Rescue of ileal Peyer's patch B cells from apoptosis is associated with the induction of Bcl-2 expression.

The major site of B-cell genesis in the sheep is the ileal Peyer's patch (PP). The B cells in the ileal PP undergo both extensive proliferation and massive death in association with an ongoing diversification of the immunoglobulin repertoire by somatic hypermutation. Most, if not all, the B-cell death in the ileal PP is due to apoptosis. When placed in culture, ileal PP B cells undergo rapid apoptosis. Here, we investigated the expression of the proto-oncogene bcl-2 in ileal PP cells in situ and in culture. Bcl-2 expression has been correlated with the prevention of apoptosis in many cell types. Western blotting, using anti-Bcl-2 monoclonal antibodies, revealed that a Bcl-2-reactive protein of 26,000 MW was expressed in ileal mesenteric lymph node cells, splenocytes and thymocytes from sheep, but was barely detectable in ileal PP B cells in situ or in culture. However, Bcl-2 expression could be markedly induced in ileal PP B cells cultured with phorbol ester and Ca2+ ionophore, a procedure that is known to rescue these cells from apoptosis. We hypothesize that those few B cells that survive a selection event in the ileal PP may begin to express elevated levels of Bcl-2 as they escape from the apoptotic pathway.     

Psoriasis in humans is associated with down-regulation of galectins in dendritic cells

We have investigated the expression and role of galectin‐1 and other galectins in psoriasis and in the Th1/Th17 effector and dendritic cell responses associated with this chronic inflammatory skin condition. To determine differences between psoriasis patients and healthy donors, expression of galectins was analysed by RT‐PCR in skin samples and on epidermal and peripheral blood dendritic cells by immunofluorescence and flow cytometry. In the skin of healthy donors, galectin‐1, ‐3 and ‐9 were expressed in a high proportion of Langerhans cells. Also, galectins were differentially expressed in peripheral blood dendritic cell subsets; galectin‐1 and galectin‐9 were highly expressed in peripheral myeloid dendritic cells compared with plasmacytoid dendritic cells. We found that non‐lesional as well as lesional skin samples from psoriasis patients had low levels of galectin‐1 at the mRNA and protein levels, in parallel with low levels of IL‐10 mRNA compared with skin from healthy patients. However, only lesional skin samples expressed high levels of Th1/Th17 cytokines. The analysis of galectin‐1 expression showed that this protein was down‐regulated in Langerhans cells and dermal dendritic cells as well as in peripheral blood CD11c⁺ DCs from psoriasis patients. Expression of galectin‐1 correlated with IL‐17 and IL‐10 expression and with the psoriasis area and index activity. Addition of galectin‐1 to co‐cultures of human monocyte‐derived dendritic cells with autologous T lymphocytes from psoriasis patients attenuated the Th1 response. Conversely, blockade of galectin binding increased IFNγ production and inhibited IL‐10 secretion in co‐cultures of monocyte‐derived dendritic cells with CD4⁺ T cells. Our results suggest a model in which galectin‐1 down‐regulation contributes to the exacerbation of the Th1/Th17 effector response in psoriasis patients. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

KIR down-regulation on NK cells is associated with down-regulation of activating receptors and NK cell inactivation

We previously reported that killer cell immunoglobulin-like receptors (KIR) could be down-regulated from the surface of T cells. Here, we show that KIR down-regulation is also induced on the surface of natural killer (NK) cells upon ligand binding. Common down-regulation characteristics are found on these two cell types: a slow kinetics and a phenomenon observed for long inhibitory forms only. Importantly, KIR down-regulation on NK cells is associated with a down-regulation of activating receptors (CD16, CD2 and 2B4) as well as with a lack of cell responsiveness (antibody-dependent and natural killing activities). This unresponsive state was not observed for MHC-restricted T cells. Our data implicate that, in addition to prevention of the immediate target cell lysis, KIR-MHC class I interactions may also regulate the subsequent NK cell cytotoxic activity. This observation opens new perspectives in the understanding of NK cell regulation.     

Human Mast Cell Apoptosis Is Regulated Through Bcl-2 and Bcl-XL

It is well established that human mast cell proliferation and maturation are regulated by kit ligand (stem cell factor). Little is known, however, about how these two processes are negatively regulated and thus, how mast cell number is controlled in normal and pathologic conditions. We therefore first hypothesized that SCF-dependent human mast cells would undergo programmed cell death (apoptosis) on removal of SCF as has been shown for growth factor-dependent rodent mast cells. We then examined whether SCF acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. As hypothesized, elimination of SCF from primary peripheral blood-derived human mast cell cultures resulted in a significant apoptotic process. During apoptosis, down-regulation of the two apoptosis-regulatory proteins Bcl-2 and Bcl-X_Lwas observed. Moreover, a deregulated expression of these two proteins was found in two human mast cell lines which are SCF-independent. Thus, SCF functions as a survival factor by repressing apoptosis of human mast cells through Bcl-2 and Bcl-X_L. Deregulated expression of these antiapoptotic proteins may contribute to proliferation and accumulation of mast cells in certain forms of systemic mast cell disorders.     

