The lectin jacalin induces phosphorylation of ERK and JNK in CD4+ T cells

The CD4 molecule plays an essential role in mediating the transduction of intracellular signals by functioning as a coreceptor for the complex T cell receptor/CD3 and also acts as the primary receptor for human immunodeficiency virus (HIV). Several authors have shown evidence that jacalin, a plant lectin, binds to CD4 and inhibits in vitro HIV infection. We analyzed jacalin-induced intracellular signaling events in CD4(+) T cells and have shown that cell activation resulted in tyrosine phosphorylation of intracellular substrates p56(lck), p59(fyn), ZAP-70, p95 (vav), phospholipase C-gamma1, and ras activation, as assessed by conversion of ras guanosine 5'-diphosphate to ras guanosine 5'-triphosphate. We further examined extracellular regulated kinase (ERK) and c-jun NH(2)-terminal kinase (JNK) phosphorylation following stimulation with jacalin. The data indicate that the kinetics of JNK phosphorylation is delayed. Optimum phosphorylation of ERK2 was observed by 10 min, and that of JNK was observed by 30 min. Pretreatment with gp120 followed by stimulation with jacalin resulted in marked inhibition of all of the aforementioned intracellular events. The data presented here provide insight into the intracellular signaling events associated with the CD4 molecule-jacalin-gp120 interactions and HIV-induced CD4(+) T cell anergy. Jacalin may be used as a possible tool for the study of CD4-mediated signal transduction and HIV-impaired CD4(+) T cell activation.     

TRPV4 induces apoptosis via p38 MAPK in human lung cancer cells

Lung cancer is one of the most common cancers worldwide. TRPV4 belongs to the ‘transient receptor potential'' (TRP) superfamily. It has been identified to profoundly affect a variety of physiological processes, including nociception, heat sensation, and inflammation. Unlike other TRP superfamily channels, its roles in cancers are unknown. Here, we elucidated the effects of TRPV4 and molecular mechanisms in human lung cancer cells. The levels of TRPV4 were detected in human lung cancer tissues and the paired paracarcinoma tissues by real-time PCR and western blotting analysis. The proliferation of human lung cancer cells was determined by MTT assay. Cell apoptosis was determined by FACS assay. The results demonstrated that low levels of TRPV4 were detected in clinical lung carcinoma specimens. Over-expression of TRPV4 induced cell death and inhibited cell proliferation and migration in A549 cells and H460 cells. Moreover, over-expression of TRPV4 enhanced the activation of p38 MAPK signal pathway. Inhibition of p38 MAPK abolished the effects of TRPV4 on cell proliferation, apoptosis, and migration in A549 cells. Collectively, our findings indicated that TRPV4 induced apoptosis via p38 MAPK in human lung cancer cells and suggested that TRPV4 was a potential target for therapy of human lung cancers.     

血管紧张素Ⅱ激活巨噬细胞株p38MAPK信号通路并诱导其增殖

目的：探讨血管紧张素Ⅱ(AngⅡ)激活巨噬细胞p38MAVK信号通路的模式变化及其对巨噬细胞株增殖的影响。方法：用Western blot测定细胞p38MAPK磷酸化表达；用细胞免疫组化观察细胞p38MAPK激活后核移位；用MTT法观察细胞增殖。结果：AngⅡ(1μmol／L)可诱导RAW264．7细胞p38MAVK磷酸化表达，15～30分钟达到高峰，随时间呈峰形变化。AngⅡ呈剂量依赖性诱导RAW264．7细胞p38MAPK磷酸化。p38MAPK特异性抑制剂SB202190可显著抑制RAW264．7细胞p38MAVK磷酸化，并呈剂量依赖性。AngⅡ可诱导RAW264．7细胞增殖，SB202190可显著抑制AngⅡ诱导的RAW264．7细胞增殖。结论：AngⅡ可激活RAW264．7巨噬细胞株p38MAPK信号通路，并通过p38MAPK信号通路调控RAW264．7细胞增殖。     

