TNF and MAP kinase signalling pathways

The binding of tumour necrosis factor α (TNFα) to cell surface receptors engages multiple signal transduction pathways, including three groups of mitogen-activated protein (MAP) kinases: extracellular-signal-regulated kinases (ERKs); the cJun NH₂-terminal kinases (JNKs); and the p38 MAP kinases. These MAP kinase signalling pathways induce a secondary response by increasing the expression of several inflammatory cytokines (including TNFα) that contribute to the biological activity of TNFα. MAP kinases therefore function both upstream and down-stream of signalling by TNFα receptors. Here we review mechanisms that mediate these actions of MAP kinases during the response to TNFα.     

p38 MAP kinase regulates RANTES production by TNF-alpha-stimulated human pulmonary vascular endothelial cells

BACKGROUND: RANTES plays an important role in the production of allergic inflammation of the airway through its chemotactic activity for eosinophils. However, the intracellular signal regulating RANTES expression in human pulmonary vascular endothelial cells has not been determined. In the present study, therefore, we examined the role of p38 mitogen-activated protein (MAP) kinase in RANTES production by tumor necrosis factor (TNF)-alpha-stimulated pulmonary vascular endothelial cells in order to clarify the signal transduction pathway regulating RANTES production by pulmonary vascular endothelial cells. METHODS: We examined p38 MAP kinase activation, and the effect of SB 203580, as the specific inhibitor for p38 MAP kinase, on p38 MAP kinase activity and RANTES production by TNF-alpha-stimulated human pulmonary vascular endothelial cells. RESULTS: The results showed that TNF-alpha induced RANTES production and p38 MAP kinase activity in human pulmonary vascular endothelial cells. Abrogation of p38 MAP kinase activity by SB 203580 repressed TNF-alpha-induced p38 MAP kinase activity and RANTES production. CONCLUSIONS: These results indicate that p38 MAP kinase plays an important role in the TNF-alpha-activated signaling pathway which regulates RANTES production by human pulmonary vascular endothelial cells.     

Regulation by intracellular glutathione of TNF-alpha-induced p38 MAP kinase activation and RANTES production by human pulmonary vascular endothelial cells

BACKGROUND: We have previously shown that p38 mitogen-activated protein (MAP) kinase regulates tumor necrosis factor-alpha (TNF-alpha)-induced RANTES production by human pulmonary vascular endothelial cells, and that sensitivity to TNF-alpha is inversely correlated with cellular reduction and oxidation (redox) state. However, a regulatory role of intracellular glutathione (GSH) in TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production has not been determined. In the present study, therefore, we extended our previous studies and focused on redox regulation on p38 MAP kinase activation. METHODS: Human pulmonary vascular endothelial cells were exposed to N-acetylcysteine (NAC) or buthionine sulfoximine (BSO), and then TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production were determined. RESULTS: The results showed that 1) NAC attenuated TNF-alpha-induced p38MAP kinase activation and RANTES production 2) SB 203580 as the specific inhibitor of p38 MAP kinase activity attenuated TNF-alpha-induced RANTES production 3) BSO facilitated TNF-alpha-induced p38 MAP kinase activation and RANTES production 4) SB 203580 attenuated BSO-mediated facilitation of TNF-alpha-induced RANTES production. CONCLUSIONS: These results indicated that TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production by human pulmonary vascular endothelial cells are inversely regulated by intracellular GSH levels.     

Intracellular glutathione regulates tumour necrosis factor-α-induced p38 MAP kinase activation and RANTES production by human bronchial epithelial cells

