Fas expression and apoptosis in human B cells

Mechanisms of B cell apoptosis are critical in reducing aberrant B cell proliferations such as those that arise in autoimmune disease and in B cell malignancies. The physiologic interaction of CD4+ helper T cells and B lymphocytes has been extensively studied over the past two decades. Although CD4+ T cells are considered primarily to offer positive costimulatory signals for B cell differentiation into active immunoglobulin-secreting cells, recent studies have shown that CD4+ T cells are crucial in downregulating the humoral immune response. In the course of cognate interaction between CD40 ligand (CD40L)-bearing CD4+ T cells and CD40-expressing germinal center B cells, CD40 ligation results in augmented Fas expression at the B cell surface. Like CD40L, Fas ligand is expressed on activated CD4+ Th1 cells and when bound to Fas receptor on the B cell surface, initiates an apoptotic signal in that cell. Thus, CD4+ T cells limit the growth of autologous germinal center B cells by first inducing Fas expression and then instigating a death signal via Fas ligand. In this work, we will consider these observations about CD4+ T-cell-induced, Fas-mediated B cell death in the context of other factors that affect apoptosis in B cells, normal and malignant.     

Cellular and molecular factors that regulate the differentiation and apoptosis of germinal center B cells. Anti-Ig down-regulates Fas expression of CD40 ligand-stimulated germinal center B cells and inhibits Fas-mediated apoptosis

To investigate the molecular and cellular mechanisms underlying the selection, differentiation, and apoptosis of germinal center (GC) B cells, we have established a culture system containing a follicular dendritic cell (FDC) line, HK. The mAb, 3C8, which is specific to HK cells and recognizes dendritic network in the GC, was developed and provided additional evidence that HK cells are related to FDC by sharing a unique surface Ag. The roles for CD40 ligand (CD40L) and T cell-derived cytokines in the differentiation of GC B cells were investigated in our culture system. We show that there are two distinct stages of GC B cell differentiation. In the early stage, GC B cells undergo spontaneous apoptosis unless they are stimulated by CD40L. In the secondary stage, IL-10 directs GC B cell differentiation toward the generation of plasma cells, while the absence of IL-10 stimulation leads to the generation of memory B cells. The major function of CD40L was found in the enhancement of cell recovery and the augmentation of memory B cell generation. Although GC B cells are Fas+, GC B cells are at first resistant to, but then become sensitive to, anti-Fas killing after 24 h in culture with CD40L, which coincides with the gradual increase in Fas expression on GC B cells. Furthermore, anti-Ig down-regulated Fas expression on CD40L-stimulated GC B cells, suggesting that Ag receptor engagement down-regulates Fas expression and prevents Fas-mediated apoptosis of GC B cells. Our data imply that GC T cells have an important role in the differentiation and apoptosis of GC B cells. GC T cells expressing both CD40L and Fas ligand have a dual function on GC B cells, helper or killer, depending on the status of target B cells. In the early stage, GC T cells stimulate the extensive proliferation of GC B cells, ensuring a large repertoire of B cells for selection. In the later stage, GC T cells kill B cells via Fas-Fas ligand interactions unless GC B cells are positively selected by Ags present on FDC.     

Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.     

Fas/APO-1 (CD95) expression in myelodysplastic syndromes

Increased apoptosis of myeloid precursors appears to contribute to the pathophysiology of cytopenias in myelodysplastic syndromes (MDS). Fas /APO-1(CD95) is a cell surface protein inducing an apoptotic signal after its binding to Fas ligand or to a functional anti-Fas antibody. Here we studied Fas expression by immunocytochemistry on marrow slides from 30 cases of MDS. Increased Fas expression in erythroblasts and/or immature granulocytes, compared to controls, was seen in 12 (40%) of the cases. In addition, in 16 of the 18 cases with > or = 5% marrow blasts, a variable proportion of blasts expressed Fas. Increased apoptosis was found by morphological analysis and/or TUNEL technique in marrow cells from 8 of the 26 cases analyzed (31%) The ability of Fas antigen to trigger apoptosis was studied after addition of a functional anti Fas antibody in 5 of the patients with Fas overexpression. Addition of this antibody, however, only lead to mild increase of apoptosis in immature granulocytes (but not other myeloid cells) in 2 of the 5 cases. Thus, increased Fas expression is seen in myeloid and/or blast cells in the majority of MDS cases. However, the relationship between this finding and increased apoptosis in MDS still remains to be established.     

