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J Bertoglio

Publications and source records attributed to J Bertoglio.

At least 55 records · Page 3Linked to original sources
PubMed1995

Structure-activity relationships in glucocorticoid-induced apoptosis in T lymphocytes.

Glucocorticoid-induced apoptosis in the murine interleukin-2-dependent T-cell line CTLL-2 and in freshly isolated thymocytes was studied. It was demonstrated here that in CTLL-2 cells, dexamethasone (methyl in position 16 alpha) was more efficient in inducing apoptosis than betamethasone (methyl in position 16 beta) or triamcinolone (hydroxyl in position 16). In contrast, no such difference between these three molecules was found in murine thymocytes. In addition, we showed that glucocorticoid-induced apoptosis on the two models was mediated through interaction with the glucocorticoid receptor and did not occur in the presence of inhibitors of transcription, translation or an endonuclease-inhibitor. Furthermore, in CTLL-2 cells, apoptosis took place in the presence of EGTA whereas it was prevented in murine thymocytes, thus indicating that calcium plays a different role in these two models. Finally, higher concentrations of interleukin-2 were needed to protect CTLL-2 cells against dexamethasone-induced apoptosis than that induced by betamethasone or triamcinolone. Thus, structural differences at position 16 of the steroid nucleus correlate with a different apoptosis-inducing activity by glucocorticoids which, however, is only evidenced in the calcium-independent CTLL-2 model.

Animals↗
PubMed1993

Characterization of a monoclonal antibody specific for the Ras-related GTP-binding protein Rho A.

The Rho family of small GTP-binding proteins is one of the three subgroups which, together with the Ras and Rab families, constitute the Ras-related superfamily. The Rho subgroup contains at least seven highly homologous members including 4 Rho proteins (RhoA, RhoC, RhoB, and RhoG), the Rac1 and Rac2 proteins, and CDC42Hs, which are involved in various aspects of cytoskeleton organisation and cell polarity. We have raised antibodies to individual members of the Rho family, and we report here the characterization of a monoclonal antibody (26C4) specific for RhoA. When used in western blot experiments, the 26C4 antibody recognizes the recombinant RhoA protein but not the almost identical RhoC or the RhoG, Rac and CDC42Hs proteins. Furthermore the 26C4 antibody identifies the natural RhoA protein in human lymphocyte cell extracts and was used to study the level of RhoA expression in several lymphoblastoid cell lines, and its association with the cell membrane.

Amino Acid Sequence↗
PubMed1993

Regulation of p56lck kinase expression and control of DNA synthesis in activated human B lymphocytes.

In this study, we analyzed the expression and regulation of src kinase p56lck expression during human B lymphocyte activation. We show that upon mitogenic stimulation with anti-IgM antibodies and interleukin-2, specific mRNA for p56lck becomes detectable in B cells after 24 h of activation and is followed by an increase in p56lck protein expression on days 2 and 3. This up-regulation is specific for p56lck since expression of other src kinases such as fyn, lyn, or yes was not modified. Furthermore, immune complex kinase assays show that p56lck protein expressed on day 2 is associated with kinase activity. Experiments using lck-specific antisense oligonucleotides show that the G0-G1 transition does not require p56lck, whereas DNA synthesis is dependent upon its expression. Thus, our data demonstrate that p56lck expression is up-regulated during human B cell stimulation and this kinase plays an important role during the control of late steps of B lymphocyte activation such as S phase entry.

B-Lymphocytes↗
PubMed1993

An immunologist's look at the Rho and Rab GTP-binding proteins.

The ras superfamily of small GTP-binding proteins contains three major branches: the Ras, Rho and Rab protein subfamilies. Recent advances in the field of ras-related small GTP-binding proteins suggest that it may be worthwhile to look at this superfamily from the standpoint of immunology. The subject of this review is to outline briefly the areas of lymphocyte function which may implicate small G proteins, with special emphasis on the established or possible roles of proteins of the Rho and Rab subfamilies in cytoskeleton organization and antigen presentation.

Animals↗
PubMed1993

A CD2+ subset of non-malignant peripheral blood lymphocytes from patients with Sézary syndromes overexpress the low-molecular-weight GTP-binding protein Rab2.

The Rab branch of the Ras-related GTP/GDP-binding proteins currently includes at least 25 related members which are involved in the intracellular vesicular transport along the secretory and endocytic pathways in eukaryotic cells. The overexpression of the Rab2 protein in peripheral mononuclear cells is demonstrated from 13 out of 17 patients exhibiting a Sézary syndrome. Moreover, this phenomenon is detectable in other lymphoid and myeloid malignancies. Several lines of evidence are shown suggesting that the Rab2 overexpression can be related not to leukemic cells but to a subset of peripheral lymphocytes with a CD2+ phenotype. Our results provides strong evidence for the implication of a small GDP/GTP-binding protein in immunological events associated with neoplastic states. The precise cellular population involved in this process remains to be determined.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1993

Regulation of interleukin-2 and interleukin-4 receptor expression in human and ape lymphoid cell lines.

