Severe impairment of B cell function in lpr/lpr mice expressing transgenic Fas selectively on B cells.

Transgenic lpr/lpr mice expressing functional Fas selectively on B cells were produced in an attempt to elucidate the role of Fas on B cells in the regulation of autoantibody production. The homozygous lpr/lpr mice carrying the transgene did not produce anti-double-stranded DNA antibodies throughout their lives, whereas the development of abnormal lpr T cells (double negative, B220(+)) was not suppressed. Further analyses, however, revealed that the expression of the transgenic Fas on B cells of lpr/lpr homozygous mice resulted in severe impairment of the B cell function. The defect was characterized by a decrease in the number of mature peripheral B cells, a reduction in the serum Ig level and the total failure of B cells to mount antibody responses to stimulations of T-dependent as well as T-independent antigens. Such a defect was prominent only when the transgene was expressed on the lpr/lpr homozygous background. On the contrary, B cells of the transgenic lpr/lpr mice were shown to be capable of producing Ig when stimulated with anti-CD40 in the presence of IL-4 and IL-5. Furthermore, lpr/lpr T cells showed enhanced non-specific cytolytic activity. These observations suggested that the observed B cell defect was probably attributable to the destruction of activated B cells expressing transgenic Fas by aggressive lpr/lpr T cells.     

Requirement of Fas expression in B cells for tolerance induction.

Fas is a death receptor that belongs to the tumor necrosis factor receptor family and is expressed in various cell types, in particular, in lymphoid cells. A loss-of-function mutation in the Fas gene (lpr mutation) causes lymphadenopathy and splenomegaly, and accelerates autoimmune diseases in some strains of mice such as MRL. In this report, Fas cDNA driven by murine lck distal promoter was used to establish transgenic MRL-lpr mouse lines. The transgenic mice expressed functional Fas in mature T cells and B cells. The lymphadenopathy and splenomegaly caused by accumulation of abnormal T cells in the lpr mice were rescued in the transgenic mice. The number of B cells in the periphery as well as the serum IgG level were significantly reduced, and the autoimmune symptoms and mortality were ameliorated. These results indicate that both mature B cells and T cells must undergo Fas-mediated apoptosis to prevent the development of autoimmune diseases.     

Significance of Fas receptor protein expression in epithelial ovarian cancer

Fas receptor (FasR) is a cell surface receptor that, when activated, triggers apoptosis. It has been postulated that this receptor may be involved in the clearance of benign ovarian epithelial inclusion cysts (IC). In this study, we test the hypothesis that the expression of FasR changes among IC, cystadenoma (AD), tumors of low malignant potential (LMP), and invasive cancer (cystadenocarcinoma, CA). Formalin-fixed paraffin-embedded sections from 53 oophorectomy specimens representing 26 IC, 17 AD, 17 LMP, and 24 CA were stained using the immunohistochemical avidin-biotin-peroxidase method. We used a mouse antihuman monoclonal antibody Apo-1/Fas (Dako), at 1:5 dilution, after antigen retrieval. The stain was semiquantitatively scored by 3 observers evaluating the intensity of the stain and percentage of positive tumor cells. Statistical analysis was performed using the Wilcoxon rank sum test. Strong (score 6+/7+) and diffuse (>75%) luminal FasR stain was identified in 22 (85%) of 26 IC and 16 (94%) of 17 AD, but only in 6 (35%) of 17 LMP and 1 (4%) of 24 CA. Conversely, weak (score 2+/3+) and focal (<25%) FasR staining was observed in 7 (29%) of 24 CA, but in none of the IC, AD, or LMP. These differences were statistically significant (P < .05). The decreased expression of FasR in malignant ovarian epithelial neoplasms as compared with benign ovarian epithelial lesions suggests that a decreased sensitivity to Fas-mediated apoptosis may be involved in ovarian epithelial carcinogenesis.     

