Roles of HSP60 in Helicobacter pylori infection

我们之前报道过热休克蛋白60kDa （HSP60）是粘膜相关淋巴组织（MALT）淋巴瘤发病机制中的重要抗原。为了研究宿主免疫和HSP60的关系，我们分析了HSP60刺激后患者外周血单个核细胞（PBMCs）中DC40配体（CD40L）和细胞因子的产生。在细胞因子（IL-4）和GM-CSF)存在的情况下，用重组hsp60或幽门螺杆菌细胞裂解物刺激胃炎和MALT淋巴瘤患者和健康志愿者的PBMCs。用含有1100个基因的cDNA芯片分析mRNA表达。在细胞因子和/或HSP60抗原刺激的MALT淋巴瘤患者外周血中，CD4阳性细胞CD40L的表达增加。MALT淋巴瘤患者外周血培养细胞中IL-4的产生增加；然而，IFN-_γ的产生处于低水平。基因芯片分析表明，更多的ed增加了HLA-DR和整合素的mRNA水平。在低级别MALT淋巴瘤病例中，针对HSP60的适应性免疫反应可能会被宿主因素（如抗原呈递和T细胞活化）增强，导致B细胞增殖，这可以在幽门螺杆菌感染期间得到证实。为了研究HSP60在幽门螺杆菌感染中的免疫作用，我们对幽门螺杆菌感染小鼠进行了HSP60免疫。整个HSP60和两个部分的HSP60被表达为gst融合蛋白。三个重组蛋白被命名为rHSPw、rHSP2和rHSP4-5。rHSPw表达为完整的hsp60 （MeT_1-Met_<545 bb0）。rHSP2 （Glu_<101>-Ser_<200>）包含一个domain细胞表位簇（Lys_<159>-Tnr_<178>）。rHSP4-5所含的结构域存在于幽门螺杆菌和人hsp60之间的T细胞epilope cluster （Asp_<396>-Gly_<4l2>）及其上游区域（Ser_<356>-Asp_<392>）中。重组大肠杆菌热不稳定肠毒素（rLT）也被用作鼻免疫佐剂。3组小鼠（BALB/c、C3H/He、C57BL/6）每周注射10^8 CRJ活的幽门螺杆菌（SS -1），每周2次，在清洁的隔离箱内保存2周。将幽门螺杆菌裂解物或rHSPs （10μg/小鼠）与rLT （10μg/小鼠）在1个月内注射4次鼻黏膜免疫。末次免疫后1个月处死小鼠，进行组织病理学和抗体检测。幽门螺杆菌裂解物和rHSPs粘膜免疫诱导BALB/c和C57BL/6小鼠重度胃炎。rHSP4-5免疫组的炎症程度以aft组最高。hsp60粘膜免疫加速IgG和IgA的产生。这些结果表明HSP60与幽门螺杆菌引起的胃炎症密切相关。少

We have previously reported that heat shock protein 60kDa (HSP60) is an important antigen in the pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphoma. To investigate association with host immunity and HSP60, DC40 ligand (CD40L) and cytokine production in peripheral blood mononuclear cells (PBMCs) from the patients were analyzed following stimulation with HSP60. PBMCs obtained from patients with gastritis and MALT lymphoma, and those from healthy volunteer were stimulated with recombinant-HSP60 or H. pylori cell fysate in the presence of cytokines (IL-4) and GM-CSF). The mRNA expression was also analyzed by a cDNA microarray containing 1100 genes. The expression of CD40L on the CD4 positive cells was increased in the PBMCs from patients with MALT lymphoma stimulated by cytokines and/or HSP60 antigens. The production of IL-4 in PBMCs cultures was increased in patients with MALT lymphoma ; however, the production of IFN-_γ was at low levels. A cDNA microarray analysis indicat … More ed increased mRNA levels of HLA-DR and integrin. In cases of low-grade MALT lymphoma, adaptive immune responses against HSP60 may be enhanced by host factors, such as antigen presentation and T cell activation, resulting in B cell proliferation, which can be demonstrated during H. pylori infection.To study immunological roles of HSP60 in H. pylori infection, we immunized HSP60 to H. pylori infected mice. Whole HSP60 and two partial regions of helicobacterial hsp60 were expressed as GST-fusion proteins. Three recombinant proteins were designated rHSPw, rHSP2, and rHSP4-5. rHSPw was expressed as whole hsp60 (MeT_1-Met_<545>). rHSP2 (Glu_<101>-Ser_<200>) contained a domainT cell epitope cluster (Lys_<159>-Tnr_<178>). The rHSP4-5 contained a domain existing in both T cell epilope cluster (Asp_<396>-Gly_<4l2>) and its upstream of region (Ser_<356>-Asp_<392>) in common between H. pylori and human hsp60. Recombinant heat-labile enterotoxin (rLT) of E. coli was also employed as adjuvant for nasal immunization. Three different mouse groups (BALB/c, C3H/He, C57BL/6) were injected 10^8 CRJ of live H. pylori (SS -1) at two times a week and kept in a clean isolator for 2 weeks. Those mice were immunized H. pylori lysate or rHSPs (10μg/mouse) with rLT (10μg/mouse) by injecting into nasal mucosa four times in 1 month. Mice were sacrificed at 1 momtn after last immunization, and then histopathology and antibodies were investigated. Mucosal immunization by H. pylori lysate and rHSPs induced severe gastritis in BALB/c and C57BL/6 mice. Inflammation grades in those two groups immunized by rHSP4-5 were highest in aft groups. Mucosal immunization by hsp60 accelerated production of IgG and IgA.These -results indicated that HSP60 was closely associated with H. pylori induced gastric inflammation. Less