In this study, we have analyzed the expression and regulation of receptors for IL-2 (alpha and beta chains) and IL-4 in four lymphoid cell lines established from leukemic cells. The gibbon ape cell line MLA 144 was the only one to express constitutively the IL-2R beta chain and IL-4R, whereas the NK-like YT cells express only IL-2R beta. The two other cell lines in this study, PEER and HSB2, are derived from T lymphocytes, and express neither IL-4R, IL-2R beta, nor IL-2R alpha unless stimulated. We report here that those receptors that are constitutively expressed, i.e., IL-2R beta on YT cells and IL-2R beta or IL-4R on MLA cells, are down-regulated by stimulation with PHA + PMA. In contrast, RNase protection experiments showed that PHA + PMA stimulation of T cell lines induces mRNA for all three receptors in PEER cells, and only IL-2R alpha and IL-4R in HSB-2. Thus each of these three receptors is subjected to a different regulation, which in addition varies depending on the lineage (or differentiation stage) of the cells. This was further supported by the finding that IL-1 alpha or TNF-alpha regulates these receptors differently. These two cytokines have no effect on IL-2R beta and IL-4R in MLA and YT, but induce IL-2R alpha in YT. In contrast, they do not induce either chains of the IL-2R in the T cell lines PEER or HSB-2, but TNF induces IL-4R mRNA in HSB2 cells, and IL-1 does so in both cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A possible role for the Ras-related Rab2 protein in the immunological events associated with hematological malignancies.

The Rab branch of the Ras-related guanine nucleotide (GTP/GDP)-binding proteins currently includes at least thirty related members which are involved in the intracellular vesicular transport along the secretory and endocytic pathways in eukaryotic cells. We have demonstrated the overexpression of the Rab2 protein in peripheral blood mononuclear cells from patients exhibiting Sézary syndromes and other lymphoid and myeloid malignancies. Several lines of evidence suggest that the Rab2 overexpression can be related not to leukemic cells but to a subset of peripheral lymphocytes with a CD2+ phenotype. Our results provide strong evidences for the implication of a small GDP/GTP binding protein in immunological events associated with neoplastic states. The precise cellular population involved in this process remains to be determined.

GTP-Binding Proteins↗
PubMed1992

Inhibition of superoxide production in B lymphocytes by rac antisense oligonucleotides.

Rac1 and Rac2 gene products are small GTP-binding proteins showing 92% homology to each other. According to recent studies performed in cell-free systems, Rac1 and Rac2 proteins may be involved in the activation of NADPH-oxidase, the superoxide-generating enzymatic complex active in phagocytes. Epstein-Barr virus (EBV) transformed B lymphocytes, which express rac1 and rac2 genes, also efficiently release superoxide anions when triggered by various cell surface stimuli. To investigate the regulatory role of Rac proteins in living cells, we analyzed superoxide production in response to cross-linking of surface immunoglobulins or phorbol ester treatment in human EBV-transformed B lymphocytes pretreated with Rac sense and antisense oligonucleotides. We report here that (i) the rac protein content estimated by immunoblotting can be decreased by 60% in Rac antisense pretreated cells and (ii) a strong (50-60%), dose-dependent inhibition of superoxide production is observed in antisense pretreated cells whereas cells pretreated with sense oligonucleotide are unaffected. The data presented show, for the first time in whole cells, that superoxide production is modulated by the Rac protein content, thus demonstrating the physiological role of Rac proteins in the regulation of NADPH-oxidase.

B-Lymphocytes↗
PubMed1992

ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity.

The Rho proteins are identified as a subgroup of the Ras superfamily of low molecular weight GTP-binding proteins. We have studied the expression of these proteins in human cytotoxic natural killer cells and found that RhoA is the most abundantly expressed member of the Rho family. The Rho proteins are specific substrates for ADP-ribosylation catalyzed by the C3 exoenzyme from Clostridium botulinum. We report here that introduction of recombinant C3 in electropermeabilized natural killer cells or in cytotoxic T lymphocytes resulted in a dose-dependent inhibition of their cytolytic function. Furthermore, a single substrate is efficiently ADP-ribosylated by C3 in extracts from cytotoxic cells. Biochemical analyses indicate that this substrate is RhoA, and subcellular fractionation experiments demonstrate that it is essentially present in the cytosol of the cells. Western blot analysis, however, revealed that a small proportion of the Rho protein can be found associated with the cell membrane as well as with the cytotoxic granules. These results indicate that the low molecular weight GTP-binding protein RhoA is present in cytotoxic lymphocytes and plays a critical role in cell-mediated cytotoxicity.

ADP Ribose Transferases↗
PubMed1992

In vitro responsiveness to interleukins and theophylline of CD16+, CD56- natural killer cells in a patient with chronic granular lymphocyte disorder.

