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Biomedical subjects

M Sarfati

Publications and source records attributed to M Sarfati.

At least 19 recordsLinked to original sources
PubMed1992

Interleukin 4 protects chronic lymphocytic leukemic B cells from death by apoptosis and upregulates Bcl-2 expression.

B chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of slow-dividing and long-lived monoclonal B cells arrested at the intermediate stage of their differentiation. We previously showed that interleukin 4 (IL-4) not only inhibits but also prevents the proliferation of B-CLL cells. We report here that IL-4 protects the B-CLL cells from death by apoptosis (programmed cell death [PCD]). IL-4 inhibits spontaneous and hydrocortisone (HC)-induced PCD of highly purified B cells from 12 unselected CLL patients, as shown by sustained cell viability and lack of DNA fragmentation. IL-1, -2, -3, -5, -6, -7, tumor necrosis factor alpha, and transforming growth factor beta have no protective effect. The in vitro rescue from apoptosis by IL-4 is reflected by an increased expression of Bcl-2 protein, a proto-oncogene directly involved in the prolongation of cell survival in vivo and in vitro. Hence, IL-4-treated B-CLL cells express significantly more Bcl-2 than unstimulated, HC-treated, or fresh B-CLL cells. Furthermore, subcutaneous injection of IL-4 into one CLL patient enhances Bcl-2 protein expression in the leukemic B cells. These data may suggest that IL-4 prevents apoptosis of B-CLL cells using a Bcl-2-dependent pathway. Given our recent observations that fresh T cells from B-CLL patients express IL-4 mRNA, we propose that IL-4 has an essential role in the pathogenesis of CLL disease, by preventing both the death and the proliferation of the malignant B cells.

Adult↗
PubMed1992

Ligation of CD23 triggers cAMP generation and release of inflammatory mediators in human monocytes.

Transduction through the CD23 molecule (Fc epsilon RII) was analyzed in normal human monocytes using monoclonal antibodies to CD23 (MHM6 and 135) and IgE/anti-IgE immune complexes. Monocytes expressing an increased amount of CD23 molecules were obtained by stimulation with IL-4 (30 U/ml). Anti-CD23 mAb as well as IgE/anti-IgE immune complexes were unable to induce any significant calcium mobilization [Ca2+]i in CD23-bearing monocytes whereas they elicited [Ca2+]i increase in B lymphocytes of the same donors. Despite their failure to induce calcium mobilization, the same CD23 ligands triggered a dose-dependent increase of intracellular cAMP, with a maximum 20 to 30 min after the onset of stimulation. This effect is mediated via CD23 inasmuch as: 1) F(ab)'2 fragments are as active as intact anti-CD23 mAb and 2) it is not observed in CD23- monocytes. The increase in cAMP was only partially altered in the presence of 1 microM indomethacin suggesting that it was not due to the release of PG. The possible role of CD23 in the activation of human monocytes was next documented by showing that anti-CD23 mAb and IgE/anti-IgE immune complexes induced the generation of IL-6 and of thromboxane B2 by CD23+ but not by CD23- monocytes. In addition, the IgE/anti-IgE-induced IL-6 production was potentiated in the presence of cAMP inducer such as the beta 2-adrenoceptor agonist salbutamol. These results indicate that ligation of CD23 induces cAMP generation in CD23+ human monocytes and that CD23 may regulate the IgE-dependent functions in normal human monocytes.

Acute-Phase Reaction↗
PubMed1992

IL-4 induces conformational change of CD20 antigen via a protein kinase C-independent pathway. Antagonistic effect of anti-CD40 monoclonal antibody.

