共信号分子CD40/CD40L质粒标准品的构建

背景:目前,对CD40/D40L的研究主要集中在可溶性和蛋白方面,对其在转录水平上的研究文献不多,可能与其无商品化质粒标准品有关.目的:构建检测CD40/CD40L mRNA表达水平差异的标准品.设计、时间及地点:单一样本实验,于2009-04在苏州大学附属第一医院检验科实验室完成.材料:取手术切除的子宫肌瘤组织,经患者知情同意.方法:以手术切除的子宫肌瘤组织细胞的总RNA为模板、Random 6mers为引物反转录合成cDNA,用该cDNA为模板PCR扩增人CD40/CD40L基因相应的cDNA片断,构建PMD-18-CD40/CD40L重组质粒,鉴定测序后,用荧光定量PCR制作标准曲线.主要观察指标:①RNA质量检测结果.②扩增的目的片段电泳结果.③CD40/CD40L基因测序结果.④标准品的扩增情况.结果:组织提取的总RNA完整性良好,构建的CD40/CD40L质粒经PCR扩增后分别得到136 bp,200 bp的清晰条带,测序结果与目的片段完全一致,且质粒的原始浓度为2.66×10~(13)、2.45×10~(13) copies/mL,倍比稀释至2.0×10~6 copies/mL均能得到良好的标准曲线(R~2=1).结论:实验成功构建了CD40/CD40L基因荧光定量PCR标准质粒.     

Cd40 but not CD154 knockout mice have reduced inflammatory response in polymicrobial sepsis: a potential role for Escherichia coli heat shock protein 70 in CD40-mediated inflammation in vivo

The CD40-CD154 system controls various aspects of the host inflammatory response in models of cellular and humoral immunity. Recently, we described a role for CD40 in the innate immune response in polymicrobial sepsis. However, recent data suggests that CD40 maybe activated by CD154 or directly via bacterial heat shock protein (HSP) 70. Therefore, we decided to test the mechanism of CD40 activation in murine polymicrobial sepsis. Wild-type (WT), CD40, and CD154 underwent cecal ligation and puncture (CLP). Compared with WT mice, CD40 had improved survival in association with attenuated production of IL-12, TNF-alpha, and IL-6. In contrast, CD154 mice behaved similar to WT mice with regard to mortality and cytokine production. The differential response of CD40 and CD154 mice to CLP was not due to a general attenuated response to inflammatory stimuli, as all three strains had similar survival after LPS administration, and CD40 macrophages had normal production of IL-12 in response to lipopolysaccharide. In contrast, CD40 macrophages had attenuated IL-12 production in response to Escherichia coli HSP70 (DnaK). Furthermore, intraperitoneal administration of DnaK resulted in a 4-fold increase in IL-12 in WT mice, which was absent in CD40 mice. This data demonstrates CD154-independent CD40 activation in polymicrobial sepsis and suggests that bacterial HSP70 is capable of stimulating CD40 in vitro and in vivo.     

Cyclosporin A inhibits CD40 ligand expression in T lymphocytes.

The ligand for CD40 is expressed on activated T lymphocytes and delivers contact-dependent activation signals to B lymphocytes. The mechanisms regulating CD40 ligand gene expression are largely unknown. Optimal expression of CD40 ligand required activation of protein kinase C and a rise in intracellular calcium concentration. CD40 ligand expression was inhibited by pretreatment of T cells with cyclosporin A. Cyclosporin A analogues inhibited CD40 ligand expression with a potency mirroring the ability of each compound to inhibit calcineurin activity, indicating that calcineurin plays a key role in CD40 ligand gene expression. Cyclosporin A inhibited IL-4-driven CD40 ligand-dependent IgE isotype switching in PBMC but did not inhibit IgE synthesis induced by CD40 mAb plus IL-4. PBMC derived from transplant patients receiving cyclosporin A failed to express CD40 ligand upon stimulation. These results suggest that patients receiving cyclosporin A may be deficient in CD40 ligand-dependent T cell help.     