抗CD28单抗和IL-15对CIK增殖和杀肿瘤作用的研究

CIK是肿瘤过继性细胞免疫治疗中的免疫效应细胞。为使CIK在实验室里能被更有效地诱导增殖并赋予其更强的杀肿瘤效应,我们在CIK常规培养环境中加入抗CD28单抗和IL-15,探讨抗CD28单抗和IL-15对CIK增殖和杀肿瘤效应。取人外周血单个核细胞(PBMC),预先以常规方法诱导CIK,然后加入抗CD28单抗和IL-15与CIK共培养。用全自动五分类血液分析仪计数CIK增殖率;用流式细胞术测定CIK中粒酶B、穿孔素和CD107a等分子的表达率;用ELISA方法检测CIK分泌IL-10、IL-12、INF-γ和TNF-α水平;用乳酸脱氢酶释放法测定CIK对人肺癌细胞株(A549)、乳腺腺癌细胞株(MFC-7)和人黑素瘤细胞株(HME1)的杀伤活性。PBMC经常规CIK诱导培养以后再加入抗CD28单抗和IL-15与对照组比较,前者细胞增殖率明显增强(P<0.05);在CIK培养体系中加入抗CD28单抗和IL-15可促进颗粒酶B、穿孔素和CD107a等分子的表达率进一步增强(P<0.05);加入抗CD28单抗和IL-15,培养8d后CIK对A549、MFC-7和HME1细胞杀伤活性分别为82.2%、59.3%和70.6%,与对照组(分别为60.9%、49.6%和48.4%)相比差异有统计学意义(P<0.05);在培养体系中加入抗CD28单抗和IL-15,培养8d后其细胞因子IFN-γ、TNF-α分泌水平显著高于对照组(P<0.05),组间IL-10和IL-12的分泌量未见显著差异(P>0.05)。实验说明在CIK培养体系中加入抗CD28单抗和IL-15可增加CIK增殖率并提高其抗肿瘤效应。     

Pathogenic monoclonal antibody against desmoglein 3 augments desmoglein 3 and p38 MAPK phosphorylation in human squamous carcinoma cell line

Pemphigus vulgaris is an autoimmune blistering disease characterized by cell-cell detachment of epidermal cells. Autoantibody against desmoglein (Dsg) 3, a transmembrane glycoprotein that mediates the association of desmosomes, plays a major role in blistering in pemphigus vulgaris (PV). The mechanisms of autoantibody-induced acantholysis have not been clarified. We previously reported that PV-IgG induces phosphorylation of Dsg3, decreases Dsg3 on the cell surface and forms Dsg3-depleted desmosomes in cultured keratinocytes, and that cell treatment with a potent pathogenic monoclonal antibody against Dsg3 (AK23 mAb) decreases the amount of Dsg3 in cultured keratinocytes. Although the precise mechanisms remain unclear, we have proposed the involvement of intracellular signal transduction resulting from the binding of autoantibodies to Dsg3. In this study, we examined whether AK23 mAb augments phosphorylation of Dsg3 and p38 mitogen-activating protein kinase (MAPK) in a human squamous cell line, DJM-1 cells. AK23 mAb increased serine phosphorylation of Dsg3 and augmented activation levels of p38 MAPK. These results indicate that antibodies bind to Dsg3, but not other antigens, in the IgG fraction and can induce activation of signal transduction.     

