小鼠心脏移植CD40的表达变化及意义

目的 检测小鼠颈部心脏移植后排斥反应过程中CD40的变化及意义.方法 建立小鼠颈部心脏异位移植模型.实验分3组,即同系移植组、同种移植组和同种环孢菌素A（CsA）治疗组.各组于术后1 d、3 d和7 d动态检测供心的病理改变,免疫组织化学方法测供心CD40表达情况,并记录移植心脏存活时间.结果 同系移植组移植心脏存活时间最长,移植后排斥反应最轻.术后第7天,同种移植组CD40表达水平明显高于同品系组（P〈0.05）.结论 移植心脏CD40分子表达与排斥反应的发生密切相关,动态监测CD40对移植心脏的预后评估具有重要意义.     

CD40 ligand expression on stimulated T-helper lymphocytes in patients with common variable immunodeficiency

Common variable immunodeficiency (CVID) is the most common symptomatic primary antibody deficiency, characterized by reduced serum immunoglobulins levels and increased susceptibility to recurrent pyogenic infections. In this study, we evaluated CD40 ligand expression on stimulated versus unstimulated T-helper lymphocytes of nine Common variable immunodeficient patients in comparison with fifteen normal controls. Phorbol myristate acetate (PMA) and Ionomycin were used to stimulate cells in vitro. After six hours stimulation, the cells were subjected to surface staining with three-color staining procedure. Events were analyzed by flow cytometer, using FloMax software. Results were reported as the percentage of lymphocytes expressing CD markers. We did not find any significant statistical difference in CD40 ligand expression between patients and controls (p > 0.05), despite having stimulation documented by CD69 expression as activation marker in each run. The results of this study are in agreement with some other studies, indicating that CD40 ligand expression on stimulated T-helper lymphocytes of Common variable immunodeficiency patients is similar to normal controls.     

Increases in circulating T lymphocytes expressing HLA-DR and CD40 ligand in patients with dilated cardiomyophthy

Inflammatory and immunological mechanisms are implicated in the development of idiopathic dilated cardiomyopathy (DCM). Since activated T lymphocytes express surface HLA-DR antigens, an increased level of these cells in the circulation could indicated an ongoing immune response. While the role of activated T lymphocytes in experimental myocarditis has been elucidated, the contribution of T lymphocyte activation in clinical DCM remains unclear. We therefore examined the role of T-cell activation in peripheral blood samples obtained from 10 patients with DCM (mean age, 49 ± 12 years) and from 10 age-matched healthy controls. Citrated whole blood was mixed with fluorescein isothiocyanate- or phycoerythrin-conjugated specific monoclonal antibodies and analyzed using a fluorescence-activated cell sorter (FACS). The ratio (%) of histocompatibility leukocyte antigen (HLA)-DR positive cells in the FACS gated lymphocyte population was significantly higher in DCM patients than in controls (7.9% ± 5.3% vs 2.0% ± 0.9%; P < 0.01). The expression of CD40L on T cells determined as mean fluorescence intensity (MFI) was also significantly higher in DCM patients than in controls (3.6 ± 2.1 vs 1.8 ± 0.4 MFI; P < 0.05). Furthermore, the ratios of T cells expressing HLA-DR and serum brain natriuretic peptide (BNP) levels closely correlated (P = 0.0008). We showed that HLA-DR on peripheral T cells significantly correlated with serum BNP levels and that high CD40L expression on T cells was concomitant with increased BNP levels (P < 0.05). Therefore the magnitude of T-cell expression, such as increased expression of HLA-DR and CD40L, contributes to myocardial dysfunction in DCM.     

Prevention of rejection of murine islet allografts by pretreatment with anti-dendritic cell antibody.