Soluble CD40 ligand can replace the normal T cell-derived CD40 ligand signal to B cells in T cell-dependent activation

We have constructed a soluble chimeric fusion protein between the mouse CD8 alpha chain and the mouse CD40 T cell ligand. This protein binds to both human and mouse B cells. By itself it induced a modest degree of B cell proliferation, but together with anti-immunoglobulin (anti-Ig) antibody it greatly stimulated B cell proliferation, as determined by both [3H]thymidine uptake and increase in cell numbers. These data are evidence that the CD40 ligand on T cells provides a signal that drives B cell proliferation. This signal is synergistic with that delivered by anti-Ig antibody.     

Glucocorticoids upregulate CD40 ligand expression and induce CD40L-dependent immunoglobulin isotype switching

IL-4 and CD40 ligation are essential for IgE synthesis by B cells. We have shown previously that hydrocortisone (HC) induces IgE synthesis in IL-4-stimulated human B cells. In this study we demonstrate that HC fails to induce IgE synthesis in B cells from CD40 ligand-deficient (CD40L-deficient) patients. Disruption of CD40L-CD40 interactions by soluble CD40-Ig fusion protein or anti-CD40L mAb blocked the capacity of HC to induce IgE synthesis in normal B cells. HC upregulated CD40L mRNA expression in PBMCs and surface expression of CD40L in PBMCs as well as in purified populations of T and B cells. Upregulation of CD40L mRNA in PBMCs occurred 3 hours after stimulation with HC and was inhibited by actinomycin D. Upregulation of CD40L mRNA and induction of IgE synthesis by HC were inhibited by the steroid hormone receptor antagonist RU-486. These results indicate that ligand-mediated activation of the glucocorticoid receptor upregulates CD40L expression in human lymphocytes.     

Increased soluble CD40 ligand levels in cystic fibrosis

Summary. Chronic inflammation represents a key pathogeneric event in the progression of lung disease in cystic fibrosis (CF). To identify novel mechanisms of the inflammatory reaction in CF and analyze its relation with coagulative activation, we carried‐out a cross‐sectional study to evaluate circulating levels of the inflammatory mediators soluble (s) CD40L, C‐reactive protein (CRP), interleukin (IL)‐1β, the coagulation markers activated factor VII (FVIIa) and prothrombin fragment (F) 1+2, as well as urinary 11‐dehydro‐thromboxane (TX)B₂, an index of in vivo platelet activation, in 34 CF patients and 34 matched healthy subjects. We observed that CF patients displayed significantly increased circulating levels of sCD40L compared to controls [2.8 (0.4–15.6) vs 1.1 (0.2–2.7) ng mL^?1 ,P = 0.0003]. sCD40L levels inversely correlated with forced expiratory volume at 1 second (FEV₁) (ρ = ?0.788, P = 0.0001), whereas it directly correlated with CRP and IL‐1β levels (ρ = 0.621, P = 0.0004; and ρ = 0.745, P = 0.0001, respectively), which were also elevated in CF patients. CF patients had also enhanced levels of FVIIa and F1+2 compared to controls [39.2 (22.6–69.8) vs 22.3 (16.2–32.4) mU mL^?1, P = 0.0001; 0.60 (0.30–1.80) vs 0.17 (0.10–0.40) nmol L^?1, P = 0.0001, respectively]. A direct correlation was observed between sCD40L and both plasma FVIIa (ρ = 0.691, P = 0.0001) and F1+2 (ρ = 0.545, P = 0.0017) as well as between sCD40L and urinary 11‐dehydro‐TXB₂ (ρ = 0.433, P = 0.0129). Our findings suggest that in CF patients, sCD40L could represent a biochemical link between the inflammatory state, and endothelial damage and coagulative activation, leading to progressive impairment of pulmonary function.     