不可分型流感嗜血杆菌通过p38 MAPK和NF-κB依赖的方式上调IL-8的表达

目的：探讨不可分型流感嗜血杆菌（NTHi）致肺泡上皮细胞炎症反应的分子机制。方法：A549肺泡上皮细胞株与NTHi（感染复数：10）共孵育15 min、30 min后收集细胞,用Western blotting检测p38丝裂原活化蛋白激酶（p38 MAPK）的磷酸化;4 h后用流式细胞仪检测胞内NF-κB p65亚单位的表达。预先用p38 MAPK抑制剂（SB203580）和NF-κB抑制剂（PDTC）与A549细胞共孵育1 h,然后加入NTHi,24 h后收集上清,以酶联免疫吸附法检测白介素8（IL-8）的水平。结果： NTHi能迅速地诱导p38 MAPK通路的磷酸化。 NTHi刺激4 h后A549细胞胞内NF-κB p65的表达较与未加细菌组明显增加（P<0.05）。NTHi刺激A549细胞24 h后上清中的IL-8较未加细菌组显著增加,差异显著（P<0.05）。与细菌攻击组比较,阻断p38 MAPK或NF-κB通路,能显著地降低A549细胞生成IL-8（P<0.05）。结论：NTHi 以p38 MAPK和NF-κB依赖的方式诱导肺泡上皮细胞的炎症反应。     

Pathogenic monoclonal antibody against desmoglein 3 augments desmoglein 3 and p38 MAPK phosphorylation in human squamous carcinoma cell line

Pemphigus vulgaris is an autoimmune blistering disease characterized by cell–cell detachment of epidermal cells. Autoantibody against desmoglein (Dsg) 3, a transmembrane glycoprotein that mediates the association of desmosomes, plays a major role in blistering in pemphigus vulgaris (PV). The mechanisms of autoantibody-induced acantholysis have not been clarified. We previously reported that PV-IgG induces phosphorylation of Dsg3, decreases Dsg3 on the cell surface and forms Dsg3-depleted desmosomes in cultured keratinocytes, and that cell treatment with a potent pathogenic monoclonal antibody against Dsg3 (AK23 mAb) decreases the amount of Dsg3 in cultured keratinocytes. Although the precise mechanisms remain unclear, we have proposed the involvement of intracellular signal transduction resulting from the binding of autoantibodies to Dsg3. In this study, we examined whether AK23 mAb augments phosphorylation of Dsg3 and p38 mitogen-activating protein kinase (MAPK) in a human squamous cell line, DJM-1 cells. AK23 mAb increased serine phosphorylation of Dsg3 and augmented activation levels of p38 MAPK. These results indicate that antibodies bind to Dsg3, but not other antigens, in the IgG fraction and can induce activation of signal transduction.     

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.     

Inducible IL-23p19 expression in human microglia via p38 MAPK and NF-kappaB signal pathways

Activated microglia can release a variety of proinflammatory cytokines that play a crucial role in the pathogenesis of multiple sclerosis (MS). IL-23, a novel proinflammatory cytokine, is required for the induction of experimental autoimmune encephalomyelitis. Previously we demonstrated that IL-23 is expressed in MS lesions and that microglia are one cellular source of IL-23 in MS patients. In the present study we investigated the inducible expression and regulation of p19, a key subunit of IL-23, in human microglia. We demonstrated the inducible expression of IL-23p19 by lipopolysaccharide-stimulated microglial cells. Using signaling pathway-specific inhibitors, we showed that blocking p38 MAP kinase or NF-kappaB signaling pathway significantly reduced p19 expression in microglia. The regulatory role of p38 MAP kinase in p19 expression was further confirmed by decreased expression in microglia transduced with dominant-negative p38. We concluded that the p38 MAP kinase and NF-kappaB signaling pathways play an important role in regulation of IL-23p19 expression on human microglia, and are thus potential therapeutic targets in the treatment of MS.     

Protease Allergens Induce the Expression of IL-25 via Erk and p38 MAPK Pathway

Allergic diseases, including asthma, are characterized by T helper type 2 (Th2) cell-mediated inflammations, coupled with tissue infiltration by eosinophils. In this study, we demonstrate that multiple protease allergens, including papain and DerP1, efficiently induce interleukin (IL)-25 and thymic stromal lymphopoietin (TSLP) gene expression, and this phenomenon is dependent on the protease activities of these allergens. The IL-25 cytokine level in bronchial alveolar lavage (BAL) was also profoundly and significantly increased after treatment with papain. Additionally, the levels of Th2 cytokines were significantly increased, as compared to those in the OVA-only treatment group. The various protease allergens triggered the expression of IL-25 and TSLP mRNA in mouse lung epithelial cells (MLE12) and primary mouse lung epithelial cells; these effects were inhibited by the deactivation of the protease activity of papain. The allergen papain activates the ErK and p38 MAP pathways; the inhibition of these pathways, but not the NFκB or PI-3 kinase pathways, impairs the induction of IL-25 and TSLP expression by proteases. In this study, we demonstrate that the protease allergens induce IL-25 and TSLP via the MAP kinase signal pathways, and their protease activities are essential to this pathway.     