Existence of a small population of IL-2Rβhi TCRint cells in SCG and MRL-lpr/lpr mice which produce normal Fas mRNA and Fas molecules from the lpr gene

Mice carrying the lpr gene, SCG and MRL-lpr/lpr mice, were used to characterize the phenotype and lpr gene of abnormally proliferating T cells in these mice. A major population which expanded in these mice were T cells expressing intermediate (int) levels of T cell receptor (TCR) (and CD3) and the phenotype of interleukin-2 receptor (IL-2R)β^lo α^? (possibly abnormal TCR^int cells). The levels of TCR^hi cells of thymic origin (generated through the mainstream of T cell differentiation in the thymus) profoundly decreased after the onset of disease. However, a small population of normal TCR^int cells (i.e. IL-2Rβ^hi α^?) were also found to exist in all tested organs. For example, the majority of abnormal IL-2Rβ^lo TCR^int cells were CD4^?8^? CD2^?, while normal IL-2Rβ^hi TCR^int cells were a mixture of single-positive cells (mainly CD8⁺), CD4^?8^? cells and CD2⁺ cells. Moreover, normal TCR^int cells preferentially produced normal Fas mRNA and Fas molecules from the lpr gene. This phenomenon explains the leaky appearance of normal Fas mRNA and Fas molecules in mice carrying the lpr gene. It is suggested that a small population of IL-2Rβ^hiTCR^int cells are resistant to the lpr genetic abnormality.     

gld/gld mice are unable to express a functional ligand for Fas

Mice homozygous for either the lpr or gld genes develop phenotypically identical autoimmune disorders. The gene responsible for the pathology in lpr/lpr mice encodes the Fas antigen, a protein associated with the induction of programmed cell death. To determine if the defect associated with gld represents a mutation in the ligand for Fas, we have assessed the ability of lymphoid cells from homozygous gld/gld mice to lyse target cells in a Fas-dependent manner. Using an antagonistic antibody to Fas, we demonstrate that activated T cells from normal and lpr mice are capable of inducing Fas-mediated lysis of tumor target cells. In contrast, activated T cells from gld/gld mice fail to induce lysis of tumor targets, although cells from gld mice are able to lyse specific allogeneic targets following mixed lymphocyte culture. In addition, activated T cells from gld/gld homozygous animals are not capable of binding to a Fas.Fc fusion protein at high levels, whereas activated T cells from normal and lpr/lpr animals bind Fas.Fc efficiently. These data indicate that mice homozygous for gld are unable to express a functional ligand for Fas.     

Abnormal distribution of IL-6 receptor in aged MRL/lpr mice: elevated expression on B cells and absence on CD4+ cells.

A mAb against murine IL-6 receptor (IL-6R), KMH7, was obtained by immunization of hamster with recombinant soluble murine IL-6R. Flow cytometry analysis of IL-6R distribution on lymphocytes in BALB/c showed that IL-6R was expressed on peripheral lymph node (LN) T cells of either CD4+ or CD8+ phenotype, and Peyer's patch IgA+ B cells, but not on splenic B cells and thymocytes. A similar distribution was observed in 5 week old MRL/lpr and 16-week-old MRL/n mice. In contrast, in 16 week old MRL/lpr mice of both sexes, IL-6R was expressed on splenic IgM+ cells. Peripheral LN CD4+ T cells in 16 week old female MRL/lpr mice did not express IL-6R. Thymocytes in any population with a phenotype of CD4+ or CD8+, double negative, and double positive were not stained with KMH7 in both BALB/c and MRL/lpr mice. In both strains, IL-6R was induced in CD4+ or CD8+ thymocytes after 2 days of culture, suggesting that CD4+ thymocytes in MRL/lpr have a potential to express IL-6R. Our results suggest that overexpression of IL-6R on B cells and absence of IL-6R on peripheral CD4+ cells are concurrent with, or may contribute to, B cell hyperreactivity and T cell abnormality in this strain.     