BACKGROUND: RANTES plays an important role in the production of allergic inflammation of the airway through its chemotactic activity for eosinophils. The cellular reduction and oxidation (redox) changes are involved in the activation of p38 mitogen-activated protein (MAP) kinase and the induction of cytokine expression. It has previously been shown that tumour necrosis factor (TNF)-MA activates p38 mitogen-activated protein (MAP) kinase to produce cytokine, including RANTES, that N-acetylcysteine (NAC) attenuates cytokine production by human bronchial epithelial cells (BECs), and that sensitivity to TNFalpha is inversely correlated with cellular redox state. However, a role of cellular redox regulated by intracellular glutathione (GSH) in TNFalpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production by human BECs has not been determined. OBJECTIVE: Human BECs were exposed to NAC or buthionine sulfoximine (BSO). TNFalpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production by human BECs were then examined in order to clarify these issues. RESULTS: The results showed that: NAC attenuated TNFalpha-induced p38 MAP kinase activation and RANTES production; SB 203580 as the specific inhibitor of p38 MAP kinase activity attenuated TNF-alpha-induced RANTES production; BSO facilitated TNF-alpha-induced p38 MAP kinase activation and RANTES production; SB 203580 attenuated BSO-mediated facilitation of TNF-alpha-induced RANTES production; and the intracellular GSH increased in NAC-treated cells, whereas the intracellular GSH was reduced in BSO-treated cells. CONCLUSIONS: These results indicate that cellular redox regulated by GSH is critical for TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production by human BECs.     

p38蛋白激酶对支气管哮喘大鼠Th2类细胞因子表达的调控

目的：探讨p38蛋白激酶（p38 MAPK）对支气管哮喘大鼠Th2类细胞因子IL-4、IL-5表达的调控作用。 方法： 分离培养正常和哮喘大鼠外周血T淋巴细胞。采用蛋白质印迹方法检测细胞核蛋白p38蛋白激酶，采用原位分子杂交方法检测IL-4、IL-5的mRNA，采用ELISA方法检测上清液中IL-4、IL-5的蛋白质浓度。 结果： 哮喘对照组T淋巴细胞核蛋白p38蛋白激酶、IL-4、IL-5的mRNA表达阳性细胞数百分比以及上清液中IL-4、IL-5的蛋白质浓度均显著高于正常对照组（P<0.01）。加入递增浓度的SB203580处理后，上述指标均较哮喘对照组显著降低（P<0.05）。T淋巴细胞核蛋白p38蛋白激酶含量与IL-4、IL-5 mRNA原位分子杂交染色阳性细胞的百分比均呈显著正相关（r=0.71、0.63，P<0.01），与培养上清液中IL-4、IL-5蛋白质含量之间亦均呈显著正相关（r=0.68、0.57，P<0.01）。 结论： p38蛋白激酶在转录和翻译水平调控支气管哮喘IL-4和IL-5的表达。p38蛋白激酶抑制剂可望成为临床治疗哮喘的新途径。     

泌乳素促进类风湿关节炎患者外周血单个核细胞分泌白细胞介素-6

目的:探讨类风湿关节炎(RA)患者血清泌乳素(PRL)水平与疾病活动程度的关系,以及PRL促进外周血单个核细胞(PBMCs)分泌白细胞介素-6(IL-6)的机制。方法:收集我院2015年3月至9月40例初治RA患者临床及实验室资料。采用化学发光免疫分析法(CLIA)检测血清PRL水平,ELISA检测IL-6水平,RT-q PCR检测泌乳素受体(PRLR)mRNA的表达,Western blot法检测MAPK通路相关蛋白p-p38的蛋白水平。结果:RA患者血清PRL水平明显升高(P0.01),活动期RA患者PRL水平明显高于非活动期RA患者(P0.01)。PRL水平与DAS28评分、ESR和CRP呈正相关(P0.01)。RA患者PBMCs中PRLR水平明显升高(P0.01)。PRL可诱导PBMCs分泌IL-6,siRNA沉默PRLR或采用MAPK通路抑制剂可抑制IL-6的产生。结论:RA患者血清PRL升高与DAS28评分、ESR和CRP呈正相关,PRL可作为预测RA严重程度的指标。PRL通过与PRLR相互作用,激活p38 MAPK通路,从而促进IL-6分泌。     