ORF61 protein of Varicella-zoster virus influences JNK/SAPK and p38/MAPK phosphorylation

Recently, it was demonstrated that the Varicella-zoster virus (VZV) infection led to an activation of MAP kinases. The viral protein encoded by ORF61 is a major effector of JNK/SAPK and p38/MAPK phosphorylation. ORF61 shows homology to HSV-1 ICP0, a multifunctional protein that influences the activity of c-Jun in infected cells. Stable expression of ORF61 in a MeWo derived cell line gave rise to two specific effects: (i) a major decrease of VZV replication and (ii) a strongly elevated basal JNK/SAPK phosphorylation but a reduced p38/MAPK phosphorylation, which were both altered following infection. A dose-dependent inhibition of JNK/SAPK in MeWo/61 cells resulted in a step-by-step increase of VZV replication. These findings indicate (i) that ORF61 is responsible for the elevated JNK/SAPK phosphorylation and (ii) that the VZV replication and the JNK/SAPK phosphorylation are related inversely. Compared to MeWo cells, the basal phosphorylation of downstream targets c-Jun and ATF-2 was reduced following ORF61 expression but restored after infection. Subsequent cascades to induce inflammatory responses were activated insignificantly; cascades to activate apoptotic events also remained silent. These data point towards an important role of ORF61 in the fine-regulation of activation of the MAPK pathways and their downstream targets to optimize the availability of cellular factors involved in VZV gene expression.     

Infectious bursal disease virus infection induces macrophage activation via p38 MAPK and NF-kappaB pathways

In the present study, we show that infection with infectious bursal disease virus (IBDV) causes activation of macrophages, the key cells involved in inflammatory and immune-regulatory functions. Exposure of cultured spleen macrophages (SM) from SPF chickens to IBDV resulted in the production of nitric oxide (NO). In addition, there was upregulation of mRNA expression of inducible nitric oxide synthase (iNOS), IL-8 and cyclooxygenase-2 (COX-2). The signal transduction pathways involved in macrophage activation were examined. The role of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) was tested by using specific pharmacological inhibitors. Addition of p38 MAPK inhibitor, SB-203580 and NF-kappaB inhibitor Bay 11-7082, suppressed IBDV-induced NO production and mRNA expression of iNOS, IL-8 and COX-2. The results suggest that IBDV uses cellular signal transduction machinery, in particular the p38 MAPK and NF-kappaB pathways, to elicit macrophage activation. The increased production of NO, IL-8 and COX-2 by macrophages may contribute to bursa inflammatory responses commonly seen during the acute IBDV infection.     

Cytotoxic difference of T cells expanded with anti-CD3 monoclonal antibody in the presence and absence of anti-CD 28 monoclonal antibody

Bulk T cells can be expanded by CD3 stimulation alone (CD3-Ts) or by CD3/CD28 dual stimulation (CD3/CD28-Ts) of peripheral blood mononuclear cells (PBMC). However, few reports have described the difference of features between CD3-Ts and CD3/CD28-Ts. PBMC were stimulated with anti-CD3 monoclonal antibody (mAb) alone or co-stimulated with anti-CD3/CD28 mAbs immobilized on plastic plates, in the presence of rhIL-2 for 4 days, subsequently cultured in the presence of rhIL-2 with no antibody then analyzed. The expansion rate was significantly lower for CD3-Ts (965 + 510-fold, n=5) than CD3/CD28-Ts (2263 + 856-fold, n=5) (p<0.05). The CD4/CD8 ratio, the percentage of CD28(+) cell, and the percentage of T cells with no ability to generate intracytoplasmic interleukin-4 (IL-4) or interferon-gamma (IFN-gamma) were all significantly higher, but, phenotypically, memory cells were lower in CD3/CD28-Ts than in CD3-Ts. The levels of activity of both natural killer (NK) and lymphocyte-activated killer (LAK) cells were lower in CD3/CD28-Ts than CD3-Ts. In comparison to CD3-Ts, CD3/CD28-Ts showed impaired migration toward RANTES. In conclusion, T cells expanded with anti-CD3 and anti-CD28 mAbs differ from those expanded with anti-CD3 alone with proliferation, cytotoxicity, chemotaxis, and phenotype. These differences may exert profound influences on the therapeutic potential of output cells.     