Previously we have demonstrated that islets of Langerhans treated with donor-specific anti-Ia serum and complement survive when transplanted across the major histocompatibility complex of the mouse. In this study, using immunofluorescence, we demonstrate two morphologically distinct populations of Ia-positive cells scattered within the Ia-negative islet tissue. A large irregularly shaped Ia-positive subset of cells were identified as dendritic cells by using the 33D1 antibody specific for a mouse dendritic cell antigen. The other small, round Ia-positive subset was 33D1 negative. Islets pretreated with anti-dendritic cell antibody and complement prior to transplantation survived in their histoincompatible recipients for greater than 200 days. Rejection of stable islet allografts promptly occurred when transplant recipients were challenged with 1 X 10(5) donor dendritic cells 60 days after transplantation. These results demonstrate an important in vivo role for donor dendritic cells in the stimulation of allograft rejection.     

Coexpression of CD40 and CD40 ligand in B-cell lymphoma cells

CD40 ligand (CD40L) is involved in the T-cell-dependent regulation of B-cell growth and survival and can rescue normal germinal centre B cells and several types of malignant B cells from apoptosis in vitro . We have previously reported that serum of patients with chronic lymphocytic leukaemia contained elevated levels of biologically active soluble CD40L (sCD40L). Whether an augmented CD40L pathway exists in patients with other types of B-cell lymphoid malignancies and the source of native sCD40L in these patients is currently unknown. Using a sensitive ELISA assay, soluble CD40L (sCD40L) was detected in the sera of both healthy individuals and patients with haematological malignancies; however, its level was significantly elevated only in patients with B-cell lymphomas ( P   < 0.0001). Several types of malignant B cells coexpressed CD40 and CD40L proteins, and CD40L mRNA was detected in purified resting malignant B cells. The dual expression of CD40 and CD40L in B cells and the presence of native sCD40L in human serum suggest that a direct T–B-cell contact may not be required for CD40L delivery to B cells. This data raises the possibility that an autocrine cytokine loop involving CD40L may contribute to the growth regulation of benign and malignant B cells in vivo .     

H2-mismatched transplantation with repetitive cell infusions and CD40 ligand antibody infusions without myeloablation

Graft rejection and graft-versus-host disease are major problems in mismatched marrow transplants along with toxicity from standard myeloablative host treatments. We have established a tolerization model, using 1 Gy irradiation, which reduces stem cell capacity to < 10% of control while causing minimal myelosuppression, donor antigen pre-exposure (spleen cells), CD40-ligand antibody blockade and high levels of marrow (40 x 106 cells), which allows for stable long-term multilineage engraftment in H2-mismatched murine marrow transplants. We now show that the establishment of 'microchimaerism' (0.5-3.8%) sets the stage for macrochimaerism, with subsequent marrow infusions in H2-mismatched mice with CD40-ligand blockade only. Neither irradiation nor spleen cell exposure were necessary. When 40 x 106 bone marrow cells were infused on weeks 0, 12, 14 and 16, blood engraftment was about seven times the single 40 x 106 control. When marrow cells were given on weeks 0, 3, 4, 5 and 6, engraftment at 24 weeks post transplant was 17.9 +/- 1.2%, compared with 2.7 +/- 0.8% for the single 40 x 106 control (P = 0.009). We have shown stable, long-term multilineage chimaerism and established that the schedule of marrow administration, not the total cell dose, is critical for tolerization. This approach indicates that microchimaerism can tolerize for subsequent marrow infusions and produce macrochimaerism. This strategy could be applied in clinical human transplants.     