LPS影响p38蛋白在单核细胞内定位

目的： 探讨 LPS作用下p38蛋白激酶激活的动力学特点及其在细胞内超微结构中的定位。方法： 应用激酶活性测定、胶体金标记的免疫电镜技术观察LPS刺激前后p38蛋白激酶的动力学特点及在单核细胞株Raw264.7中的分布特征。结果： 动力学检测结果显示，LPS作用后15 min，p38磷酸化活性明显升高，30 min达到高峰，2 h达基线水平；p38在LPS浓度为100 μg/L时达最大激活效应。超微定位结果显示，未受刺激的及EGF刺激的细胞，p38在胞浆和胞核中金颗粒呈弥散性分布，金颗粒弥散在细胞的各个部分，如细胞质中线粒体、内质网、溶酶体；单核细胞株受到LPS刺激后，细胞核区的金颗粒明显增多，而胞浆区域的金颗粒显著减少。结论： 单核细胞株Raw264.7受LPS刺激后，其p38蛋白激酶由胞浆移位到胞核。     

糖皮质激素通过抑制p38MAPK信号通路缓解大鼠内毒素性急性肺损伤

目的： 研究糖皮质激素对内毒素导致的急性肺损伤的影响及作用机制。方法: 成年雄性SD大鼠被随机分为6组：对照组（control 组,n=6）;LPS组（n=24）;地塞米松＋LPS组（Dex＋LPS组,n=24）;糖皮质激素受体拮抗剂RU486组（RU486组,n=6）;RU486+LPS组（n=24）以及RU486＋Dex＋LPS组（n=24）。除对照组和RU486组外,其余4组在注射LPS后1、3、6、12 h又被分为4个亚组。分别检测各组大鼠支气管肺泡灌洗液（BALF）中TNF-α和IL-6的浓度,肺组织的病理变化,以及肺组织中p38MAPK的活化状态和MKP-1的表达情况。另外又分别比较了LPS组与RU486＋LPS组、Dex＋LPS组与RU486＋Dex＋LPS组大鼠48 h的死亡率。结果: 注射LPS后,BALF中TNF-α 和IL-6的浓度明显升高（P<0.05）,HE染色显示肺组织内广泛炎症反应。而这些现象在应用RU486后变得更加严重,并且RU486＋LPS组的死亡率也明显高于LPS组(P<0.05)。地塞米松能明显缓解LPS导致的肺损伤,糖皮质激素受体(GR)参与此作用。另外,在注射LPS后,肺组织中磷酸化的p38MAPK的表达明显升高,而MKP-1的表达则明显受到抑制。地塞米松能显著降低p38MAPK的磷酸化,这一作用也是GR依赖的。结论: 糖皮质激素活化GR诱导肺组织中MKP-1的表达,进而抑制p38MAPK的活化,从而发挥其抗炎作用,缓解LPS诱导的肺损伤。     

Elevated TNF receptor plasma concentrations in patients with rheumatoid arthritis

Summary Two types of tumor necrosis factor receptors have been characterized, both capable of transmitting the signal and exerting the biological functions of TNF and lymphotoxin. We measured the plasma concentrations of two types of TNF binding proteins (sTNFR-A and sTNFR-B) in patients with rheumatoid arthritis (RA) and spondylarthropathies (SpA) using an enzyme-linked binding assay. In normal controls (n = 43), mean plasma concentrations were 1030 ± 55 and 1461 ±59 pg/ml for sTNFR types A and B, respectively. In 67 patients with moderate RA, mean levels were 1422 ± 82 pg/ml (type A) and 2088 ± 109 pg/ml (type B); in 34 patients with severe RA, 2588 ±279 pg/ml and 4494 ± 550 pg/ml, respectively, were measured (P < 0.0001 compared to normal controls). Concentrations of both type A and type B sTNFR were highly correlated in severe RA (R² = 0.7) but not in SpA or normal controls. T lymphocytes in synovial fluid of patients with RA expressed predominantly type A TNF receptors on their surface; in some patients a weaker expression of type B receptors was also detectable. Soluble TNF binding proteins in patients with RA were able to neutralize TNF in a cytotoxiity assay, demonstrating their ability to act as TNF-inhibiting factors. We conclude that both types of TNF receptors are parameters of disease activity in RA and may also act as TNF antagonists.Abbreviations TNF tumor necrosis factor - TNFR tumor necrosis factor receptor - sTNFR soluble tumor necrosis factor receptor - RA rheumatoid arthritis - SpA Spondylarthropathy - ELIBA Enzyme linked binding assay - mAb monoclonal antibody     

p38 MAP kinase regulates TNFα-, IL-1α- and PAF-induced RANTES and GM-CSF production by human bronchial epithelial cells