Modulation of Bcl-2 family proteins in primary endothelial cells during apoptosis

We studied the expression of Bcl-2 family proteins during cytokine- and verotoxin (VT)-induced apoptosis in primary human umbilical vein endothelial cells (HUVECs). Our experiments demonstrated that high initial expression of Bcl-2 protein was significantly downregulated in HUVECs treated with IFN-gamma whereas TNF-alpha gave a less pronounced decrease in Bcl-2 level. Treatment with the combination of cytokines was more efficient in downregulating Bcl-2 protein. HUVECs pretreated with cytokines and incubated with VT gave a further significant decrease in Bcl-2 level. Simultaneous measurement of Bcl-xl level did not reveal any significant changes. Bax protein was upregulated in HUVECs stimulated with TNF-alpha alone or in combination with IFN-gamma. However, addition of VT did not give any further increase in Bax level suggesting that Bax upregulation is more important for cytokine- rather than VT-mediated apoptosis. Total endothelial cell growth factor deprivation gave a significant increase in apoptosis accompanied by a decrease of Bcl-2 in apoptotic cells while Bcl-xl and Bax levels were unaffected. Our data indicate that anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax are reciprocally regulated during apoptosis, whilst Bcl-xl is essentially unaffected. This implies that Bcl-2/Bax ratio rather than Bcl-xl controls apoptosis in primary endothelial cells.     

Staurosporine-induced apoptosis in astrocytes is prevented by A1 adenosine receptor activation

Astrocyte apoptosis occurs in acute and chronic pathological processes at the central nervous system and the prevention of astrocyte death may represent an efficacious intervention in protecting neurons against degeneration. Our research shows that rat astrocyte exposure to 100 nM staurosporine for 3 h caused apoptotic death accompanied by caspase-3, p38 mitogen-ed protein kinase (MAPK) and glycogen synthase kinase-3β (GSK3β) activation. N⁶-chlorocyclopentyladenosine (CCPA, 2.5–75 nM), a selective agonist of A₁ adenosine receptors, added to the cultures 1 h prior to staurosporine, induced a dose-dependent anti-apoptotic effect, which was inhibited by the A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. CCPA also caused a dose- and time-dependent phosphorylation/activation of Akt, a downstream effector of cell survival promoting phosphatidylinositol 3-kinase (PI3K) pathway, which in turn led to inhibition of staurosporine-induced GSK3β and p38 MAPK activity. Accordingly, the anti-apoptotic effect of CCPA was abolished by culture pre-treatment with LY294002, a selective PI3K inhibitor, pointing out the prevailing role played by PI3K pathway in the protective effect exerted by A₁ receptor activation. Since an abnormal p38 and GSK3β activity is implicated in acute (stroke) and chronic (Alzheimer's disease) neurodegenerative diseases, the results of the present study provide a hint to better understand adenosine relevance in these disorders.     

Bcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cancer cells

To dissect apoptotic genes governing the survival of colorectal carcinoma cells, we employed RNAi to silence Bcl-2 and Bcl-x(L) in isogenic clones of p53+/+ and p53-/- cells, and of Bax+/- and Bax-/- cells. We identify a novel proapoptotic function of p53 that does not require activation by genotoxic agents and that appears to be constitutively suppressed by Bcl-2. Silencing of Bcl-2 induced massive p53-dependent apoptosis. The "Bcl-2/p53 axis" requires Bax and caspase 2 as essential apoptotic mediators. This newly discovered Bcl-2/p53 functional interface represents a key regulator of apoptosis which can be activated by targeting Bcl-2 in colorectal carcinoma cells.     

Clonal Th2 cells associated with chronic hypereosinophilia: TARC-induced CCR4 down-regulation in vivo

We analyzed the expression of chemokine receptors on clonal Th2-type CD4(+)CD3(- )lymphocytes isolated from blood of two patients with chronic hypereosinophilia. First, we observed that these Th2 cells express membrane CCR5 and CXCR4 but neither CCR3 nor CCR4 when analyzed immediately after purification. However, CCR4 appeared following culture in human serum-free medium, suggesting that it was down-regulated in vivo. Indeed, patient's serum, but not control human serum, strongly down-regulated CCR4 expression on cultured Th2 cells. As high levels of TARC, a CCR4 ligand, were detected in the serum of four hypereosinophilic patients with CD3(-)CD4(+) clonal Th2 cells, we evaluated the effect of TARC neutralization in this system. Addition of a neutralizing anti-TARC mAb inhibited CCR4 down-regulation by patient's serum, indicating that circulating TARC contributed to CCR4 down-regulation on Th2 cells in vivo. Clonal Th2 cells did not secrete high levels of TARC themselves but induced a sustained production of TARC by monocyte-derived dendritic cells, a phenomenon that was inhibited by addition of blocking mAb against IL-4 receptor. We conclude that high circulating levels of TARC in serum of patients with chronic hypereosinophilia, most likely derived from antigen-presenting cells stimulated by Th2-type cytokines, induce down-regulation of CCR4 on Th2 cells in vivo.     