Antimycotics suppress interleukin-4 and interleukin-5 production in anti-CD3 plus anti-CD28-stimulated T cells from patients with atopic dermatitis]

It is reported that antimycotic agents are effective for the treatment of patients with atopic dermatitis (AD). We studied in vitro effects of antimycotics on T helper-1 and T helper-2 cytokine production in anti-CD3 plus anti-CD28-stimulated T cells from AD patients and normal donors. The amounts of interleukin-4 (IL-4) and IL-5 secreted by anti-CD3/CD28-stimulated T cells were higher in AD patients than in normal donors. Azole derivatives, ketoconazole, itraconazole, miconazole and non-azole terbinafine hydrochloride and tolnaftate reduced IL-4 and IL-5 secretion without altering that of IFN-gamma and IL-2 in anti-CD3/CD28-stimulated T cells from both AD patients and normal donors. The azole derivatives were more inhibitory than non-azole antimycotics. These antimycotics reduced the anti-CD3/CD28-induced mRNA expression and promoter activities for IL-4 and IL-5. The cAMP analogue dibutyryl cAMP reversed the inhibitory effects of the antimycotics on IL-4 and IL-5 secretion, mRNA expression, and promoter activities. Anti-CD3/CD28 transiently (< or = 5 min) increased intracellular cAMP in T cells, and the increase was greater in AD patients than in normal donors. The increase of cAMP by anti-CD3/CD28 correlated with IL-4 and IL-5 secretion by anti-CD3/CD28. The transient cAMP increase was suppressed by antimycotics, and azole derivatives were more suppressive than non-azoles. Azole derivatives inhibited the activity of cAMP-synthesizing adenylate cyclase while terbinafine hydrochloride and tolnaftate enhanced the activity of cAMP-hydrolyzing cyclic nucleotide phosphodiesterase in AD and normal T cells. These results suggest that the antimycotics may suppress IL-4 and IL-5 production by reducing cAMP signal, and strengthen the concept of their potential use for the suppression of T helper-2-mediated allergic reactions.     

Nitration of p38 MAPK in the placenta: association of nitration with reduced catalytic activity of p38 MAPK in pre-eclampsia

Peroxynitrite, a potent pro-oxidant formed from the interaction of superoxide and nitric oxide, has been widely reported to be nitrating tyrosine residues in proteins resulting in the formation of nitrotyrosine. Biological nitration of tyrosine, a footprint of oxidative injury, has been found to occur in various pathological states including pre-eclampsia, a leading cause of maternal mortality and increased perinatal mortality. Oxidative stress is a major contributor to endothelial dysfunction in pre-eclampsia. Previously, we have demonstrated increased nitrotyrosine immunostaining in placental villous vascular endothelium, surrounding vascular smooth muscle and villous stroma from pre-eclamptic or diabetic pregnancies. Immunoprecipitation (IP) with antinitrotyrosine antibodies followed by immunoblot analysis identified increased nitration of phospho-p38 mitogen-activated protein kinase (MAPK) in the pre-eclamptic placenta. The catalytic activity of p38 MAPK and concentration of phospho-p38 MAPK was also found to be reduced in placentae from pre-eclamptic pregnancies. Comparison of peptide masses of a 42-kDa protein obtained by mass spectrometry with masses of a theoretical tryptic digest of p38 MAPK that was modified by phosphorylation and nitration identified the protein to be p38 MAPK.     