The CD20 molecule is a unique phosphoprotein exclusively expressed on B cells during most stages of B cell ontogeny. We here report that rIL-4 down-regulates the expression of CD20 with anti-Leu-16 mAb (clone L27) on both unstimulated and anti-mu preactivated normal and leukemic B cells. None of the other recombinant lymphokines tested (IL-1, IL-2, IL-3, IL-6, IFN-alpha, and IFN-gamma, granulocyte/macrophage-CSF, transforming growth factor-beta, TNF-alpha, and lymphotoxin) decreased CD20 expression. Incubation of unstimulated or anti-mu preactivated B cells with IL-4 did not affect the steady state CD20 mRNA, suggesting that IL-4 exerted its effect mainly at a nontranscriptional level. Hence, IL-4 selectively down-regulates the CD20 epitope recognized by clone L27 without affecting seven other different epitopes, indicating that IL-4 acts by modifying the conformation of the CD20 molecule rather than by inhibiting its production or inducing its internalization. IL-4 most likely utilizes a protein kinase C-independent signal transduction pathway to modify CD20 molecule inasmuch as staurosporine, an inhibitor of protein kinase C, antagonizes phorbol esters (PMA) but not IL-4-induced CD20 down-regulation. In contrast, anti-CD40 mAb reverses the IL-4 but not the PMA inhibitory effect on CD20 expression. Given that CD20 may be part of a Ca2+ ion channel and plays a role in B cell activation and proliferation, it is proposed that the ability of anti-CD40 mAb to maintain the CD20 molecule in a given epitopic configuration on IL-4-stimulated B cells may be related to the long term proliferation of normal B cells that are strictly dependent on the presence of IL-4 and cross-linked anti-CD40 mAb for their continuous growth.

Antibodies, Monoclonal↗
PubMed1992

The interleukin-1 receptor in normal and osteoarthritic human articular chondrocytes. Identification as the type I receptor and analysis of binding kinetics and biologic function.

OBJECTIVE: To identify and investigate the kinetic binding properties of interleukin-1 receptors (IL-1R), and examine the abilities of the 2 IL-1 isoforms to stimulate metalloprotease synthesis, in normal and osteoarthritic (OA) chondrocytes. METHODS: Receptor affinity and density were determined using radioligand binding experiments and flow cytometry. Immunocytochemical analysis and affinity cross-linking studies were performed for characterization of IL-1R. RESULTS: While no difference in receptor affinity between normal and OA chondrocytes was noted in binding studies (Kd approximately 30 pM), a 2-fold increase in receptor density was found in OA chondrocytes as compared with normal chondrocytes (mean 4,069 sites/cell versus 2,315 sites/cell). Flow cytometry experiments also showed a significant increase in receptor density in OA cells, as well as an enhancement in the percentage of positive cells in diseased cartilage compared with normal. Binding data for both IL-1 isoforms revealed a single class of binding sites and receptor specificity. Factors such as IL-2, interferon-gamma, tumor necrosis factor alpha, and bovine insulin did not compete with IL-1 beta. By covalent ligand cross-linking and electrophoretic analysis, only type I IL-1R, a protein of 80 kd, was detected on chondrocytes. By immunocytochemical analysis, IL-1R was identified at the cell membrane level, in both normal and OA chondrocytes. The presence of nuclear staining was also observed, but only in OA chondrocytes. Recombinant human IL-1 (alpha and beta) induced the secretion of stromelysin and collagenase in a dose-dependent manner. The IL-1 concentration required for half-maximal metalloprotease stimulation was 3-4 times lower in OA chondrocytes than in normal cells. CONCLUSION: These results indicate that OA chondrocytes have a higher sensitivity to the stimulation of metalloprotease synthesis by IL-1 than do normal cells. This could be related to the increased levels of IL-1R expressed in the OA cells. The implications of these findings with regard to the possible roles of IL-1 and IL-1R in the pathogenesis of OA are discussed.

Aged↗
PubMed1992

Low-molecular weight B cell growth factor (BCGF-12KD) as an autocrine growth factor in B cell chronic lymphocytic leukemia.

The role of cytokines in the growth and spreading of human hematologic malignancies has been underlined in recent years. Here we report evidence that a human 12-kDa B cell growth factor (BCGF-12KD) may function as a growth stimulatory factor in B cell chronic lymphocytic leukemia (B-CLL). First, recombinant BCGF-12KD induced dose-dependent DNA synthesis in neoplastic B cells of four B-CLL patients tested. Second, seven different B-CLL clones secreted a BCGF-12KD-like product that accounted for their proliferation. A neutralizing monoclonal antibody (mAb; Ac8) directed against a BCGF-12KD synthetic peptide inhibited the spontaneous growth of the leukemic B cells. The same mAb blocked DNA synthesis in normal tonsillar B lymphocytes induced by the culture supernatant of spontaneously proliferating B-CLL cells. Finally, BCGF-12KD mRNA was expressed in freshly isolated (two of three patients) as well as in vitro proliferating B-CLL cells (three of three patients). These findings strongly suggest that BCGF-12KD can modulate the growth of B-CLL cells in vivo as well as in vitro. They may offer significant insights into the biology of this frequent B lymphoproliferative disorder.