Upregulation of CD40--CD40 ligand system in patients with diabetes mellitus

BACKGROUND: Diabetes is associated with an increased risk of cardiovascular disease and atherosclerosis. Increasing evidence shows that CD40-CD40L interaction plays a crucial role in the pathogenesis of atherosclerosis and coronary artery disease. The purpose of this study was to assess whether CD40 system expressions were disrupted in patients with diabetes. METHODS: Sixteen normal controls and 72 patients including 20 with type 2 diabetes mellitus (DM), 15 with type 1 DM, 20 with coronary heart disease (CHD) and 17 CHD with coexisting DM were investigated. The expression of CD40 and CD40L on platelet was analyzed by indirect-immunofluorescence flow cytometry and serum-soluble CD40L level was determined by a commercially available ELISA. Serum of AGE was detected by fluorescence spectroscopy. RESULTS: Type 1 DM, type 2 DM, CHD and CHD Patients with coexisting diabetes showed a significant increase of CD40 (81.8 +/- 11.7, 70.7 +/- 11.6, 68.5 +/- 10.2, 79.9 +/- 11.9 MIF, respectively) and CD40L (18.4 +/- 5.1, 13.9 +/- 4.1, 13.5 +/- 3.7, 16.7 +/- 4.7 MIF, respectively) coexpression on platelets as well as sCD40L (15.6 +/- 3.5, 14.1 +/- 3.3, 12.2 +/- 3.5, 13.5 +/- 3.6 ng/ml, respectively) compared with controls (p < 0.01). A positive correlation was found between serum AGE levels in patients with DM and CD40-CD40L system. We also observed a significant correlation between hemoglobinA1c (HbA1c) concentration and CD40L on platelets (r = 0.71, p < 0.001) as well as sCD40L (r = 0.69, p < 0.001), but not for CD40 on platelets. CONCLUSIONS: Patients with diabetes show increased coexpression of CD40 system, especially CD40L, which may create a proinflammatory and prothrombotic milieu for aggravating the development of atherosclerosis.     

A critical role for CD40-CD40 ligand interactions in amplification of the mucosal CD8 T cell response.

The role of CD40 ligand (CD40L) in CD8 T cell activation was assessed by tracking antigen-specific T cells in vivo using both adoptive transfer of T cell receptor transgenic T cells and major histocompatibility complex (MHC) class I tetramers. Soluble antigen immunization induced entry of CD8 cells into the intestinal mucosa and cytotoxic T lymphocyte (CTL) differentiation, whereas CD8 cells in secondary lymphoid tissue proliferated but were not cytolytic. Immunization concurrent with CD40L blockade or in the absence of CD40 demonstrated that accumulation of CD8 T cells in the mucosa was CD40L dependent. Furthermore, activation was mediated through CD40L expressed by the CD8 cells, since inhibition by anti-CD40L monoclonal antibodies occurred after adoptive transfer to CD40L-deficient mice. However, mucosal CD8 T cells in normal and CD40(-/-) mice were equivalent killers, indicating that CD40L was not required for CTL differentiation. Appearance of virus-specific mucosal, but not splenic, CD8 cells also relied heavily on CD40-CD40L interactions. The mucosal CTL response of transferred CD8 T cells was MHC class II and interleukin 12 independent. The results established a novel pathway of direct CD40L-mediated CD8 T cell activation.     

Increased expression of CD40 ligand on systemic lupus erythematosus lymphocytes.