狼疮肾炎患者肾组织CD40及其配体表达的免疫组织化学分析

目的　探讨CD40 CD40配体 (CD40L)相互作用在狼疮肾炎 (LN)发病机制中的可能作用。方法　用免疫组织化学方法对 2 0例LN患者肾组织CD40和CD40L的表达进行检测 ,并对其与肾脏病变的相关性进行分析。结果　Ⅲ、Ⅳ型LN肾组织CD40表达较正常对照组显著上调 (P <0 0 1) ,除系膜细胞、内皮细胞和肾小管上皮细胞CD40表达增强外 ,还出现CD40阳性的间质浸润细胞。Ⅲ、Ⅳ型LN患者肾组织CD40L表达亦显著增强 (P <0 0 1) ,其分布范围与CD40一致。Ⅱ、Ⅴ型LN患者肾组织CD40表达范围和强度与正常肾组织大致相似。LN肾组织CD40表达与病变活动指数相关 (r =0 78,P <0 0 1)。结论　CD40 CD40L在肾脏局部的相互作用可能在LN发生和发展中起重要作用。     

Increased concentration of soluble CD40 ligand in preeclampsia.

Preeclampsia has been associated with increased platelet activation detected before disease onset. Platelets are involved in hemostasis and also directly initiate an inflammatory response of the vessel wall. Inappropriate activation of platelets may be involved in pathogenesis in preeclampsia by promoting coagulation and thrombosis, and also as a mediator of inflammation. Platelets may release inflammatory mediators such as soluble CD40 ligand. The plasma level of soluble CD40 ligand was investigated during preeclamptic (n =20) and normal pregnancies (n = 20) to emphasize inflammatory response in preeclampsia. The mean soluble CD40 ligand levels were 1.08 +/- 0.43 ng/mL in patients with preeclampsia and 0.76 +/- 0.24 ng/mL in healthy pregnant women, which was statistically significant (P = .01). To clarify whether inflammation may cause inappropriate endothelial cell activation or inappropriate endothelial cell activation may start this inflammatory response, future studies are needed in a larger study population.     

CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis.

We investigated the role of CD40-CD40 ligand (CD40L) interactions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). Activated helper T cells expressing CD40L (gp39) surface protein were found in MS patient brain sections, but not in brain tissue sections of normal controls or patients with other neurological disease. CD40L-positive cells were co-localized with CD40-bearing cells in active lesions (perivascular infiltrates). Most of these CD40-bearing cells proved to be of the monocytic lineage (macrophages or microglial cells), and relatively few were B cells. To functionally evaluate CD40-CD40L interactions, EAE was elicited in mice by means of proteolipid-peptide immunization. Treatment with anti-CD40L monoclonal antibody completely prevented the development of disease. Furthermore, administration of anti-CD40L monoclonal antibody, even after disease onset, shortly before maximum disability score was reached led to dramatic disease reduction. The presence of helper T cells expressing CD40L in brain tissue of MS patients and EAE animals, together with the functional evidence provided by successful experimental prevention and therapy in an animal model, indicates that blockade of CD40-CD40L-mediated cellular interactions may be a method for interference in active MS.     

Induction of antigen-specific tolerance to bone marrow allografts with CD4+CD25+ T lymphocytes

Thymus-derived regulatory T lymphocytes of CD4(+)CD25(+) phenotype regulate a large variety of beneficial and deleterious immune responses and can inhibit lethal graft-versus-host disease in rodents. In vitro, CD4(+)CD25(+) T cells require specific major histocompatibility complex (MHC)/peptide ligands for their activation, but once activated they act in an antigen-nonspecific manner. In vivo, regulatory T cells are also activated in an antigen-specific fashion, but nothing is known about antigen specificity of their suppressor-effector function. Here we show that CD4(+)CD25(+) regulatory T lymphocytes isolated from naive mice and activated in vitro with allogeneic antigen-presenting cells (APCs) induced specific long-term tolerance to bone marrow grafts disparate for major and minor histocompatibility antigens; whereas "target" bone marrow was protected, third-party bone marrow was rejected. Importantly, in mice injected with a mix of target and third-party bone marrows, protection and rejection processes took place simultaneously. These results indicate that CD4(+)CD25(+) regulatory T cells can act in an antigen-specific manner in vivo. Our results suggest that CD4(+)CD25(+) regulatory T cells could in the future be used in clinical settings to induce specific immunosuppression.     

Soluble CD40 ligand in acute and chronic heart failure.