BACKGROUND: RANTES and granulocyte macrophage-colony stimulating factor (GM-CSF) play an important role in the production of allergic inflammation of the airway through their chemotactic activity for eosinophils. Recent studies have indicated that p38 mitogen-activated protein (MAP) kinase regulates cytokine expression in various cells; however, the role of p38 MAP kinase in RANTES and GM-CSF production in human bronchial epithelial cells (BECs) has not yet been determined. OBJECTIVE: In the present study, we examined serine phosphorylation of MKK3 and MKK6 which is the upstream regulator of p38 MAP kinase and p38 MAP kinase activation in tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha and platelet-activating factor (PAF)-stimulated BECs and the effect of SB 203580 as the specific inhibitor for p38 MAP kinase activity on RANTES and GM-CSF expression in order to clarify the intracellular signal regulating RANTES and GM-CSF production by human BECs. RESULTS: The results showed that TNF alpha, IL-1 alpha and PAF induced serine phosphorylation of MKK3 and MKK6, and p38 MAP kinase activation in BECs. SB 203580 inhibited p38 MAP kinase activity and RANTES and GM-CSF production by TNF alpha-, IL-1 alpha- or PAF-stimulated human BECs. CONCLUSIONS: These results indicate that p38 MAP kinase plays an important role in TNF alpha-, IL-1 alpha- or PAF-activated signalling pathway which regulates RANTES and GM-CSF production by BECs and that the specific inhibitor for p38 MAP kinase activity might be useful for the treatment of allergic inflammation of the airway.     

Lactobacillus rhamnosus reduces CD8+T cell mediated inflammation in patients with rheumatoid arthritis

BackgroundThe T cells, components of adaptive immunity participate in immune pathology of the autoimmune inflammatory disorder called rheumatoid arthritis (RA). The presence of TLRs on the surface of the CD8⁺ T cells and their ability to recognize bacterial moieties adds to the inflammatory burden in case of RA. It has been reported that the gut microbiome is necessary for the crucial shift in the balance between proinflammatory and anti-inflammatory cytokines. The altered gut microbiome and the presence of TLRs emphasizes on the microbiome driven inflammatory responses in case of RA.MethodsEighty-nine RA patients participated in this study. Clinical variations like disease duration, number of actively inflamed joints, number and type of bone deformities, CRP, RF, Anti-CCP, ESR, DAS 28 score were recorded for each patient. Co-culture of CD8⁺T cells and bacteria has been performed with proper culture condition. TLRs and inflammatory mediators’ expression level were checked by both qPCR and flow cytometry analysis.ResultsWe observed in the suppression of pro-inflammatory molecules like Granzyme B and IFNƳ and expression of TLR2 in CD8 + T cells upon treatment with Lactobacillus rhamnosus (L. rhamnosus). Moreover, L. rhamnosus activated CD8⁺T cells such that they could induce FOXP3 expression in CD4⁺T cells thereby skewing T cell population towards a regulatory phenotype. On the contrary, TLR4 engagement on CD8⁺T cell by Escherichia coli (E.coli) increased in inflammatory responses following ERK activation.ConclusionsThus, we conclude that L. rhamnosus can effectively suppress CD8⁺T cell mediated inflammation by a simultaneous decrease of Th1 cells that may potentiate better treatment modalities for RA.     

