察培养前后细胞形态变化,并行免疫细胞化学鉴定。结果：培养前脐血MNCs胞体小呈圆形,nestin阳性细胞、AP2阳性细胞散在分布(阳性率为1．5％和3．4％)无GFAP阳性细胞着色。培养14d后,细胞群中相邻细胞突起连成网状;AP2、GFAP染色阳性细胞成片状分布(阳性率33．5％和24．6％),未见nestin阳性细胞。结论：脐血细胞中可能有多能干细胞,经体外培养后能分化为具有一定形态的神经细胞。     

Bioenergetic analysis of human peripheral blood mononuclear cells

Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)−1β, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses.     

DHMEQ, a novel nuclear factor-kappaB inhibitor, induces selective depletion of alloreactive or phytohaemagglutinin-stimulated peripheral blood mononuclear cells, decreases production of T helper type 1 cytokines, and blocks maturation of dendritic cells

Dehydroxymethylepoxyquinomicin (DHMEQ), a novel nuclear factor κB (NF-κB) inhibitor, has been shown to be active against variety types of solid tumours as well as haematological malignant cells. This study explored the anti-inflammatory effects of DHMEQ in vitro. DHMEQ inhibited the proliferation of phytohaemagglutinin (PHA)-stimulated or alloreactive peripheral blood mononuclear cells (PBMC) in mixed lymphocyte cultures as measured using a 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. In contrast, DHMEQ did not affect the viability of resting PBMC. In addition, real-time polymerase chain reaction showed that DHMEQ decreased PHA-stimulated expression of T helper type 1 (Th1) cytokines, including interleukin-2, interferon-γ, and tumour necrosis factor α, in PBMC as well as Jurkat T-lymphoblastic leukaemia cells, and also decreased levels of p65 isoforms of NF-κB in the nucleus. Furthermore, we found that DHMEQ inhibited the endocytic capacity of dendritic cells (DCs) and down-regulated the expression of cell surface antigen CD40, suggesting that DHMEQ blocked the maturation as well as the function of DCs. Taken together, the results suggest that DHMEQ may be useful for treatment of inflammatory diseases, including graft-versus-host disease after allogenic haematopoietic stem cell transplantation.     

Interleukin-12 secretion by peripheral blood mononuclear cells is decreased in normal pregnant subjects and increased in preeclamptic patients

PROBLEM: It has been reported that T-helper (Th) 2 dominance in normal pregnancy shifts to Th1 dominance in preeclampsia. Peripheral blood mononuclear cell (PBMC) production of interleukin (IL)-12, which induce Th1 responses, has not been compared between these clinical states. METHOD OF STUDY: Peripheral blood mononuclear cell from 35 non-pregnant women, 35 healthy pregnant women, 12 mildly preeclamptic patients, and 15 severely preeclamptic patients were cultured for 24 hr. IL-12 secretion was determined by enzyme-linked immunosorbent assay (ELISA). Th1/Th2 ratios in PBMC were determined flow-cytometrically, and the amounts of HLA-DR and CD14 expression on the monocytes were obtained by flow cytometry. RESULTS: Peripheral blood mononuclear cell from healthy pregnant subjects secreted less IL-12 than non-pregnant women. PBMC from severely preeclamptic patients secreted more IL-12 than those from healthy pregnant subjects, while IL-12 secretion in mild preeclampsia resembled secretion in normal pregnancy. Th1/Th2 ratios correlated were positively with IL-12. Increased HLA-DR antigens and reduced CD14 expression, suggesting monocyte activation, were observed in preeclamptic patients, although monocyte counts were unchanged. CONCLUSION: Decreased IL-12 secretion by PBMC may cause Th2 dominance in normal pregnancy, while increased IL-12 secretion by activated monocytes may cause Th1 dominance in preeclampsia.     