Adult↗
PubMed1992

Cytokine effects of CD23 are mediated by an epitope distinct from the IgE binding site.

Human CD23 and its soluble forms (sCD23) display various biological activities, in addition to their IgE binding function (IgE/BF). The IgE binding domain was recently mapped to residues between Cys163 and Cys282 but its involvement in IgE-independent, CD23 functions remains unknown. In order to clarify this point, a series of N-terminal, C-terminal and internal deletion mutants of CD23 or sCD23 were expressed in CHO cells and tested for their ability (i) to bind to IgE, (ii) to induce colony formation by human myeloid precursor cells, (iii) to promote mature T cell marker expression by early prothymocytes, and (iv) to regulate IgE synthesis. The present study indicates that cytokine activities require the presence of Cys288, while this amino acid is not necessary for IgE/BF. Blocking experiments using various conformation-sensitive monoclonal antibodies further suggest that active epitope(s) of CD23 in cytokine assays is(are) distinct from those involved in IgE/BF.

Animals↗
PubMed1992

Expression, regulation and function of human Fc epsilon RII (CD23) antigen.

CD23, also known as the low affinity receptor for IgE (Fc epsilon RII), belongs to a novel superfamily of type-II integral membrane proteins. Fc epsilon RII expression was originally described on B cells but subsequent studies showed that CD23 is expressed on a variety of hematopoietic cells and is regulated by several cytokines (i.e., interleukin-4, interferons) in a tissue-specific manner. In some pathological conditions such as B-chronic lymphocytic leukemia, the CD23 gene is abnormally regulated resulting in CD23 overexpression. CD23 is not only an IgE receptor but also a membrane-bound precursor of soluble molecules that still bind IgE (sCD23 or IgE-binding factors). The functions of membrane CD23 are IgE-dependent and vary according to the cell types on which it is expressed. In contrast, sCD23, in addition to being an IgE regulatory molecule, displays multiple cytokine activities that are IgE-independent.

B-Lymphocytes↗
PubMed1992

CD23 antigen regulation and signaling in chronic lymphocytic leukemia.

B lymphocytes from patients with chronic lymphocytic leukemia (B-CLLs), strongly express the CD23 antigen, a surface marker with significant prognostic importance in this disease. Because we previously reported that IL-4 shows a poor capacity for CD23 expression on B-CLLs, we first examined the possible mechanisms underlying CD23 overexpression on B-CLLs and found that mitogen-activated CLL T cells release soluble factors that are capable, in synergy with IL-4, of strongly inducing CD23. Using neutralizing Abs, we noticed that the T-cell-derived enhancing activity is entirely ascribed to the combined effects of IFN gamma (potent inhibitor of CD23 on normal B cells), TNF alpha (which has no effect on normal B cells), and IL-2 (which has a slight enhancing effect on both CLL and normal B cells). Furthermore, recombinant IFN gamma as well as IFN alpha, TNF alpha, and IL-2 (but not IL-3, IL-5, IL-6, IL-7, and lymphotoxin) significantly enhance CD23 protein and mRNA expression on B-CLLs, in the presence or absence of IL-4. Inasmuch as optimal CD23 expression absolutely requires the combination of IFN gamma, IL-2, TNF alpha (the production of which is increased in CLL disease), and IL-4, it was relevant to show that IL-4 mRNA is indeed expressed in fresh T-CLL cells. We next examined the possible role of CD23 in the regulation of B-CLL proliferation. Signaling through CD23 via ligation of the antigen by F(ab')2 anti-CD23 MAb but not Fab fragments inhibits the cytokine-induced B-CLL DNA synthesis. It is concluded that the CD23 gene is abnormally regulated in B-CLL disease and that cross-linking of CD23 molecule delivers a negative growth signal to the leukemic B cells.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1992

Native and recombinant soluble CD23 fragments with IgE suppressive activity.

