肾小球系膜细胞增生与系膜硬化机制的初步探讨

我们应用细胞培养及免疫组化的方法，探讨了肾小球系膜细胞增生及系膜硬化的机制，并进而观察了它们在肾小球硬化听作用。结果显示：系膜细胞有自分泌功能，肿瘤坏死因子和血管内皮素可促进系膜细胞增生。大量增多的系膜基质不但含有Ⅳ型胶原，也有Ⅲ型胶原。系膜细胞增生，系膜基质增多最终导致肾小球硬化。     

炎症因子对肾小球系膜细胞分泌IL—8的影响

目的：观察炎症因子ＩＬ－１及肿瘤坏死因子（ＴＮＦ）对人肾小球系膜细胞（ＨＭＣ）分泌ＩＬ－８的影响。方法：体外细胞培养技术及ＥＬＩＳＡ夹心法。结果：ＩＬ－１、ＴＮＦ均可诱导ＨＭＣ分泌ＩＬ－８，并呈剂量依赖性。结论：在一些炎症因子刺激下，ＨＭＣ可能通过分泌趋化因子ＩＬ－８参与肾脏炎症反应的启动和维持     

原发性肾小球病变患者尿液肿瘤坏死因子水平

原发性肾小球病变患者尿液肿瘤坏死因子水平〔英〕／ＯｚｅｎＳ…／／Ｎｅｐｈｒｏｎ．－１９９４，６６（３）．－２９１～２９４据报道肿瘤坏死因子（ＴＮＦ）是一种与肾小球损伤有关的细胞激素，ＴＮＦ对肾小球系膜、内皮及上皮细胞的作用可导致肾脏的炎症反应。文章拟...     

白细胞介素1刺激系膜细胞IL—1转化生长因子β基因表达

本文应用分子生物学技术证明培养的肾小球系膜细胞有 TGFβmRNA 基因表达。并且 IL—1可促进系膜细胞 IL—1、TGFβ基因表达,提示细胞因子的自分泌和旁分泌在肾小球炎症硬化过程中起着重要作用.     

MADK调节人肾小球系膜细胞和近端小管上皮细胞产生IL-6途径

IL - 6和 INF- a是多向性细胞因子 ,其在不同的免疫调节的肾脏疾病中 ,与小球、小管损伤关系密切。虽然培养的肾细胞中 TNF- a显示能剌激 IL - 6产生 ,但调节 IL - 6机制还不完全清楚。本研究目的 P38和细胞外的信号调节酶丝裂原激活蛋白酶调节在人肾小球系膜细胞和近端小管上皮细胞由肿瘤坏死因子介导产生 IL - 6途径。方法　从切除的肾组织中采取肾小球系膜细胞和近端小管上皮细胞 ,在有或无特异性 P38和 ERK1,2 MAPK抑制剂 SB2 0 35 80和 PD980 5 9存在的条件下 ,用肿瘤坏死因子处理细胞 2 4h。在培养基中 IL- 6浓度可以通过…     

NF-κB介导脂氧素A4拮抗TNF-α所致的系膜细胞白介素的合成

为研究脂氧素A4(LXA4)拮抗肿瘤坏死因子α(TNF-α)对肾小球系膜细胞的白介素(IL)-1β(IL-1β)、IL-6合成的作用。对体外培养大鼠肾小球系膜细胞,用不同浓度的LXA4预刺激,再加入TNF-α共同孵育;或单用TNF-α刺激肾小球系膜细胞。在孵育后用ELISA法检测培养上清中的IL-1β、IL-6蛋白表达量;用RT-PCR法检测IL-1β、IL-6的mRNA表达量。应用凝胶电泳迁移率试验(EMSA)测定核因子-κB(NF-κB)的DNA结合活性。结果发现,LXA4呈剂量依赖性地抑制TNF-α诱导的肾小球系膜细胞IL-1β和IL-6蛋白的合成与mRNA表达,抑制NF-κB的DNA结合活性。说明LXA4通过下调NF-κB的DNA结合活性,拮抗TNF-α对肾小球系膜细胞的IL-1β、IL-6合成的促进作用。     