外周血单个核细胞在乙肝相关性肾炎发病机制中的作用研究

目的通过观察外周血单个核细胞（PBMc）对体外培养的肾小管上皮细胞凋亡的影响,探讨PBMC在乙肝相关性肾炎发病机制中的作用。方法用流式细胞仪检测HBVDNA阳性血清与人近端肾小管上皮细胞（HK-2）培养时HK．2凋亡率作为对照,分别观察健康人及HBVDNA阳性患者PBMC对HK-2凋亡的影响。结果健康人PBMC组HK-2凋亡率明显低于对照组和HBVDNA阳性患者PBMC组（P〈0．01）。结论健康人PBMC能抑制HK-2凋亡,而HBVDNA阳性患者PBMC此抑制作用降低,提示HBVDNA阳性患者PBMCs功能缺陷,可能是乙型肝炎相关肾炎（HBV—GN）的发病机制之一。     

骨髓基质干细胞治疗实验性自身免疫性神经炎的研究

目的:探讨骨髓基质干细胞(BMSC)移植治疗实验性自身免疫性神经炎(experimentalautoimmuneneuritis,EAN)的疗效以及相应的作用机制。方法:用P0180-199多肽与弗氏完全佐剂的混合液免疫Lewis大鼠,建立EAN动物模型。治疗组在免疫后第10天,尾静脉回输荧光染料PKH26标记的BMSC(2×106个细胞/只),通过临床评估、免疫组化及ELISA等方法,研究了BMSC对EAN的治疗作用。结果:回输的BMSC能向脱髓鞘神经组织周围迁移,减轻脱髓鞘的病理改变和炎性细胞浸润。与对照组比较,治疗组CD4 和CD8 T细胞的浸润显著减少(P<0.05),血清中IFN-γ和TNF-α的水平明显降低(P<0.05),培养上清中IL-4的水平显著增加(P<0.05)。结论:BMSC静脉移植治疗EAN有一定的疗效。BMSC通过调节细胞因子的表达逆转Th1/Th2型细胞之间的失衡而发挥治疗作用,并能够抑制T细胞活化增殖。     

Estradiol enhances primary antigen-specific CD4 T cell responses and Th1 development in vivo. Essential role of estrogen receptor alpha expression in hematopoietic cells

It is widely accepted that females have superior immune responses than males, but the ways by which sex hormones may enhance T cell responses are still poorly understood. In the present study, we analyzed the effect of estrogens on CD4 T cell activation and differentiation after immunization with exogenous antigens. We show that administration of low doses of 17beta-estradiol (E2) to castrated female mice results in a striking increase of antigen-specific CD4 T cell responses and in the selective development of IFN-gamma-producing cells. Quantitative assessment of the frequency of T cells bearing a public TCR beta chain CDR3 motif demonstrated that the clonal size of primary antigen-specific CD4 T cells was dramatically increased in immune lymph nodes from E2-treated mice. By using mice with disrupted estrogen receptor (ER) alpha or beta genes, we show that ERalpha, but not ERbeta, was necessary for the enhanced E2-driven Th1 cell responsiveness. Furthermore, ERalpha expression in hematopoietic cells was essential, since E2 effects on Th1 responses were only observed in mice reconstituted with bone marrow cells from ERalpha+/+, but not ERalpha-deficient mice. These results demonstrate that estrogen administration promotes strong antigen-specific Th1 cell responses in a mechanism that requires functional expression of ERalpha in hematopoietic cells.     

The early immunological response to acute ischemic stroke: Differential gene expression in subpopulations of mononuclear cells

Peripheral blood mononuclear cells (PBMCs), i.e. lymphocytes, monocytes and macrophages are key players in the development of innate and adaptive immune responses. However, little is known about their properties in patients with acute stroke. Experimental procedures: We presently characterized the early time course of PBMC subpopulations in 19 patients with acute ischemic stroke and symptom onset below 6 h compared to 19 age-matched healthy subjects. Immediately after acute ischemic stroke, as well as 1 and 3 days thereafter, PBMC subpopulations (cluster of differentiation [CD]3+, CD14+, CD19+, CD68+) were isolated by magnetic bead system and the expression of proinflammatory (CD40, tumor necrosis factor-alpha [TNFα]), proapoptotic (caspase-3 [CPP32], poly(ADP-ribose) polymerase [PARP]) and adhesion relevant (CD38) genes was measured by quantitative polymerase chain reaction (PCR). Furthermore, besides routine parameters, plasma levels of oxidized low-density lipoproteins (oxLDL) were studied. Results: In comparison to healthy subjects, patients revealed (i) twofold elevated plasma oxLDL concentrations, (ii) decreased (15%) blood cholesterol levels, and (iii) a 40% decrease in total number of lymphocytes. Furthermore, the majority of PBMC subpopulations revealed an increased expression of proinflammatory, proapoptotic or adhesion-relevant genes. Significant positive correlations were observed between expression of most of these genes in PBMCs and individual plasma oxLDL concentrations. Conclusion: Elevated expression of proinflammatory, proapoptotic and adhesion genes in subsets of PBMCs after ischemic stroke may contribute to an immunodepressive syndrome, possibly due to increased plasma oxLDL levels.     