IL-18 enhances SCF production of melanoma cells by regulating ROI and p38 MAPK activity

It has been reported that interleukin-18 (IL-18) is secreted by B16 murine melanoma cells and that this endogenous IL-18 is involved in the immune escape of murine melanoma cells. The present study investigated whether interleukin (IL)-18 can regulate stem cell factor (SCF) expression, known to be associated with melanocyte proliferation, in B16F10 murine melanoma cells. SCF expression was examined by RT-PCR, intracellular FACS analysis, and ELISA in IL-18 antisense transfectants. Transfection with IL-18 antisense cDNA reduced SCF expression and the expression was enhanced by addition of exogenous IL-18. In addition, the effect of IL-18 was blocked by the antioxidant, N-acetyl-L-cysteine (NAC), indicating that IL-18 regulates ROI production, which is involved in SCF production. Furthermore, inhibitors of p38 mitogen-activated protein kinase (MAPK), such as SB203580, blocked enhanced SCF expression, indicating that p38 MAPK activity is required for IL-18-enhanced SCF production. Taken together, these results suggest that IL-18 plays a critical role as a regulatory factor of SCF expression via ROI and p38 MAPK activity in B16F10 murine melanoma cells.     

p38MAPK mediates IL-1-induced down-regulation of aggrecan gene expression in human chondrocytes

The pleiotropic cytokine interleukin 1 (IL-1) is considered to be the principal inducer of mediators of cartilage degradation in both, osteoarthritis (OA) and rheumatoid arthritis (RA). IL-1 activates numerous signaling pathways involved in cartilage destruction and dedifferentiation of chondrocytes. In this study, we analyzed expression and functional effects of IL-1 in human chondrocytes. We found an IL-1-induced reduction in the expression of the cartilage specific proteoglycan aggrecan as an indicator for the IL-1-mediated dedifferentiation of chondrocytes. To block the IL-1-induced signaling pathways specifically, we incubated human chondrocytes and cartilage explants with IL-1 in the presence of different signal transduction inhibitors and analyzed their effect on aggrecan mRNA expression and IL-6 secretion. IL-6 has been found to act synergistically in the IL-1-induced suppression of the proteoglycan synthesis in chondrocytes. Our results led to the identification of p38MAPK and/or PI3K/JNK as being crucial for IL-1-induced IL-6 secretion by chondrocytes. IL-1-induced down-regulation of aggrecan expression was found to be mediated by p38MAPK and/or ERK1/2. The identification and characterization of these signaling pathways will enable us to develop new modulation strategies for therapeutic use in inflammatory joint diseases.     

IL-18 together with anti-CD3 antibody induces human Th1 cells to produce Th1- and Th2-cytokines and IL-8

Although IL-18 was initially regarded as a factor that enhances IFN-gamma production from Th1 cells, later studies revealed its potential to induce Th2 cytokine production from T cells, NK cells and basophils/mast cells. Very recently, we demonstrated that passively transferred memory phenotype Th1 cells induce airway inflammation and hyperresponsiveness in a host mouse by production of Th1-, Th2-cytokines, GM-CSF and chemokines, when the transferred cells are stimulated in the host mice with nasally administered Ag and IL-18. Moreover, IL-18 is suggested to contribute to asthma exacerbation in human patients. Therefore, it is important to determine whether human Th1 cells also have the potential to produce these soluble factors when stimulated with anti-CD3 and IL-18 in vitro. Here we demonstrated that only Th1 cells, but not Th2 cells, produce IFN-gamma, IL-13, GM-CSF and IL-8 after stimulation with anti-CD3 and IL-18. Furthermore, highly purified IFN-gamma-producing Th1 cells have the same potential. Thus, human Th1 cells may become very harmful cells, when stimulated with Ag and IL-18 in vivo, and produce IFN-gamma, IL-13, GM-CSF and IL-8, which in combination might induce severe inflammation such as airway inflammation.