大鼠肾小球系膜细胞产生的生长抑制性因子－转化生长因子β

本文观察了肾小球系膜细胞产生的自分泌生长抑制因子的情况。结果显示，肾系膜细胞培养上清中含有抑制自身生长的活性物质，后者有耐酸性；ＨＰＬＣ分析洗脱液中最主要的抑制性活性物质的分子量为１６～３０ｋｄ；抗－转化生长因子β（ＴＧＦβ）抗体能中和其大部分抑制活性；生物学方法检测表明上清中含有活性ＴＧＦβ；此外，培养液中加入抗ＴＧＦβ抗体能明显增强细胞的增殖。结果表明：大鼠肾系膜细胞能产生活性ＴＧＦβ，后者是一种自分泌的生长抑制性因子。     

HMG—CoA还原酶抑制剂与肾小球系膜细胞增殖的分子机制

羟甲基戊二酰辅酶A(HMG CoA)还原酶抑制剂不但有抑制HMG CoA还原酶、调节血脂水平的作用 ,还能通过抑制肾小球系膜细胞核因子κB(NF κB)、转化生长因子 β(TGF β)、血小板衍化生长因子 (PDGF)、纤维连接蛋白 (FN)等转录因子、细胞因子和基质蛋白的合成与分泌 ,从而抑制肾小球系膜细胞增殖与细胞外基质蛋白沉积。HMG CoA还原酶抑制剂将在预防和治疗终末期肾病方面发挥重要的作用。     

脂氧素A4拮抗肿瘤坏死因子α对系膜细胞Jak1/STAT3途径的活化

目的：验证脂氧素A4(LXA4) 是否抑制肿瘤坏死因子α(TNFα) 所致的大鼠肾小球系膜细胞的增殖，并探讨其作用中信号转导的分子机制。 方法： 对体外培养的大鼠肾小球系膜细胞，用不同浓度的LXA4 预刺激，再加入TNFα共同孵育，或单用TNFα刺激系膜细胞。用MTT渗入法检测细胞的增殖。用凝胶电泳迁移率试验（EMSA）检测信号转导子和转录激活子-3（STAT3）的活性。用RT-PCR法检测细胞周期素E的mRNA表达。用Western blotting法检测细胞周期素E的蛋白表达量。结果： LXA4呈剂量依赖性地抑制TNFα诱导的肾小球系膜细胞的增殖、STAT3结合活性增加、细胞周期素E mRNA表达与蛋白合成的亢进。结论： LXA4能够抑制TNFα所致的大鼠系膜细胞的增殖，其机制可能是阻断Jak1/STAT3信号转导途径。     

大鼠肾小球和系膜细胞产生转化生长因子β的研究

用标准的生物学方法检测了肾小球和系膜细胞培养上清中转化生长因子(TGF_s)的水平。结果显示:肾小球和系膜细胞均可产生一定量的TGF_s;肾小球产生的THF_s无活性,而系膜细胞产生的TGF_s大约10％有活性。鉴于TGF_s在调节细胞生长和细胞外基质代谢中的独特作用,肾小球和系膜细胞来源的rGF_s可能在维持正常肾小球结构和功能中起重要作用。     

Tumor necrosis factor receptors in lymphoid tissues and lymphomas. Source and site of action of tumor necrosis factor alpha.

Tumor necrosis factor alpha (TNF alpha), which is produced by germinal center dendritic reticulum cells (DRC) in lymphoid tissue, plays a regulatory role in a local immune response. However no information is available on the nature and location of cells responding to this cytokine. Thus TNF receptor distribution was investigated in situ by immunohistochemistry using monoclonal antibodies directed against the p75 and p55 receptor proteins. Receptor expression was unique and restricted to the lymphoreticular tissue. The p75 receptor was found on activated lymphocytes and interdigitating reticulum cells of the T-cell area, whereas the p55 receptor was confined to the germinal center DRCs, which are the main site of TNF alpha production. The two receptor proteins were expressed on distinct cell populations of the lymphoid system and no coexpression was observed. Preliminary results indicate that TNF receptor (TNFR) expression is regulated; Upregulation of TNFR proteins was found in reactive hyperplasia together with increased TNF alpha expression. In lymphoproliferative disorders, expression of the p75 receptor and TNF alpha was found mainly in high-grade malignant non-Hodgkin lymphomas. In summary, TNF alpha produced by germinal center DRCs might regulate an in vivo immune response through autocrine and paracrine pathways. Thus TNF alpha might signal, through the distinct TNFR proteins, the p55 and p75 receptor, which are expressed on different cell types in lymphoid tissue.     