CD23-bearing cells are known to release 37,000 33,000 and 25,000 MW soluble CD23 (sCD23) fragments that were reported to display multiple biological activities, including the potentiation of IgE synthesis. We previously reported that tunicamycin treatment of RPMI-8866 cells switched the biological activity of the sCD23 released by these cells from IgE potentiation to IgE suppression. In this study we show that tunicamycin-treated cells release small CD23 fragments with a MW of 16,000. These fragments are formed by truncation of the N-terminal 160 amino acids and truncation of the carboxy-terminal end of CD23. Two observations indicate that the cleavage of surface CD23 into 16,000 MW fragments is not caused by tunicamycin-mediated inhibition of the N-glycosylation of CD23 but rather by the deletion of the carboxy terminal end of the molecule: (1) Chinese hamster ovary (CHO) transfectants expressing a CD23 mutant lacking the N-glycosylation site release 37,000-33,000 MW sCD23 unless they are treated with tunicamycin; (2) transfectants expressing a CD23 deletion mutant lacking the last 33 carboxy-terminal amino acids release 16,000 MW sCD23. Highly purified native and recombinant 16,000 MW sCD23 bind to IgE and down-regulate the ongoing and the interleukin-4 (IL-4)-stimulated synthesis of IgE.

Animals↗
PubMed1991

The in vivo expression of type B CD23 mRNA in B-chronic lymphocytic leukemic cells is associated with an abnormally low CD23 upregulation by IL-4: comparison with their normal cellular counterparts.

We recently reported that the sera of chronic lymphocytic leukemia (CLL) patients contained 3-500 times more soluble CD23 (or IgE-BF) than the sera of patients with other lymphoproliferative diseases or normal individuals and that their B cells (B-CLLs) overexpressed CD23 Ag. In the present report, we extended these studies and showed that CD5+ B cells from all CLL patients (n = 15) co-express CD23 Ag. We next identified two additional major differences between B-CLLs and normal adult B cells. First, in contrast to normal adult B cells which exclusively express type A CD23 mRNA, freshly isolated B-CLLs expressed both type B and type A CD23 mRNA. Second, although IL-4 is a potent inducer of type B CD23 mRNA on normal B cells, an optimal concentration of IL-4 infranormally upregulated CD23 on highly purified B-CLLs both at the protein and at the molecular levels. However, co-stimulation of CLL PBMC with phytohemagglutinin (PHA) and IL-4 strongly upregulated CD23 on B-CLLs, reconstituting the high level of CD23 expression observed in vivo. We next attempted to relate B-CLLs to the CD5+ B cell subpopulations present in peripheral blood mononuclear cells (PBMC, n = 3), cord blood mononuclear cells (CBMC, n = 6) and tonsillar lymphocytes (TONS, n = 3) by analysing their co-expression of CD20, CD5 and CD23 Ag and their phenotypic regulation by IL-4. Our results indicated that B-CLLs presented some features in common with the CD23+ umbilical cord blood B cells in as much as, like in B-CLLs; (i) all CD23+ cord blood cells co-expressed CD5 Ag, (ii) freshly isolated CBMC expressed both type A and type B CD23 mRNA, and finally (iii) these cells weakly re-expressed CD23 Ag upon IL-4 stimulation as compared to adult PBMC.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1991

Antiproliferative effect of interleukin-4 in B chronic lymphocytic leukemia.

Recombinant interleukin-4 (IL-4) profoundly inhibits the proliferative response of chronic lymphocytic leukemic B cells (B-CLLs) to recombinant interleukin-2 (IL-2). In the present study, we confirmed and extended these data by showing that IL-4 strongly suppresses the [3H]thymidine incorporation by B-CLLs stimulated by recombinant tumor necrosis factor alpha, recombinant interferon alpha, IL-2, and low molecular weight B cell growth factor in the absence of costimulant. Recombinant interleukin-4 inhibits spontaneous DNA synthesis suggesting that it also interferes with the autocrine proliferation of these cells. Kinetic studies indicate that IL-4 suppresses rather than shifts the peak of cytokine-induced DNA synthesis. Moreover, IL-4 blocks the progression of B-CLLs in or into G1 stage of the cell cycle as shown by the inhibition of cytokine-induced [3H]uridine incorporation. Finally, IL-4 pretreatment of B-CLLs prevents their subsequent proliferative response to the above cytokines, indicating that IL-4 confers to the B-CLLs a state of resistance to numerous stimulatory cytokines. The antiproliferative effects of IL-4 suggest that this lymphokine may have important therapeutic implications for the B-CLL patients.

Cell Cycle↗
PubMed1991

Glucocorticoids increase the synthesis of immunoglobulin E by interleukin 4-stimulated human lymphocytes.