Aberrant integrin activation induces p38 MAPK phosphorylation resulting in suppressed Fas-mediated apoptosis in T cells: Implications for rheumatoid arthritis

Delayed Fas-mediated apoptosis in T cells is associated with inflammatory diseases including rheumatoid arthritis (RA). CD3⁺ T cells in RA synovia expressed high amounts of phospho-p38 MAPK. Exposure to RA synovial fluid or soluble collagen, a degradation product of extracellular matrix abundant in RA synovium, induced the phosphorylation of p38 MAPK in Jurkat T cells accompanied by resistance against Fas-mediated apoptosis. Blocking β1 integrin by antibody diminished this effect. In addition, ectopic expression of auto-activated β1 integrin variant in T cells profoundly induced the phosphorylation of p38 MAPK. Suppression of p38 MAPK sensitized T cells to Fas-mediated apoptosis and increased caspase-8 and caspase-3 cleavage. A physical interaction of p38 MAPK and caspase-8 was demonstrated by using confocal microscopic imaging and co-immunoprecipitation assay. RA synovial fluid markedly increased the formation of phospho-p38 MAPK/caspase-8 complex in Jurkat T cells. In conclusion, abnormal activation of p38 MAPK to prevent Fas-mediated apoptosis may represent a common survival mechanism of RA synovial T cells contributing to the persistent inflammation of affected synovium.     

p38 MAPK及基质金属蛋白酶在心力衰竭病人心肌重构中的意义

目的: 探讨不同程度心力衰竭病人心肌组织丝裂素活化蛋白激酶（p38 MAPK）、基质金属蛋白酶家族（MMP-2、3、9）、细胞外基质（ECM）纤连蛋白（FN）表达与心肌重构的关系。方法: 选择因二尖瓣关闭不全心脏病接受二尖瓣置换术的心力衰竭病人39例，正常对照8例来自意外伤亡的器官捐献者。光镜检查心肌组织病理变化;免疫沉淀法检测心肌组织p38 MAPK磷酸化，及p38 MAPK、MMP-2、3、9蛋白表达; 免疫荧光法检查心肌组织FN的分布。结果: 瓣膜病所致心力衰竭病人心肌组织呈典型的心肌重构病理改变。心力衰竭组心肌 p38 MAPK 磷酸化明显强于对照组（P<0.05），随心功能恶化，其表达逐渐增加（P<0.05或P<0.01）。 心力衰竭组心肌组织MMP-2、3、9蛋白表达明显强于正常对照组，各心力衰竭组与正常对照组相比差异显著（P<0.05或P<0.01）; 相反，心力衰竭组心肌组织FN蛋白表达明显弱于正常组，各心力衰竭组与正常对照组相比差异显著（P<0.05或P<0.01）。结论: 心力衰竭病人通过激活p38 MAPK诱导心肌细胞肥大、坏死，通过MMP-2、3、9表达量的增高降解心肌细胞外基质FN，共同参与心肌重构的病理过程而恶化心功能。     

P38激酶介导胆红素诱导的SHSY5Y细胞凋亡

观察P38激酶在胆红素诱导神经细胞凋亡中的作用 ,探讨参与此过程的信号转导通路。将胆红素 (2× 1 0 -2g L)直接作用于体外培养的人神经母细胞瘤细胞系SHSY5Y ,利用Hochest332 58染色在荧光显微镜下观察细胞核的形态是否发生凋亡样改变 ;用P38激酶抑制剂SB2 0 3580预处理细胞后 ,在倒置光显微镜下观察胆红素作用不同时间细胞形态的变化及存活情况 ;利用流式细胞仪检测细胞凋亡率的改变。结果显示SHSY5Y细胞经胆红素作用后细胞核出现典型的凋亡样改变 ;细胞经SB2 0 3580预处理 1h ,胆红素作用 3h后凋亡率为 (1 2 1± 2 4) % ,对照组为(1 9 4± 2 7) % ;4h后凋亡率为 (39 3± 4 8) % ,对照组为 (66 2± 5 2 ) % ,差异有显著性意义 (P <0 0 1 )。提示P38激酶参与了胆红素诱导SHSY5Y细胞凋亡的信号转导过程     