类风湿性关节炎患者外周血芳香烃受体表达与Th17细胞相关性研究

目的 检测类风湿性关节炎患者(RA)外周血单个核细胞芳香烃受体(AhR)的表达情况,分析其表达与Th17细胞相关性并初步探讨AhR在RA发病中的作用机制.方法 Ficoll密度梯度法分离75例RA患者(其中活动期RA患者40例,缓解期RA患者35例)及70例健康对照者外周血单个核细胞(PBMC),酶联免疫吸附实验(EUSA)检测细胞裂解上清中AhR蛋白表达.流式细胞术检测75例RA患者Th17细胞表达比例.分析RA患者AhR蛋白表达与Th17细胞比例及其他临床指标的相关性.结果 RA患者PBMC内AhR表达[(91.25±15.27) ng/L]高于健康对照组[(41.81 ±9.72) ng/L],差异有统计学意义(t=3.191,P＜0.01).活动期RA患者AhR表达与缓解期RA患者表达差异无统计学意义(P＞0.05).RA患者PBMC中AhR水平与Th17细胞表达呈正相关(r=0.361,P=0.0015).RA患者PBMC中AhR表达与血清IL-17水平呈正相关,与其他临床指标无相关性(r=0.324,P=0.006).结论 RA患者PBMC中AhR表达升高,且与Th17细胞和血清中IL-17水平呈正相关,推测RA中AhR可能正向调控Th17细胞分化,促进IL-17表达.     

脱氧雪腐镰刀菌烯醇对人外周血单个核细胞TAP-1表达的抑制作用

目的: 探讨脱氧雪腐镰刀菌烯醇 (DON)对人外周血单个核细胞 (PBMC)中TAP- 1表达的影响。方法: 采用流式细胞仪(FCM)和半定量RT- PCR方法, 从蛋白和mRNA水平上分析不同浓度的DON对体外培养的人PBMC中TAP- 1分子表达的影响及量 效关系。结果: FCM定量检测表明, 用不同浓度的DON处理, 均可一定程度地抑制人PBMC中TAP -1蛋白的表达, 且二者呈显著的负相关 (r= -0. 865,P<0. 01)。半定量RT -PCR检测显示, 不同浓度的DON处理均可抑制人PBMC中TAP -1mRNA的表达。结论: DON可剂量依赖地抑制体外培养的人PBMC中TAP- 1蛋白和其mRNA的表达, 对阐明食管癌高发区被DON污染的粮食与食管癌发生的关系具有重要的意义。     

In vitro production of cytokines by peripheral blood mononuclear cells from hydatid patients.

The role of cytokines in human hydatidosis (Echinococcus granulosus infection) was evaluated in immunoassays determining production of IL-4, IL-10 and interferon-gamma (IFN-gamma) in peripheral blood mononuclear cell (PBMC) cultures from 30 hydatid patients and 14 uninfected controls. In cell cultures from hydatid patients parasite and non-parasite antigen stimulation significantly increased IL-4 production (P < or 0.005). Spontaneous and mitogen-driven IL-4 production was similar in patients and controls. IL-10 and IFN-gamma production did not differ statistically in the two groups, even though some hydatid patients produced these cytokines in large amounts. Notably, antigen-driven IFN-gamma concentrations were invariably higher in patients than in uninfected controls. Data analysis showed a relationship between IgE and IgG4 responses and parasite-driven cytokine production. High IgE and IgG4 responders produced high IL-4 and IL-10 concentrations. High IgE responders showed decreased IFN-gamma production, but high IgG4 responders had IFN-gamma levels slightly higher than those of low responders. Cytokine response patterns did not relate to the clinical stage of disease. The significantly increased IL-4 and the high IL-10 concentrations found in PBMC from many hydatid patients in this study are consistent with Th2 cell activation in human hydatidosis. The presence of antigen-driven IFN-gamma production in patients with E. granulosus infection implies concurrent intervention of the Th1 or Th0 cell subset.