This study indicates that hydrocortisone (HC) markedly increases the synthesis of immunoglobulin E (IgE) by interleukin 4 (IL-4)-stimulated human lymphocytes. The effect is glucocorticoid specific and is obtained with low concentrations of HC (0.1-10 microM). In both the early and the late phase of the IL-4-induced response HC exerts its effects which are respectively IL-4 dependent and IL-4 independent. The IgE potentiation cannot be explained by the inhibition of interferon-gamma (IFN-gamma) production since it is observed in the absence of endogenous secretion of IFN-gamma. HC inhibits the production of IgE-binding factors (soluble CD23) and the expression of the low-affinity receptor for IgE, also known as the (Fc epsilon RII) CD23 antigen; however, the residual expression of Fc epsilon RII by IL-4- and HC-treated peripheral blood mononuclear cells (PBMCs) is important since the IgE response of these cells is markedly inhibited by anti-CD23 monoclonal antibody. HC acts mainly by amplifying the cellular interactions between monocytes and lymphocytes; indeed, HC has no effect on monocyte-depleted PBMCs, and moreover, monocytes cannot be replaced by soluble factors. Most importantly, T cells are not required for the induction of IgE synthesis by costimulation with IL-4 and HC. However, the IgE response of rigorously T cell-depleted PBMCs may be further increased by the addition of T cells. Further analysis of the permissive effect of HC on the synthesis of IgE by T cell-depleted PBMCs suggests that HC acts in synergy with IL-4 to trigger the activation and the differentiation of B cells into IgE-producing cells.

Antigens, CD↗
PubMed1990

1,25(OH)2 vitamin D3 inhibits the CD23 gene expression by human peripheral blood monocytes.

A biologically active form of vitamin D, 1,25(OH)2D3, and a leukocyte surface molecule, CD23, play important roles in immune regulation. The effect of 1,25(OH)2D3 on the CD23 gene expression was examined in this study. The results show that 1,25(OH)2D3 suppresses spontaneous and IL-4-stimulated CD23 synthesis by peripheral blood monocytes. The inhibition occurs at both the protein and mRNA levels. However, 1,25(OH)2D3 has no inhibitory effect on CD23 production by either resting or activated tonsillar B cells. Our results suggest a possible role of 1,25(OH)2D3 in the regulation of certain immune and inflammatory responses via its effect on CD23 production.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1990

Monoclonal anti-CD23 antibodies induce a rise in [Ca2+]i and polyphosphoinositide hydrolysis in human activated B cells. Involvement of a Gp protein.

Transduction through the CD23 molecule (Fc epsilon RII) was analyzed in human activated B lymphocytes using anti-CD23 mAb. B cell blasts expressing an increased amount of surface CD23 molecule were obtained by stimulation of normal peripheral blood B lymphocytes with Staphylococcus aureus strain Cowan I and IL-4. Anti-CD23 mAb were found to trigger polyphosphoinositide hydrolysis in these cells (and also in tumoral B cells expressing spontaneously CD23) and a rise in [Ca2+]i which could be attributed to mobilization from cytoplasmic pools. This increase in [Ca2+]i could be mimicked, with a comparable time-course, by the addition of InsP3 to permeabilized B cell blasts indicating that the increase in inositol phosphate accumulation induced by the antibodies was due to a preferential attack of phosphatidylinositol-bisphosphate by a specific phosphoinositidase C (PIC). In permeabilized cells, raising the free calcium concentration above 3 microM was found to induce polyphosphoinositides hydrolysis and to activate directly the PIC. Addition of 100 microM GTP-tetralithium salt, a non-hydrolyzable analogue of GTP, also resulted in an increased accumulation of inositol phosphates. A Ca2(+)-dependent PIC, linked to a GTP-binding protein (Gp protein), can thus be activated in B cell blasts. Addition of anti-CD23 antibodies to permeabilized B cells in the presence of a physiologic concentration of Ca2+ (100 nM) evoked, within 10 min, a rise in the various inositol phosphates. This ability of anti-CD23 antibodies to activate PIC was enhanced in the presence of GTP-tetralithium salt 100 microM. By contrast, preincubation with GDP-trilithium salt, a nonhydrolyzable analogue of GDP, caused a marked reduction in the release of inositol phosphates. Preincubation of B cell blasts with Pertussis toxin resulted in a total inhibition of the capacity of the toxin to ADP-ribosylate a 41-kDa protein, probably of the Gi type; in these conditions, no modification of anti-CD23-elicited polyphosphoinositide hydrolysis could be detected. These results suggest that the CD23 molecule may be coupled to the phosphoinositide signaling pathway by a GTP-dependent component that is insensitive to Pertussis toxin.

Adenosine Diphosphate Ribose↗