含蟾酥胶囊血清诱导人肝癌BEL-7402细胞凋亡并下调Bcl-2的表达

目的 探讨含蟾酥胶囊血清诱导人肝癌BEL-7402细胞凋亡的作用机制.方法 用中药血清药理学方法,不同浓度含药血清加入体外培养的人肝癌BEL-7402细胞,分别孵育24h、48h,显微镜下观察细胞形态学变化,MTT比色法检测细胞存活率,流式细胞仪检测细胞凋亡率,琼脂糖凝胶电泳测定DNA梯状条带,免疫细胞化学染色检测Bc...     

The interaction of Bcl-2 and Bax regulates apoptosis in biliary epithelial cells of rats with obstructive jaundice

 A complex molecular network controls cell homeostasis by inducing apoptosis or proliferation. The balance of Bcl-2 and Bax, members of a protein family, determines whether a cell will become immortal (Bcl-2) or will undergo apoptosis (Bax). To determine the role of Bcl-2 and Bax during proliferation of biliary epithelial cells (BEC) after bile duct ligation (BDL) and their regression after biliary decompression we induced hyperplasia of BEC by BDL in male rats. Regression of hyperplastic BEC by way of apoptosis was induced by biliary decompression through a Roux-en-Y biliodigestive anastomosis. To quantify apoptosis a modified TUNEL assay was used. Expression of Bcl-2 and Bax was visualized by immunohistochemistry and quantified stereologically. BEC increased from <1% to >20% after BDL; this increase was associated with overexpression of Bcl-2 in up to 30% of hyperplastic BEC. After biliodigestive anastomosis, apoptotic BEC increased from <0.1% to a peak of 5.4% after 1 day to reach baseline again 1 week after decompression. This was associated with de novo appearance of Bax. The interaction between Bcl-2 and Bax triggers apoptosis in BEC and acts as a cell rheostat in BEC hyperplasia and its involution after biliary decompression. Received: 9 July 1998 / Accepted: 16 December 1998     

Enhanced B cell survival in familial macroglobulinaemia is associated with increased expression of Bcl-2.

A family with three cases of macroglobulinaemia of undetermined significance (MGUS), and one case each of immunoblastic lymphoma, Waldentröm's macroglobulinaemia and multiple myeloma was first described 20 years ago. We have previously identified 10 out of 35 healthy family members tested whose lymphocytes produced abnormally high amounts of immunoglobulins in culture. In the present study lymphocyte subpopulations of these hyper-responders have been further characterized and lymphocyte reactivity and survival in vitro have been studied. No differences were detected in the proportions of resting B lymphocytes (CD19⁺) co-expressing CD5, CD10, CD11b, or CD38, and the CD4/CD8 ratio of T cells was normal before and after stimulation with pokeweed mitogen (PWM). The initial rate of response in terms of immunoglobulin production was not increased, but immunoglobulin levels continued to rise during the second week of culture whereas the production peaked at 8 days in control cultures. This was associated with significantly greater survival of lymphocytes and at 14 days surviving B cells could only be identified in samples from hyper-responders. A lymph node removed because of tuberculosis from a family member 23 years before the diagnosis of multiple myeloma showed very marked Bcl-2 expression in a B cell follicle. This was not seen in a tuberculous lymph node from an unrelated subject. Stimulated cultures from three hyper-responders tested demonstrated significantly higher retention of Bcl-2 in B cells compared with one family control and six unrelated controls. We conclude that the increased production of immunoglobulins previously observed in this family with an inherited tendency for benign and malignant B cell proliferation is the result of enhanced B cell survival, which is associated with increased expression of Bcl-2 following stimulation.     

Measles virus-induced down-regulation of CD46 is associated with enhanced sensitivity to complement-mediated lysis of infected cells

CD46, the major component of the measles virus (MV) receptor complex and a member of the regulators of complement activity (RCA) gene cluster, is down-regulated in MV-infected cells. We investigated whether the reduction of surface CD46 correlates with enhanced sensitivity of lymphoid and monocytic cells to lysis by activated complement. On human U937 cells, acutely or persistently infected with MV-Edmonston (ED) vaccine strain, infection-dependent down-regulation of CD46 confers sensitivity to activated complement, regardless of the pathway of activation and the specificity of the activating antibodies. Interestingly, down-regulation of CD46 alone is sufficient to confer susceptibility of cells to complement lysis despite the continued surface expression of other RCA proteins such as CD35 and CD55. In primary cultures, both peripheral blood lymphocytes and macrophages are efficiently lysed in the presence of complement activated via the alternative pathway after MV infection. In contrast to the MV-ED infection, infection of cells with the lymphotropic MV wild-type strain WTF does not down-regulate CD46. Cells infected with MV-WTF do not exhibit enhanced susceptibility to complement lysis. These data suggest that MV strains similar to WTF that do not down-regulate CD46 may have an enhanced potential for replication and dissemination within the human host, whereas complement-mediated elimination of cells infected with CD46-down-regulating strains of MV, such as ED, may limit the spread of MV infection, and could thus represent an attenuating factor for MV.     

Bcl-2 blocks peroxynitrite-induced apoptosis in HL-60 cells,an association with reactive oxygen species

Inflammation Research -