Cross-linking CD21/CD35 or CD19 increases both B7-1 and B7-2 expression on murine splenic B cells

Activation of the complement cascade and ligation of complement C3 receptors on B cells represent an important bridge between innate and Ag-specific acquired immunity. We show here that cross-linking of mouse CD21 (complement receptor type 2, CR2, C3d receptor) and CD35 (complement receptor type 1, CR1, C3b/C4b receptor) or co-cross-linking of CD21/CD35 and surface IgM rapidly up-regulates both B7-1 and B7-2 expression on murine resting splenic B cells. CD21/CD35-mediated up-regulation of both B7-1 and B7-2 expression is observed within 14 h, while other stimuli up-regulate only B7-2 but not B7-1 at this early time point. Consistent with the increase in B7 levels, BALB/c B cells on which surface IgM and CD21/CD35 have been co-cross-linked stimulate C57BL/6 T cells more effectively than controls. This CD21/CD35-enhanced allogeneic MLR is blocked nearly completely by anti-B7-2 mAbs and partially by anti-B7-1 mAbs. In addition, cross-linking of CD19, which is physically associated with CD21/CD35, leads to increased B7-1 and B7-2 expression. These data suggest that CD21/CD35 ligation results in enhanced B cell Ag presentation using costimulatory mechanisms shared with other activators and thus works cooperatively in this process. Rapid up-regulation of B7-1 expression, a unique response to CD21/CD35 and CD19 cross-linking, may be a particularly important effect of C3-containing ligands. We propose that CD21/CD35- and CD19-mediated B7-1 and B7-2 up-regulation is an important mechanism by which complement activation links innate and acquired immunity.     

Functional expression of Fas antigen (CD95) on hematopoietic progenitor cells

We investigated the expression of an apoptosis-associated antigen (Fas) (CD95) on hematopoietic progenitor cells in the presence or absence of interferon-gamma (IFN-gamma) and/or tumor necrosis factor-alpha (TNF-alpha). CD34+ cells freshly isolated from bone marrow did not express Fas. However, IFN-gamma and/or TNF-alpha induced the expression of both the mRNA of Fas and Fas itself in a dose-dependent fashion on the surface of CD34+ cells after 48 hours of serum-free culture. IFN-gamma and TNF-alpha had a synergistic effect on the induction of Fas, when both cytokines were added to the culture. The TNF-alpha-induced Fas expression is mediated by p55 TNF-alpha receptor. CD34+ cells cultured in medium alone or with stem cell factor (SCF) showed some slight expression of Fas. When anti-Fas antibody (IgM) was added to CD34+ cells after the induction of Fas expression, CD34+ cells underwent apoptosis, as shown by a decrease in the number of viable cells, morphologic changes, the induction of DNA fragmentation, and a decrease in the number of colony-forming cells (CFC) including colony-forming unit granulocytes/macrophages (CFU-GM) and burst-forming unit erythroids (BFU-E). These observations indicate that IFN-gamma and/or TNF-alpha, well known as negative hematopoietic regulators, induce functional Fas on hematopoietic progenitor cells. The suppression of hematopoiesis by negative hematopoietic regulators may be mediated in part by Fas induction.     