T-cell expression of CD91 - a marker of unresponsiveness to anti-TNF therapy in rheumatoid arthritis

The aim of this study was to investigate the expression of thrombospondin-1 (TSP-1) and its receptors, lipoprotein receptor-related protein/cluster of differentiation (CD)91, calreticulin (CRT), and CD47, on T cells and monocytes from patients with rheumatoid arthritis (RA) treated with anti-tumor necrosis factor (TNF) therapy. The surface expression of CD91 and associated components on CD3- and CD14-positive cells was examined using flow cytometry in 12 patients with established RA before and after beginning therapy and compared with that of 9 healthy controls and 12 patients with early RA treated with conventional therapies. CD3-positive cells from anti-TNF non-responders showed significantly greater expression of CD91 expression than those from responders (p<0.05) after 6 weeks and when all measurements were pooled (p<0.001). CD91 expression on CD3-positive cells from non-responders to other therapies was at the same level as in healthy controls. In contrast, CD14-positive cells showed no differences in CD91 expression between patients and controls or between responders and non-responders to anti-TNF therapy. The expression of TSP-1, CRT, and CD47 showed no differences between responders and non-responders. The results suggest T-lymphocyte expression of CD91 to be a biomarker that signifies unresponsiveness to anti-TNF therapy in patients with RA and may be used to identify potential responders and non-responders.     

Increased Tim-3 expression on peripheral lymphocytes from patients with rheumatoid arthritis negatively correlates with disease activity

Tim-3 has been reported as an important regulatory molecule and plays a pivotal role in several autoimmunity diseases. Here, we demonstrated the increased expression of Tim-3 on peripheral CD4⁺ T, CD8⁺ T, NKT cells and monocytes from RA patients compared to those from healthy controls. Percentage of Tim-3⁺ cells in peripheral blood mononuclear cells (PBMCs) showed an inverse correlation with disease activity score 28 (DAS28) and plasma TNF-α level. Similar negative correlations were found between disease activity and Tim-3 levels on CD4⁺ T, CD8⁺ T and NKT cells. Consistently, Tim-3 expression on CD3⁺ T cells was further increased in patients with disease remission after treatment. Tim-3 expression on CD8⁺ T and NKT cells negatively correlates with plasma TNF-α. Our results suggest that Tim-3 might participate in the proceeding of RA by its negative regulation on various T cell subsets. Tim-3 might be a potential new marker for assessing severity of RA.     

p38MAPK信号通路在钙调磷酸酶促心肌凋亡中的作用

目的：探讨钙调磷酸酶（CaN）在缺氧/复氧和肾上腺素能刺激诱导心肌凋亡中的作用及p38 MAPK在CaN调节心肌凋亡中的作用。方法:采用体外培养新生Wistar大鼠心肌缺氧/复氧（H/R）模型，模拟在体缺血再灌注损伤，将心肌细胞随机分为3组：正常对照组（N组）、H/R+异丙基肾上腺素组（Ao组）、H/R+异丙基肾上腺素+环孢菌素A组（A1组）。采用流式细胞仪检测细胞凋亡，RT-PCR 检测CaN mRNA的表达， Western 免疫印迹法检测CaN及p38MAPK、p-p38 MAPK蛋白表达。结果:H/R和异丙基肾上腺素（Iso）共同作用心肌后，心肌细胞凋亡率明显增加，给予CaN抑制剂环孢菌素（CsA）后，细胞凋亡率显著少于干预前(P<0.05)。H/R和Iso共同作用下，心肌CaN mRNA及蛋白表达水平均明显上调，同时p38 MAPK的活化状态p-p38 MAPK蛋白表达也显著增加，CsA干预后，CaN表达并无明显变化，但p-p38 MAPK蛋白表达显著减少(P<0.05)。结论:CaN 促进缺氧/复氧和肾上腺素能刺激诱导心肌细胞凋亡，可通过活化p38 MAPK而发挥促凋亡的作用。