Staphylococcal enterotoxin B up-regulates interleukin-2 receptor β chain expression on tonsillar B cells

The superantigen staphylococcal enterotoxin B (SEB) selectively up-regulates the interleukin-2 receptor (IL-2R) β chain (p70) without up-regulating the IL-2R a chain (CD25) on a human tonsillar B cell population depleted of T cells. This action of SEB, probably mediated by binding to major histocompatibility complex class II, renders B cells sensitive to T cell-derived IL-2 and is sufficient for induction of vigorous DNA synthesis with low concentrations of IL-2. This explains one of the mechanisms by which bacterial superantigens activate large numbers of B cells and may reflect a similar mechanism operative in cognate helper T cell/B cell interactions.     

B-lineage cell deficits in bone marrow of lpr/lpr mice

Analyses of bone marrow (BM) lymphocytes in C57BL/6 mice homozygousfor the lpr mutation (BS.Ipr) disclosed low numbers of pre-Band B cells, as compared with age-matched control B6 mice. BMdepletion in B6.lpr mice was selective for B-lineage cells,appeared in young adults, and developed markedly with age anddisease progression, contrasting with the peripheral lymphocytehypercellularity. Normalization of pre-B and B cellularity inBM of B6.lprmice was observed after administration of polyclonalIg, that also markedly improved the clinical condition. Isolatedpre-B (B220⁺ IgM^–) cells from B6 or B6.lpr mice, however,showed essentially the same rates of IL-7-dependent proliferationand differentiation to B (lgM⁺) cells in culture, indicatingthat the BM B-lineage deficit is not the result of an intrinsicdefect inB cell generation.     

Toll-like receptor 2 induces mucosal homing receptor expression and IgA production by human B cells

There is a need for developing vaccines that elicit mucosal immunity. Although oral or nasal vaccination methods would be ideal, current strategies have yielded mixed success. Toll-like receptor 2 (TLR2) ligands are effective adjuvants and are currently used in the Haemophilus influenzae type B vaccine. Induction of humoral immunity in the mucosa is critical for effective vaccination; thus, we sought to determine the effects of TLR2 ligands on human mucosal B cell differentiation. We demonstrate that TLR2 ligands induce CCR9 and CCR10 expression by circulating B cells and increased chemotaxis to cognate chemokines CCL25 and CCL28 suggesting that TLR2 induces B cell homing to the gastrointestinal tract. TLR2 stimulation of B cells also induced J chain and IgA production demonstrating the induction of mucosal-like antibody secreting cells. These observations suggest that vaccines containing TLR2-ligands as adjuvants could induce mucosal B cell immunity even when delivered in a non-mucosal manner.     

不稳定型心绞痛病人血清sFas、sFasL及sIL-2R水平的临床价值

目的　探讨血清sFas、sFasL和sIL 2R水平与不稳定型心绞痛 (Unstableanginapectoris,UAP)之间的关系。方法　应用酶联免疫吸附双抗体夹心 (ELISA)法 ,测定了 32例UAP病人 (UAP组 )和 2 0例对照组受试者血清sFas、sFasL和sIL 2R水平。结果　UAP组病人血清sFas和sIL 2R水平均明显高于对照组 (P <0 .0 1) ,而 2组sFasL水平无显著性差别 (P >0 .0 5 )。UAP组病人sFas水平与sIL 2R水平存在显著正相关 (r=0 .4 4 7,P <0 .0 5 )。结论　高水平的血清sFas、sIL 2R与UAP有关。高水平的血清sFas可能通过维持自身免疫炎性反应而导致UAP的发生发展     