The specificity of T cell help for B cell activation and differentiation is maintained by the brief expression on the T cell surface, following T cell receptor-mediated triggering, of CD40 ligand (CD40L). Interaction of T helper (Th) cell CD40L with B cell CD40 induces B cell activation, cell surface expression of activation antigens, proliferation, and initiation of immunoglobulin isotype switch. We predicted that in patients with systemic lupus erythematosus (SLE), in whom Th cell-dependent production of autoantibodies results in immune complex-mediated tissue damage, CD40L expression might be augmented, prolonged, or abnormally regulated. Baseline expression of CD40L was increased in some SLE patients studied, when compared with control subjects. While Th cells from normal subjects (n = 14) and rheumatic disease control patients (n = 9) showed maximal expression of CD40L, after in vitro activation with phorbol myristate acetate (PMA) and ionomycin, at 6 h of culture with diminished levels observed at 24 and 48 h, Th cells from SLE patients (n = 19) maintained high level cell surface expression of CD40L through 24 and 48 h of culture. The prolonged expression of CD40L was functionally significant, as 24 h-activated SLE T cells, when cocultured with target B cells, induced greater B cell surface CD80 (B7-1) expression than did 24 h-activated normal T cells. These results document impaired regulation of CD40L expression in SLE T cells and identify an important potential target for therapy in this systemic autoimmune disease.     

Functional CD40 ligand is expressed by T cells in rheumatoid arthritis.

CD40 ligand (CD40-L), a member of the tumor necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. Interactions between CD40-L and CD40 induce B cell immunoglobulin production as well as monocyte activation and dendritic cell differentiation. Since these features characterize rheumatoid arthritis (RA), the expression and function of CD40-L in RA was examined. Freshly isolated RA peripheral blood (PB) and synovial fluid (SF) T cells expressed CD40-L mRNA as well as low level cell surface CD40-L. An additional subset of CD4+ RA SF T cells upregulated cell surface CD40-L expression within 15 min of in vitro activation even in the presence of cycloheximide, but soluble CD40-L was not found in SF. CD40-L expressed by RA T cells was functional, since RA PB and SF T cells but not normal PB T cells stimulated CD40-L-dependent B cell immunoglobulin production and dendritic cell IL-12 expression in the absence of prolonged in vitro T cell activation. In view of the diverse proinflammatory effects of CD40-L, this molecule is likely to play a central role in the perpetuation of rheumatoid synovitis. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.     

Exacerbation of antigen-induced arthritis in urokinase-deficient mice.

In rheumatoid arthritis, synovial expression of urokinase (uPA) activity is greatly increased (Busso, N., V. Péclat, A. So, and A. -P. Sappino. 1997. Ann. Rheum. Dis. 56:550- 557). We report the same effect in murine antigen-induced arthritis. uPA-mediated plasminogen activation in arthritic joints may have deleterious effects via degradation of cartilage and bone matrix proteins as well as beneficial effects via fibrin degradation. We evaluated these contrasting effects in vivo by analyzing the phenotype of uPA-deficient (uPA-/-) and control mice during antigen-induced arthritis. Joint inflammation was comparable in both groups up to day 3 and subsequently declined in control mice, remaining significantly elevated in uPA-/- mice on days 10 and 30 after arthritis onset. Likewise, synovial thickness was markedly increased in uPA-deficient mice persisting for up to 2 mo, whereas it subsided in control animals. Bone erosion was exacerbated in uPA-/- mice on day 30. By contrast, no difference in articular cartilage proteoglycan content was found between both groups. Significantly increased accumulation of fibrin was observed by day 30 in arthritic joints of uPA-/- mice. We hypothesized that synovial fibrin deposition plays a role in joint inflammation. Accordingly, defibrinogenation of uPA-/- mice by ancrod significantly decreased the sustained joint inflammation. All the above observations were reproducible in plasminogen-deficient (Pln-/-) mice. In conclusion, synovial fibrin deposition plays a role as a nonimmunological mechanism which sustains chronic arthritis.     

Increased levels of CD40-CD40 ligand system in patients with essential hypertension