重组白细胞介素1对系膜细胞产生与表达白细胞介素6的影响

采用ＩＬ－６依赖型细胞株ＫＤ８与Ｎｏｒｔｈｅｍｂｌｏｔ方法观察了重组ＩＬ－１对人胎肾小球系膜细胞产生与表达ＩＬ－６ｍＲＮＡ的影响，结果表明，重组ＩＬ－１加入系膜细胞培养体系中，ＩＬ－６活性与ｍＲＮＡ表达均明显高于对照组，提示重组ＩＬ－１可促进系膜细胞产生与表达ＩＬ－６。     

Inhibition of IKK down-regulates antigen + IgE-induced TNF production by mast cells: a role for the IKK-IkappaB-NF-kappaB pathway in IgE-dependent mast cell activation

Mast cells (MC) are major effector cells for allergic diseases. Cross-linking of immunoglobulin E (IgE) and its high-affinity receptor, FcepsilonRI, by antigen initiates a cascade of signaling events leading to nuclear factor (NF)-kappaB activation and tumor necrosis factor (TNF) production. Here, we demonstrated that inhibition of inhibitor of kappaB (IkappaB) kinase (IKK) by a peptide IKK inhibitor or by four individual chemical IKK inhibitors including 15-deoxy-prostaglandin J(2), BMS-345541, SC-514, or sulindac significantly blocked IgE + trinitrophenyl (TNP)-induced TNF production by mouse bone marrow-derived MC (BMMC). Moreover, IgE + TNP induced a rapid phosphorylation of IKKalpha but not IKKbeta in BMMC. IgE + TNP-induced phosphorylation of IKKalpha was accompanied with phosphorylation and degradation of IkappaBalpha, subsequent NF-kappaB activation, and TNF production. Inhibition of IKK by sulindac decreased IKKalpha phosphorylation, IkappaBalpha phosphorylation and degradation, NF-kappaB activation, and TNF production by BMMC. It is interesting that IgE + TNP stimulation also induced a prominent synthesis of IKKalpha and IkappaBalpha. Inhibition of NF-kappaB activity by pyrrolidine dithiocarbomate (PDTC) blocked IgE + TNP-induced IkappaBalpha synthesis. NF-kappaB activity and TNF production were also inhibited when PDTC was used even after IgE + TNP stimulation, suggesting a potential role for the newly synthesized IkappaBalpha in MC activation. In addition, IgE + TNP-induced IKKalpha and IkappaBalpha phosphorylation was inhibited by a protein kinase C (PKC) inhibitor Ro 31-8220. Taken together, our results support a role for the IKK-IkappaB-NF-kappaB pathway, which likely involves PKC in IgE-dependent TNF production by MC. Thus, IKK may serve as a new target for the regulation of MC function in allergy.     

Stimulation of TNFα expression by hyperosmotic stress

Hyperosmotic stress is known to induce apoptotic cell death, an effect previously attributed to seemingly ligand-independent clustering of tumour necrosis factor alpha (TNF alpha) receptors. An alternative explanation for the clustering of TNF alpha receptors may be stimulation of TNF alpha production, with subsequent autocrine or paracrine stimulation of the receptors. The present study was performed to test for an effect of exposure to hyperosmotic extracellular fluid on cellular TNF alpha production. In both the macrophage cell line U937 and the B lymphocyte cell line LCL721, an increase of extracellular osmolarity to 500 mosmol/l indeed increased TNF alpha expression, an effect reversed by the p38 kinase inhibitor SB203580. In both cell types hyperosmotic stress triggered apoptosis, which in U937 cells was significantly inhibited by neutralizing antibodies against TNF alpha and by SB203580 and was similarly elicited by exogenous addition of TNF alpha. In contrast, osmotically induced apoptosis of LCL721 cells was only slightly blunted by anti-TNF alpha antibodies and rather increased by SB203580. In conclusion, through activation of p38 kinase hyperosmotic stress stimulates the expression of TNF alpha which at least in U937 macrophages may participate in the triggering of subsequent apoptotic cell death. However, the observations in LCL721 cells point to other, TNF alpha-independent, mechanisms mediating apoptotic cell death following an excessive increase of extracellular osmolarity.     