AIMS: Inflammatory cytokines may play a pathogenic role in heart failure (HF). CD40-CD40 ligand (CD40L) interactions are important in atherogenesis and based on its role in inflammation we sought to evaluate the role of CD40L in human HF. METHODS AND RESULTS: Serum levels of soluble (s) CD40L were measured in 236 patients with acute HF following myocardial infarction, treated with either angiotensin-converting enzyme (ACE)-inhibition or angiotensin II blockade and followed for 2 years, and in 116 patients with chronic HF. Our main findings were: (i) patients with acute HF had increased sCD40L levels, particularly those with severe HF, diabetes, or hypertension; (ii) when these patients were followed longitudinally, persistently raised sCD40L levels were found throughout the observation period with no effect of captopril or losartan; (iii) the increase in sCD40L during follow-up was not seen in patients receiving warfarin therapy; (iv) patients with chronic HF also had raised sCD40L, significantly correlated with clinical severity, neurohormonal dysregulation, and left ventricular dysfunction; (v) studies from different blood compartments suggest that the vasculature of lower extremities and the failing myocardium itself may produce and secrete sCD40L in chronic HF. CONCLUSION: Our findings may suggest a pathogenic role for enhanced CD40-CD40L interactions in human HF.     

Soluble CD40 ligand, platelet surface CD40 ligand, and total platelet CD40 ligand in atrial fibrillation: relationship to soluble P-selectin, stroke risk factors, and risk factor intervention

BACKGROUND: Abnormal levels of soluble CD40 ligand (sCD40L) have been reported in patients with hypertension, coronary artery disease, diabetes mellitus, heart failure, and stroke, all of which are conditions that are associated with nonvalvular atrial fibrillation (AF). We hypothesized the following: (1) CD40 ligand (CD40L)-related indexes (ie, platelet surface expressed CD40L, the soluble fragment of CD40L [sCD40L], and the total amount of CD40L per platelet [pCD40L]) are elevated in patients with AF compared to control subjects; (2) these indexes correlate with soluble P-selectin (sP-selectin), which is an established platelet marker; and (3) these indexes differentiate "high-risk" from "low-risk" subjects. METHODS: We performed a case-control study of 121 AF patients, 71 "disease control subjects," and 56 "healthy control subjects." Peripheral venous levels of platelet surface-expressed CD40L were analyzed by flow cytometry, while levels of sCD40L, pCD40L, and sP-selectin were measured by enzyme-linked immunosorbent assay. RESULTS: AF patients had significantly higher sCD40L levels compared to healthy control subjects (p = 0.042), with no difference in platelet surface CD40L and pCD40L levels. A positive correlation was noted between levels of sCD40L and pCD40L, and not with sP-selectin. CD40L-related indexes failed to distinguish between high-risk and low-risk AF patients. AF patients receiving optimal antithrombotic therapy had significantly lower pCD40L levels (p < 0.001) compared to control subjects. Optimized AF management also resulted in significant reductions in the levels of sCD40L (p = 0.023) and pCD40L (p < 0.001). CONCLUSION: CD40L-related indexes are not useful in the risk stratification of AF patients, and abnormal sCD40L levels can be reduced by intense multifactorial risk management. While there is a significant, albeit modest, excess of platelet activation in AF patients (as measured by sCD40L levels) compared to healthy control subjects, this is not in excess of that seen in patients with underlying cardiovascular diseases.     

Platelet-CD40 ligand interaction with melanoma cell and monocyte CD40 enhances cellular procoagulant activity.