BACKGROUND: Although it has been hypothesized that hypertension is in part an inflammatory disorder, clinical data linking inflammation with incident hypertension are scarce. There is evidence that have shown that CD40-CD40L interaction plays a pathogenic role in inflammatory disorders. We assessed whether CD40 system expressions were altered in patients including 30 with hypertension grade 1, 80 with hypertension grade 2 and 40 with hypertension grade 3. METHODS: Twenty normal controls and 150 patients with essential hypertension were investigated. The expression of CD40 and CD40L on platelet was analyzed by indirect-immunofluorescence flow cytometry and soluble CD40L level was determined by a commercially available ELISA. C-reactive protein was also measured by ELISA. RESULTS: All patients with hypertension showed a significant increase of CD40 (67.1+/-9.6 Mean Fluorescence Intensity, MFI) and CD40L (15.3+/-5.0 MFI) coexpression on platelets as well as sCD40L levels (12.8+/-3.9 ng/ml ) compared with controls (p<0.0001). We found that CRP levels related to CD40-CD40L system. We also observed a slight correlation between sCD40L level and blood pressure. During 3 months follow-up, patients with enhanced levels of sCD40L (>15 ng/ml) indicated a tough control of blood pressure. CONCLUSION: Patients with essential hypertension show increased coexpression of CD40 system, which suggests that hypertension is in part an inflammatory disorder.     

Intra-hepatic expression of scavenger receptor and CD14 and their relationship with local inflammatory responses in endotoxemia in mice.

Our objective was to investigate the expression of scavenger receptor (SR) and CD14 in the liver and their relationship with local anti-inflammatory and proinflammatory responses in endotoxemia in order to uncover the mechanism for the liver to turn into effector organ from defense one at the level of cell receptors in sepsis. Mouse models of endotoxemia of different severity were reproduced by injection of different doses of lipopolysaccharide (LPS) via tail vein. Expression of SR and CD14 in the liver was assayed by immunohistochemistry and was then analyzed with an image analysis system. The levels of TNFalpha, IL-6, IL-4, and IL-10 in liver tissue were determined with ELISA. Expression of SR in the liver in the high-dose group was markedly decreased 1 h after injection of LPS, and also in low- and medium-dose groups at 3 h. The expression of SR in the liver in the three groups was shown to be progressively decreased with the time prolonged. There was significant difference in average optical density (OD) values of SR among the three groups. The expression of CD14 in the liver in the three groups was shown to be significantly increased 1 h after injection of LPS, and much more with the time prolonged. But there was no significant difference in OD values of CD14 among the three groups. The contents of intrahepatic proinflammatory mediators TNFalpha and IL-6 and anti-inflammatory mediators IL-4 and IL-10 were successively significantly increased after injection of LPS. The release of anti-inflammatory mediators was shown to be later than that of proinflammatory mediators. Correlation analysis indicated that there was negative correlation between expression of SR and CD14, and that changes of TNFalpha, IL-6, IL-4, and IL-10 levels in liver tissues were correlated significantly positively with OD values of CD14 and negatively with OD values of SR. Expression of SR in the liver was shown to be progressively decreased, and that of CD14 increased in endotoxemia, which was closely related to the uncontrolled inflammatory response in liver. This might be an important mechanism for the liver to turn into effector organ from defense one in sepsis.     

Nonreplicating recombinant vaccinia virus expressing CD40 ligand enhances APC capacity to stimulate specific CD4+ and CD8+ T cell responses