肺炎链球菌溶血素经死亡受体/Fas途径和线粒体途径诱导RAW264.7细胞凋亡

目的:探讨肺炎链球菌溶血素(Pneumolysin,Ply)对小鼠RAW264.7细胞的增殖抑制和诱导凋亡的作用及机制。方法:Ply蛋白加入RAW264.7细胞培养上清与细胞共孵育。倒置显微镜观察Ply对RAW264.7细胞形态的影响。MTT法检测Ply对RAW264.7细胞的增殖抑制。Annexin V法检测细胞凋亡率。分光光度法检测Caspase-3、8、9活性。免疫细胞化学法检测到Bax、Fas、Bcl-2蛋白的表达。结果:Ply对小鼠RAW264.7细胞有明显的增殖抑制作用,呈剂量和时间依赖性;1μg/ml Ply处理RAW264.7细胞24小时后,可见典型的凋亡形态学改变;1μg/ml Ply处理RAW264.7细胞1小时和3小时后,细胞凋亡率分别为32.90%和51.56%(P<0.05);1μg/ml Ply蛋白处理RAW264.7细胞24小时,Caspase-3、8、9活性均比对照组升高(P<0.05);免疫细胞化学法检测到Bax、Fas表达较对照组增强,Bcl-2表达减弱(P<0.01)。结论:Ply可诱导小鼠RAW264.7细胞凋亡,诱导凋亡的机制可能是通过死亡受体/Fas途径和线粒体途径双重机制的介导实现。     

乙型肝炎肝组织Fas，FasL和HBV抗原的表达

目的 评价Fas和FasL介导细胞凋亡在乙型肝炎肝损伤中的作用及其与HBV抗原的关系。方法 应用免疫组化方法检测了62例乙型肝炎患者肝组织内的Fas、FasL和HBsAg、HBcAg,并以6例下正常肝组织为对照。结果 Fas/FasL在正常肝组织内无表达;Fas主要在乙型肝炎患者肝细胞质内表达,58例阳性（93．5％）;FasL在肝内浸润的单个核细胞和肝细胞质内均见到,37例阳性（59．7％）。结论 Fas/FasL的表达程度与肝组织活动性炎症程度密切相关;Fas/FasL介导的细胞凋亡可能在乙型肝炎时肝损伤中起重要作用;Fas/FasL表达程度与肝内HBV抗原HBsAg、HBcAg无明显相关性。     

The role of the Fas/Fas ligand system in estrogen-induced thymic alteration

PROBLEM: Estrogen induces atrophy in the thymus by an unknown mechanism. Since the Fas/FasL system is one of the main pathways in T cell apoptosis, we tested the hypothesis that estrogen-induced thymic atrophy is mediated by the Fas/FasL system. METHODS OF STUDY: In vivo experiments were done using ovariectomized female rats treated with estrogen or saline. In vitro experiments were performed using isolated thymocytes. Estrogen receptor (ER) alpha and beta expression was characterized using flow cytometry, RT-PCR and immunofluorescence. Fas and FasL mRNA and protein expression was evaluated using RT-PCR and Western blot analysis respectively. RESULTS: ERalpha and ERbeta are present in thymocytes and stromal cells. ER expression is mainly localized in the Double Positive CD4+CD8+ thymocytes. Estrogen treatment decreases thymus size and increase FasL expression. CONCLUSION: CD4+CD8+ thymocytes and thymic stroma cells express ERalpha and ERbeta. In vivo and in vitro we showed that estrogen treatment increases FasL expression while decreasing thymus cell number. These findings support the hypothesis that estrogen-induced thymic atrophy occurs as a result of apoptosis and is mediated by estrogen-induced FasL expression.     

Differential expression of LFA-3, Fas and MHC Class I on Ad5- and Ad12-transformed human cells and their susceptibility to lymphokine-activated killer (LAK) cells

Adenovirus (Ad) E1A is a potent oncogene and has been shown to deregulate the expression of a large number of cellular genes leading to cellular transformation. Here we have analysed the expression of several immunomodulatory molecules on the surface of a set of human cell lines transformed with either Ad12 or Ad5. Human cells transformed with Ad12 demonstrated reduced expression of cell surface LFA-3, Fas and MHC class I when compared to Ad5-transformed cells. Furthermore, Ad12-transformed human cell lines demonstrated greater susceptibility to lysis by lymphokine-activated killer (LAK) cells, compared to Ad5-transformed human cell lines. In contrast, previous studies with rodent cells showed that both Ad5- and Ad12-transformed rat cells were susceptible to LAK cells. Thus, transformation of human cells with Ad5 or Ad12 results in differences in the expression of immunomodulatory molecules on the cell surface and differential recognition of these virus-transformed cells by immune effector cells.     