Platelet-tumor cell interactions are believed to be important in tumor metastasis. Tumor cell tissue factor (TF) expression enhances metastasis and angiogenesis, and is primarily responsible for tumor-induced thrombin generation and the formation of tumor cell-platelet aggregates. Activated platelets express and release CD40 ligand (CD40L), which induces endothelial TF expression by ligation to CD40. We investigated the effect of platelet-derived CD40L on the TF activity of human CD40-positive melanoma cells and monocytes by incubating supernatants from activated or resting platelets with tumor cells or monocytes, and by bringing resting or activated platelets into close apposition with tumor cell monolayers. CD40L was present on the surface of activated (but not resting) platelets and was also released following platelet activation. Both recombinant soluble CD40L (rsCD40L) and activated platelet supernatants increased procoagulant activity (PCA) and TF antigen in tumor cells and monocytes. The increase in TF activity induced by both rsCD40L and activated platelet supernatants was inhibited by anti-CD40L antibody. Furthermore, contact of activated platelets with tumor cells increased cellular PCA, and this effect was also inhibited by anti-CD40L. In malignancy, the increase in cellular TF activity via CD40 (tumor cell)-CD40L (platelet) interaction may possibly enhance intravascular coagulation and hematogenous metastasis.     

Demonstration of functional CD40 in B-lineage acute lymphoblastic leukemia cells in response to T-cell CD40 ligand

Because activated T cells were previously shown to induce proliferation of human normal B-cell precursors (BCP) via the CD40 pathway, we investigated the effects of T cells on leukemic blasts isolated from patients with B-lineage acute lymphoblastic leukemia (BCP-ALL). An anti- CD3 activated human CD4+ T-cell clone was found to induce significant call proliferation in four of nine BCP-ALL samples analyzed. In one of these cases, the T-cell effect was clearly dependent on interaction between CD40 and its ligand. Accordingly, a more thorough analysis was performed on this particular leukemia (case 461, adult early pre-B-ALL, mBCR+, Philadelphia+, i(9q)+). Thus, autologous CD4+ T cells isolated from the patient were also able to induce CD40-dependent proliferation of the leukemic blasts. Analysis of the phenotype after coculture showed that, among the CD19+ cells, a proportion gradually lost expression of CD10 and CD34, whereas some cells acquired CD23. In addition, and in contrast with normal BCP, activated T cells promoted maturation of a subset of the case 461 leukemic cells into surface IgM+ cells. The leukemic origin of the cycling and the maturing cells was assessed by the presence of i(9q), a chromosomal abnormality associated with this leukemia and evidenced by fluorescence in situ hybridization. Taken together, these results show that leukemic BCP can be activated via CD40 but that not all cases display detectable stimulation in response to T cells despite their expression of CD40. In addition, the present data suggest that CD4+ T cells could potentially play a role in the physiology of BCP-ALL.     

大鼠胰岛与睾丸细胞共移植诱导同种胰岛移植免疫豁免

目的　探究大鼠胰岛与睾丸细胞共移植后睾丸细胞Fas配体表达能否对胰岛移植物提供免疫豁免作用。方法　将同种大鼠胰岛及睾丸细胞移植于糖尿病受体,观察移植物存活情况,并体外检测睾丸Sertoli细胞对活化淋巴细胞的抑制作用以及移植物内淋巴细胞凋亡情况。结果　单纯移植胰岛组移植胰岛平均存活期为(6±1)天。睾丸细胞和胰岛细胞共移植,当睾丸细胞数量为5×10     

Inverse correlation between soluble CD40 ligand and soluble CD40 is absent in patients with unstable angina