Protein kinase C regulates Fas (CD95/APO-1) expression

Fas (CD95/APO-1) is a transmembrane protein of the TNF/neuron growth factor receptor family. Ligation of Fas by specific Abs or Fas ligand (FasL/CD95 ligand) induces rapid apoptotic cell death in a variety of cell types. Despite progress in understanding the death signals transduced from Fas, very little is known with regard to the mechanisms by which Fas expression is regulated. Using our previously established murine T cell hybridoma model A1.1, we show that specific protein kinase C (PKC) inhibitors could block activation-induced Fas expression and apoptosis. The activation of PKC with PMA or 1-oleoyl-2-acetyl-sn-glycerol could mimic the TCR signal by inducing the expression of Fas but not FasL. PKC-dependent Fas expression was also observed in several murine and human tumor cell lines. Since the inhibition of Ca2+ redistribution by an inhibitor of intracellular Ca2+ mobilization, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride, inhibited TCR-induced FasL but not Fas, the expression of Fas appears to be independent of Ca2+ mobilization. Significantly, expression of the newly identified Fas-regulatory gene, TDAG51, was found to be dependent upon the activity of PKC. PKC activation only induced Fas expression in cells expressing wild-type TDAG51. Thus, Fas expression is likely mediated by PKC through TDAG51.     

Transcription factor AP-2 regulates human apolipoprotein E gene expression in astrocytoma cells

Apolipoprotein E (apoE), one of the major plasma lipoproteins, also is expressed in a variety of cell types, including the glial cells of the nervous system. apoE is involved in processes of degeneration and regeneration after nerve lesions as well as in the pathogenesis of Alzheimer's disease (AD). Glial synthesis of apoE is activated in response to injury both in the peripheral and central nervous system. We now report that the activity of the proximal apoE promoter in astrocytes is upregulated by cAMP and retinoic acid, which act synergistically. Sequence analysis of the apoE promoter indicated the presence of several AP-2 consensus sequences that could mediate the stimulatory effect of cAMP and retinoic acid. The possible functional role of AP-2 was examined by cotransfection of AP-2-deficient HepG2 cells with an apoE promoter construct and a human AP-2 expression construct. Cotransfection with AP-2 significantly elevated apoE promoter activity. DNase I footprinting technique revealed the existence of two binding sites for recombinant AP-2 in regions from -48 to -74 and from -107 to -135 of the apoE promoter. Mutations in these regions markedly impaired the trans-stimulatory effect of AP-2. These results indicate the existence of functional AP-2 sites in the promoter region of apoE that could contribute to the complex regulation of this gene in developmental, degenerative, and regenerative processess of the nervous system.     

A combination of IL-10 and direct contact with plasma cell tumors decreases CD23 expression on splenic B cells

We and others have previously found that splenic B cells from plasma cell tumor-bearing mice exhibit decreased CD23 expression. In the present study we further examined the mechanism of CD23 down-regulation by plasma cell tumors. We show here that although direct contact is required between the tumor cells and B cells, it is not sufficient, since fixed tumor cells do not induce the same reduction in CD23 expression. We have identified IL-10, a cytokine produce by the tumors, as the sole soluble factor that contributes to decreased CD23 expression on B cells induced by plasma cell tumors because 1) Abs to IL-10 prevent the loss of CD23 induced by plasma cell tumors both in vitro and in vivo; 2) engineered IL-10 negative variants of these tumors are reduced in their ability to down-regulate CD23 expression; 3) rIL-10 alone induces partial, but significant, decreases in CD23 expression on normal splenic B cells; and 4) the addition of IL-10 and fixed tumor cells to cultures of normal splenocytes decreases CD23 expression to levels similar to those in cocultures with live tumor cells. Collectively, these results demonstrate that plasma cell tumors down-regulate CD23 expression on B cells by a coordinate mechanism of IL-10 plus contact-mediated events and reveal a novel role for IL-10 in the regulation of CD23 expression on B cells that is suggestive of host B cell activation in the presence of the tumor.     