Recombinant poxviruses expressing immunomodulatory molecules together with specific antigens represent powerful vaccines for cancer immunotherapy. Recently, we and others have demonstrated, in vitro and in vivo, that coexpression of CD80 and CD86 costimulatory molecules enhances the immunogenic capacity of a recombinant vaccinia virus (rVV) encoding different tumor-associated antigens. To further investigate the capacity of these vectors to provide ligands for different costimulatory pathways relevant in the generation of T cell responses, we constructed a recombinant virus (rVV) expressing CD40 ligand or CD154 (CD154rVV). Upon binding the CD40 receptor expressed on antigen presenting cells (APC), this molecule, physiologically expressed on activated CD4+ T cells, increases their antigen presentation and immunostimulatory capacities. Therefore, we evaluated the effects of CD154rVV infection on APC activation and its consequences on T cell stimulation. CD154rVV infection of autologous fibroblasts, monocytes, or iDC promoted the expression of a number of cytokines, including GM-CSF, TNF-alpha, and IL-15 in iDC. Most importantly, IL-12 p40 gene expression and protein secretion were induced by CD154rVV but not by wild-type VV (WT VV) in either CD14+ cells or iDC, and these effects could be blocked by anti-CD40 monoclonal antibodies. Furthermore, phenotypic characterization of CD154rVV infected iDC revealed enhanced expression of CD83 and CD86 surface markers as compared with wild-type vaccinia virus infection. As expected, VV infection triggered cytokines gene expression in cultures including APC and T cells from VV immune donors. However, cytokine genes typically expressed by T cell receptor triggered T cells such as those encoding IL-2 and IFN-gamma, or T cell proliferation, were detectable to a significantly higher extent in CD154rVV infected cultures, as compared with WT VV. Activation of specific CD8+ T cells was then investigated using MART-1/Melan-A(27-35) epitope as the model of tumor-associated antigen (TAA). In the presence of CD154rVV activated APCs, significantly higher numbers of specific cytotoxic CD8+ T cells were detected, as compared with cultures performed in the presence of WT VV or in the absence of virus. Taken together, these data indicate that functional CD154 expression from rVV infected cells promotes APC activation, thereby enhancing antigen-specific T cell generation. Such a recombinant vector might help bypass the requirement for activated helper cells during CTL priming, thus qualifying as a potentially relevant vector in the generation of CD8+ T cell responses in cancer immunotherapy.     

OX40 ligand expressed by DCs costimulates NKT and CD4+ Th cell antitumor immunity in mice

The exceptional immunostimulatory capacity of DCs makes them potential targets for investigation of cancer immunotherapeutics. We show here in mice that TNF-alpha-stimulated DC maturation was accompanied by increased expression of OX40 ligand (OX40L), the lack of which resulted in an inability of mature DCs to generate cellular antitumor immunity. Furthermore, intratumoral administration of DCs modified to express OX40L suppressed tumor growth through the generation of tumor-specific cytolytic T cell responses, which were mediated by CD4+ T cells and NKT cells. In the tumors treated with OX40L-expressing DCs, the NKT cell population significantly increased and exhibited a substantial level of IFN-gamma production essential for antitumor immunity. Additional studies evaluating NKT cell activation status, in terms of IFN-gamma production and CD69 expression, indicated that NKT cell activation by DCs presenting alpha-galactosylceramide in the context of CD1d was potentiated by OX40 expression on NKT cells. These results show a critical role for OX40L on DCs, via binding to OX40 on NKT cells and CD4+ T cells, in the induction of antitumor immunity in tumor-bearing mice.     

Efficient adenovector CD40 ligand immunotherapy of canine malignant melanoma

Cutaneous canine melanomas are usually benign in contrast to human malignant melanoma. However, the canine oropharyngeal, uveal, and mucocutaneous neoplasms are aggressive and have metastatic potential. Surgery and to a lesser extent radiotherapy and chemotherapy are widely adopted treatments but are seldom curative in advanced stages. The similarities between human and canine melanoma make spontaneous canine melanoma an excellent disease model for exploring novel therapies. Herein, we report the first 2 adenovector CD40L immunogene (AdCD40L) treatments of aggressive canine malignant melanoma. Case no. 1 was an advanced stage III oral melanoma that was cured from malignant melanoma with 2 intratumor AdCD40L injections before cytoreductive surgery. After treatment, the tumor tissue was infiltrated with T lymphocytes and B lymphocytes suggesting immune activation. This dog survived 401 days after the first round of gene therapy and was free of melanoma at autopsy. Case no. 2 had a conjunctival malignant melanoma with a rapid progression. This case was treated with 6 AdCD40L injections over 60 days. One hundred and twenty days after start of gene therapy and 60 days after the last injection, the tumor had regressed dramatically, and the dog had a minimal tumor mass and no signs of progression or metastasis. Our results indicate that AdCD40L immunogene therapy is beneficial in canine malignant melanoma and could be considered for human malignant melanoma as well.