The CD40/CD40 ligand (CD40L) system mediates inflammatory processes important in atherogenesis and plaque instability. The expression of CD40L on activated T cells was suppressed by soluble CD40 (sCD40) in vitro. However, the relationship between soluble CD40L (sCD40L) and sCD40 in unstable angina (UA) is still unknown. Thirty-seven consecutive patients with recent chest pain or discomfort were recruited. Patients with both Braunwald's class IB–IIIB and with coronary stenosis (or stenoses) of >75% were assigned to the UA group (n = 19, aged 67.2 ± 8.2 years), and the rest to the control group (n = 18, aged 63.4 ± 8.7 years). The serum levels of sCD40L and sCD40, and the plasma levels of matrix metalloproteinase (MMP)-9, were measured by enzyme-linked immunosorbent assays. A significantly inverse correlation between sCD40L and sCD40 was shown in the controls (r = −0.72, P = 0.0007), but was absent in the UA group (r = −0.16, P not significant), although there was no statistical significance between these groups in terms of serum levels of sCD40L or sCD40. The difference of the regression slopes of these regression lines was statistically significant (P < 0.01). Additionally, there was a significant correlation between sCD40 and plasma levels of MMP-9 in the patients with and without UA (r = 0.58, P = 0.0096), but no significant correlation between sCD40L and MMP-9 levels (r = 0.00, P not significant). The balance between CD40 and CD40L may be lost in patients with UA. Soluble CD40 expression may also be related to MMP-9 expression in atherosclerotic tissues.     

Depletion of alloantigen-primed lymphocytes overcomes resistance to allogeneic bone marrow in mildly conditioned recipients

OBJECTIVE: Successful implantation of allogeneic bone marrow (BM) cells after nonmyeloablative conditioning would allow to compensate for the inadequate supply of compatible grafts and to reduce mortality of graft-vs.-host disease (GVHD). Recently, we proposed to facilitate engraftment of mismatched BM by conditioning for alloantigen-primed lymphocyte depletion (APLD) with cyclophosphamide (CY). Here we summarize the experimental results obtained by this approach. MATERIALS AND METHODS: Naive or mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (day 0), treated with CY (day 1) to deplete alloantigen-primed lymphocytes, and given a second C57BL/6 BM transplant (day 2) for engraftment. Recipients were repeatedly tested for chimerism in the blood and followed for GVHD and survival. The protocol was also tested for inducing tolerance to donor tissue and organ allografts, and for treatment of leukemia, breast cancer, and autoimmune diabetes in NOD mice. RESULTS: APLD by 200 mg/kg CY provided engraftment of allogeneic BM from the same donor in 100% mildly irradiated recipients. Eighty percent chimeras remained GVHD-free more 200 days. All chimeras accepted permanently donor skin grafts and donor hematopoietic stromal progenitors. Allogeneic BM transplantation (BMT) after APLD had a strong therapeutic potential in BALB/c mice harboring malignant cells and in autoimmune NOD recipients. Tolerance-inducing CY dose could be reduced to 100 mg/kg. Conditioning for APLD resulted in engraftment of allogeneic BM after a significantly lower radiation dose than treatment with radiation and CY alone. CONCLUSION: Our results demonstrate that conditioning for APLD has a definite advantage over general immunosuppression with CY and radiation therapy.     

Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154

Reported effects of anti-CD154 treatment on autoimmunity, alloreactivity, and inflammatory events mediated by macrophages and endothelial cells indicated that it might be an ideal agent for the prevention of intrahepatic islet allograft failure. This hypothesis was tested in MHC-mismatched rhesus monkeys. Transplantation of an adequate number of viable islets resulted in engraftment and insulin independence in six of six recipients treated with anti-CD154 (hu5c8) induction plus monthly maintenance therapy (post-operative day >125, >246, >266, >405, >419, >476). Anti-CD154 (hu5c8) displayed no inhibitory effect on islet cell function. For monkeys followed for >100 days, continued improvement in graft function, as determined by first phase insulin release in response to intravenous glucose, was observed after the first 100 days post-transplant. No evidence of toxicity or infectious complications has been observed. All recipients treated with anti-CD154 became specifically nonresponsive to donor cells in mixed lymphocyte reactions. Furthermore, three monkeys are now off therapy (>113, >67, and >54 days off anti-CD154), with continued insulin independence and donor-specific mixed lymphocyte reaction hyporeactivity. In striking contrast to all previously tested strategies, transplantation of an adequate number of functional islets under the cover of anti-CD154 (hu5c8) monotherapy consistently allows for allogeneic islet engraftment and long-term insulin independence in this highly relevant preclinical model.