Circulating factors in sera or peripheral blood mononuclear cells in patients with membranous nephropathy or diabetic nephropathy.

In order to investigate the status of some circulating factors in nephrotic syndrome, we examined the secretion of monocyte chemotactic peptide (MCP)-1, tumor necrosis factor (TNF) alpha or fibronectin in sera or by peripheral blood mononuclear cells (PBMC) from patients with membranous nephropathy (MN), diabetic nephropathy (DN) or minimal change disease (MCD). Also the effects of PBMC or sera on human mesangial cells (MC) were evaluated. Serum TNF alpha levels were higher in patients with MN than in controls, but PBMC exhibited no differences in TNF alpha production between patients and controls. Serum fibronectin levels were higher in patients with MN than in controls. PBMC from diabetic patients with or without nephropathy produced more MCP-1 than cells from controls. When MC were cultured with PBMC supernatants from patients, TNF alpha levels in PBMC supernatants correlated with production of MCP-1 or fibronectin by MC. PBMC supernatants obtained from patients with MCD and MN decreased MCP-1 production by MC, but did not affect thymidine incorporation or fibronectin production by MC. Sera obtained from patients with DN and MCD reduced thymidine incorporation in MC. In summary, serum TNF alpha or fibronectin levels were increased in patients with MN that is known to progress to renal failure. MCP-1 Production was increased by PBMC obtained from diabetic patients with or without nephropathy. Also TNF alpha production by PBMC in individual patients may affect the pathophysiology of their MC.     

Activated T-lymphocytes induce growth inhibition and prostaglandin E2 release from syngeneic glomerular mesangial cells.

The concept of an active role of T lymphocytes in the initiation and development of autoimmune glomerulonephritis has gradually evolved from recent investigations. In the present study we started in a murine coculture system to directly examine cellular interactions of intrinsic glomerular mesangial cells (MC) and syngeneic T lymphocytes. Lymph node lymphocytes and, moreover, cloned T helper cells specifically affected syngeneic proliferating MC, causing growth inhibition and prostaglandin E2 (PGE2) release. The T cell specificity of mesangial cell responses was confirmed by demonstrating (i) that MC cocultured with other cell types showed no reaction and (ii) that additional activation of T lymphocytes by IL-2 or concanavalin A significantly enhanced the MC responses. Subsequently, we confirmed the presence of T cell factors in the supernatants responsible for the observed effects: interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF). Experiments with combinations of recombinant mouse IFN-gamma and human lymphotoxin or TNF-alpha showed that these lymphokines could substitute for the direct T lymphocyte effects causing a synergistic growth inhibition and PGE2 release from mouse MC. The observed lymphokine activities were not due to mesangiolysis as shown by neutral red uptake of MC. Pointing to the essential role of T helper cell-specific products, IFN-gamma antibodies abolished both the IFN-gamma and the combined IFN-gamma/TNF-alpha effect. Thus, our investigations with syngeneic MC-lymphocyte cocultures demonstrated that cultured MC specifically responded to T lymphocytes and their products.     