Dissection of pathways leading to antigen receptor-induced and Fas/CD95-induced apoptosis in human B cells

To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with enhanced BCR death sensitivity and vice versa. In contrast, clones resistant to Fas-mediated apoptosis could still undergo BCR-induced cell death. Based on the functional phenotypes of these clones, we hypothesized that both receptor-induced apoptosis pathways are initially distinct but may eventually converge. Indeed, ligation of both Fas and BCR resulted in cleavage of the IL-1beta-converting enzyme/Ced-3-like protease caspase 3 and its substrates Ac-Asp-Glu-Val-Asp-aldehyde and poly(ADP-ribose) polymerase. Markedly, qualitative differences in the caspase 3 cleavage pattern induced by Fas or BCR ligation were observed; whereas Fas ligation generated caspase 3 cleavage products of 19/20 and 17 kDa, only the latter cleavage product was found upon BCR cross-linking. The caspase inhibitor Val-Ala-Asp-fluoromethylketone blocked both Fas- and BCR-mediated apoptosis, but differentially affected caspase 3 cleavage induced by either stimulus. Finally, overexpression of a Fas-associated death domain (FADD) dominant-negative mutant protein was found to inhibit Fas-induced apoptosis but not BCR-induced apoptosis. Together our findings imply that Fas and BCR couple, via FADD-dependent and FADD-independent mechanisms, respectively, to distinct proteases upstream of caspase 3.     

Functional expression of Fas (CD95) in acute myeloid leukemia cells in the context of CD34 and CD38 expression: possible correlation with sensitivity to chemotherapy

Clinical studies of bone marrow transplantation (BMT) suggest that the immune system contributes to the eradication of acute myeloid leukemia (AML). A recent study also showed that the Fas (CD95/APO1) mediates apoptotic signal from cytotoxic T lymphocytes. Sixty-four patients with AML were studied for the expression of Fas in the context of CD34 and CD38 coexpression. The clinical relevance of Fas expression and function on AML was also investigated. Fas was expressed on 2% to 98% of AML cells (2% to 20% in 11 patients, 20% to 50% in 20 patients, 50% to 80% in 24 patients, and 80% to 98% in nine patients). Only 44.4% of patients with AML M1 (French-American-British [FAB] classification) were Fas+ (>/=20% of leukemia cells expressed Fas), whereas 89.1% of patients with AML M2, M3, M4, M5 were Fas+ (P < .01). Among 43 CD34+ patients (>/=20% leukemia cells were CD34+), 34 were Fas+, and 19 of 21 CD34- patients were Fas+ (P = NS). Thirteen cases were studied for their expression of Fas in the context of CD34 and CD38 using three-color analysis. Fas is expressed at a high level in the gated CD34+CD38+/- and CD34+CD38+ population. In 10 AML samples, Fas was expressed at a higher level in CD34+/CD38+ population than in CD34+/CD38+/- or CD34- cell populations. Fas-induced apoptosis by anti-Fas monoclonal antibody (MoAb) was determined by morphologic features and colorimetric DNA fragmentation assay. Induction of apoptosis was found in 14 of 24 cases. However, no statistically significant correlation was observed between Fas expression and induction of apoptosis. Leukemia colony-forming unit assays suggested that in some cases, Fas-induced apoptosis occurred in the clonogenic cell populations. Parameters such as laboratory and clinical data at initial diagnosis were correlated with Fas expression and only response to initial induction chemotherapy showed significant correlation with Fas expression (P < .05). We conclude that the majority of AML cells exhibit variable expression of Fas, and apoptosis could be induced by anti-Fas MoAb in some cases. Our results suggest the Fas-mediated apoptosis may be clinically relevant, whereas the issue of clonogenic leukemia cells and Fas expression needs further studies.     

HIV disease in children is associated with a selective decrease in CD23+ and CD62L+ B cells

Data on persons who died of cancer of the gastrointestinal tract in a Croatian coastal area with an asbestos-cement plant were analysed for the period 1970-1990. By poll method applied to the families of deceased subjects, additional data on occupation, lifestyle, educational level, length of resistance and cancer mortality among relatives were collected. The investigation showed that in the study area, but also in certain narrower locations within it (subarea settlements), some of the tumours studied occurred at higher rates than expected. Although not conclusive, these findings may indicate a role of environmental exposure to asbestos, particularly in the occurrence of peritoneal mesothelioma.