瘦素对缺氧复氧L02肝细胞凋亡及Fas/FasL表达的影响

目的： 观察瘦素(leptin)对缺氧复氧人正常肝细胞（L02）凋亡的影响。方法: 将L02细胞分别分为正常对照组、单纯缺氧12 h复氧组(IR组)和缺氧12 h复氧加不同浓度的瘦素（分别为100 μg/L、200 μg/L、400 μg/L、800 μg/L 和1 600 μg/L）干预组, 以流式细胞仪分析、DNA缺口末端标记 (TUNEL) 试验、荧光定量 PCR 等方法观察leptin 对L02肝细胞凋亡、Fas/FasL mRNA表达的影响。结果: （1）与正常对照组相比,IR组细胞凋亡率和TUNEL细胞阳性率增加（P<0.01）,加用不同浓度瘦素干预组的细胞凋亡率和TUNEL细胞阳性率与IR组相比明显下降（P<0.05）;（2）与正常对照组相比,IR组 L02 细胞中Fas/FasL mRNA表达明显上调(P<0.01);加用不同浓度瘦素干预组与IR组相比,Fas/FasL mRNA表达下降,以400 μg/L瘦素作用明显,结果有显著差异（P<0.05）。结论: 瘦素能减轻缺氧复氧培养L02肝细胞的凋亡,其机制可能与其下调细胞中Fas/FasL mRNA的表达有关。     

Antitumor effector B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL‐10

We have previously reported that adoptive transfer of tumor‐draining lymph node (TDLN) B cells confers tumor regression in a spontaneous pulmonary metastasis mouse model of breast cancer. In this study, we identified IL‐10‐producing cells within these B cells, and found that IL‐10 removal, either by using IL‐10^?/? TDLN B cells or by systemic neutralization of IL‐10, significantly augmented the therapeutic efficacy of adoptively transferred TDLN B cells. Depletion of IL‐10 in B‐cell adoptive transfers significantly increased CTLs and B‐cell activity of PBMCs and splenic cells in the recipient. Activated TDLN B cells express Fas ligand, which was further enhanced by coculture of these TDLN B cells with 4T1 tumor cells. Effector B cells killed tumor cells directly in vitro in an antigen specific and Fas ligand‐dependent manner. Trafficking of TDLN B cells in vivo suggested that they were recruited to the tumor and lung as well as secondary lymphoid organs. These findings further define the biological function of antitumor effector B cells, which may offer alternative cellular therapies to cancer.     

CD40 interacts directly with RAG1 and RAG2 in autoaggressive T cells and Fas prevents CD40-induced RAG expression

CD4＋ T cells expressing CD40 （Th40 cells） constitute a pathogenic T-cell subset that is necessary and sufficient to transfer autoimmune disease. We have previously demonstrated that CD40 signals peripheral Th40 cells to induce RAG1 and RAG2 expression, proteins necessary for the expression of T-cell receptor （TCR）, leading to TCR revision. The dependency of TCR expression in the thymus on RAG proteins has long been known. However, despite numerous publications, there is controversy as to whether TCR expression can be altered in the periphery, post-thymic selective pressures. Therefore, a better understanding of TCR expression in primary peripheral cells is needed. We now show that the CD40 protein itself interacts with RAG1 and RAG2 as well as with Ku70 and translocates to the nucleus in Th40 cells. This indicates that the CD40 molecule is closely involved in the mechanism of TCR expression in the periphery. In addition, Fas signals act as a silencing mechanism for CD40-induced RAGs and prevent CD40 translocation to the nucleus. It will be important to further understand the involvement of CD40 in peripheral TCR expression and how TCR revision impacts auto-antigen recognition in order to effectively target and tolerize autoaggressive T cells in autoimmune disease.