Endothelins regulate mediator production of rat tissue-cultured mucosal mast cells. Up-regulation of Th1 and inhibition of Th2 cytokines

Mast cells have been shown to produce endothelin-1 (ET-1) and to express ET receptors. Thus, we postulated that ETs modulate mast cell mediator production in an autocrine manner. Rat tissue-cultured mast cells (RCMC-1) were incubated with exogenous ET-1 or ET-3, and beta-hexosaminidase release and TNF, IL-4, IL-10, IL-12, IL-13, macrophage inflammatory protein-1alpha (MIP-1alpha), and nitric oxide (NO) production were investigated. ET-1 and -3 induced the release of beta-hexosaminidase and TNF and of mRNA expression. An antagonist of the ET(B) receptor subtype abrogated ET-stimulated TNF release, although ET(A) and ET(B) receptors have been identified by immunocytochemistry. It is interesting that ET-1 and ET-3 inhibited (25-30%) mRNA expression of Th2-type cytokines (IL-4, IL-10, and IL-13) and increased IL-12 release (39% and 41%, respectively) without affecting MIP-1alpha and NO production. Thus, our data suggest that ETs may play an important role in modulating the cytokine network by regulating Th1/Th2 cytokine production by mast cells.     

Effects of tumor necrosis factor and lymphotoxin on peripheral lymphoid tissue development

Previously, we have reported that neutralization of surface lymphotoxin (LT-alphabeta) in mice which expressed an LT-beta receptor-Fc fusion protein, driven by the cytomegalovirus promoter, resulted in an array of anatomic abnormalities. We now report that mice which express a tumor necrosis factor (TNF) receptor p60-Fc fusion protein (which neutralizes TNF and soluble LT-alpha3 activity) develop unique lymphoid abnormalities. Our data demonstrate that some aspects of peripheral lymphoid organ development require both surface LT-alphabeta and TNF interacting with their specific receptors. However, these related cytokines are also capable of signaling distinct developmental events. Splenic MAdCAM-1 expression, follicular dendritic cell localization and normal Peyer's patch development all require both surface LT-alphabeta and TNF activity. Marginal zone formation and splenic B cell localization primarily require surface LT-alphabeta-LT-beta receptor interactions. Primary follicle formation was dependent upon TNF receptor(s) engagement. Interestingly spleen, lymph nodes and Peyer's patches from TNF receptor p60-Fc-expressing mice all develop different abnormalities, suggesting distinct pathways of development in these lymphoid organs. Thymus development appears to be independent of these signaling pathways. These results demonstrate that TNF and LT are crucial for normal peripheral, but not central lymphoid organ development.      

Interferon-γ regulates the interaction of RBL-2H3 cells with fibronectin through production of nitric oxide

Interferon-gamma (IFN-gamma) is an important regulatory cytokine in cell proliferation, differentiation, adhesion, mediator release, and gene induction. This diversity of effector roles is achieved by a variety of incompletely understood mechanisms. In the mast cell (MC), IFN-gamma downregulates mediator synthesis and secretion. The present study demonstrates and characterizes for the first time IFN-gamma inhibition of adhesion of the MC analogue RBL-2H3 to the extracellular matrix protein fibronectin (FN). Inhibition requires preincubation of the cells with IFN-gamma for 20 hr, and is statistically significant at 100 U/ml IFN-gamma. Flow cytometry indicates that cell surface expression of very late antigen-4 (VLA-4), VLA-5, and the vitronectin receptor (VNR) remain constant following IFN-gamma treatment, indicating the inhibitory effect of IFN-gamma on adhesion to FN is not achieved through a reduction in integrin receptors for FN. Fluorescent labelling with Texas red phalloidin demonstrated rearrangement of the actin cytoskeleton in response to IFN-gamma was not significant. The tyrosine phosphatase inhibitor vanadate, and the nitric oxide (NO) synthase inhibitor L-NAME, reduced the IFN-gamma effect on adhesion to FN by 62 and 70%, respectively, demonstrating that the IFN-gamma effect is dependent upon the production of NO, potentially though a tyrosine phosphatase dependent mechanism. The NO donors sodium nitroprusside and S-nitrosoglutathione mimicked the effect of IFN-gamma. Thus, following stimulation with IFN-gamma, NO plays an autocrine role in the MC, and is able to modulate integrin function. This adds to the pathways NO is able to inhibit in the mast cell, shows that endogenous NO is able to inhibit these pathways, and suggests NO is impinging upon an element common to many signalling mechanisms in the MC.