We report here the case of a 55-year-old patient with chronic granular lymphocyte disorder associated with moderate neutropenia. The majority of peripheral blood lymphocytes displayed a CD3-, CD8-, CD16+, CD56(NKH1)- phenotype. The patient's cells showed high spontaneous cytotoxic activity against K562 targets and developed the ability to kill the natural killer (NK)-resistant target Daudi following activation with interleukin 2 (IL-2). Simultaneously, IL-2 induced proliferation of these cells, albeit to a low level. The effects of IL-2 are likely to be mediated through the IL-2R beta chain (p70) which is expressed on these cells in the absence of the IL-2R alpha chain (p55, Tac). IL-4 was demonstrated to be inhibitory of both the cytotoxic and proliferative effects of IL-2. Thus, despite an unusual CD56- phenotype, the expanded lymphocyte population in this patient display functional and phenotypic properties of normal, non-activated NK cells. These cells probably represent the counterpart of a minor NK cell subpopulation, present in normal individuals at a low frequency, and which has never been fully characterized functionally. In addition, we show that the cytolytic activity of this NK cell population can be blocked in vitro in the presence of a cAMP analog or of theophylline, possibly providing new means of investigating the role of NK cell cytotoxicity on the pathogenesis of associated symptoms in such patients.

Antigens, CD↗
PubMed1991

A hemopoietic specific gene encoding a small GTP binding protein is overexpressed during T cell activation.

We have isolated, from a human B cell line cDNA library, a cDNA (Gx) encoding a small G protein identical to rac 2, a member of the ras superfamily. Gx/rac 2 gene is expressed as a unique mRNA of 1,7 Kb in peripheral blood lymphocytes, in purified B and T cells, in thymus as well as in several B and T cell lines. It is not expressed in many other tissues analysed including liver, brain, lung, heart and kidney. Upon in vitro stimulation with phytohemagglutinin A, peripheral blood lymphocytes show a clear increase of the Gx/rac 2 mRNA after 6 hours; a 30-50 fold accumulation is reached at 24 hours and persists thereafter. Purified T lymphocytes exhibit a similar increase in Gx/rac 2 mRNA expression upon mitogenic stimulation. Therefore, the expression of the Gx/rac 2 gene appears to be restricted to cells of the hemopoietic lineage and to be strongly stimulated during T cell activation. Gx/rac 2 protein must fulfill a specific role in activated T cells that could provide a new model for studying the function of small G proteins.

Amino Acid Sequence↗
PubMed1989

Interleukin 2 stimulates tyrosine phosphorylation in T cell membrane fractions.

Interleukin 2 is a growth factor secreted by T lymphocytes upon antigenic stimulation and inducing the proliferation of T cells bearing at their surface the heterodimeric high-affinity form of its receptor. No enzymatic function has so far been demonstrated in the receptor subunits. In an attempt to elucidate the biochemical pathway of signal transduction, we investigated the capacity of interleukin 2 to modulate tyrosine phosphorylation in T cell membranes. Membrane-rich fractions from T cells were tested for their ability to phosphorylate tyrosine in the presence or absence of added recombinant interleukin 2. Using as substrate a synthetic polymer of glutamic acid and tyrosine, we demonstrated a 3-4-fold stimulation of tyrosine phosphorylation in the presence of interleukin 2; this stimulating effect appeared to be well correlated with interleukin 2 function since (a) it was not observed in insensitive cells, (b) it required the presence of the high-affinity form of the receptor and (c) it was dose-dependent. Confirmatory results were obtained by phosphorylating membrane-rich fractions with [gamma-32P]ATP and by analysing the resulting phosphoproteins: only in fractions from cells with the high-affinity form of the receptor were several membrane proteins specifically phosphorylated on tyrosine residues in response to interleukin 2. At least two proteins of 115 and 58 kDa were consistently hyperphosphorylated on tyrosine in an interleukin-2-dependent manner. This stimulation was strongly dependent on the presence of the protein tyrosine phosphatase inhibitor, sodium orthovanadate. Thus, we propose that interleukin 2 enhances tyrosine phosphorylation by stimulating a tyrosine kinase activity. The nature of the enzyme involved remains to be determined.

Cell Fractionation↗
PubMed1989

Functional interactions of IL2 and TNF in the differentiation of LGL into LAK effectors.

We have previously reported on the synergistic effect of IL2 and TNF on the differentiation of LGL into LAK effectors. In the present study, we have further investigated the molecular basis of this synergistic mechanism and examined the role of TNF in the induction of LAK activity. We show that the generation of optimal LAK activity by low doses of IL-2 in the presence of TNF involves the induction of high-affinity IL2 receptors on LGL and occurs without promoting significant proliferation, suggesting a functional activation rather than a proliferative expansion of LAK precursors. Using blocking studies with anti-Tac and with an anti-IL2 (IHII), which specifically inhibits the binding of IL2 to the p75 IL2 receptor component, we also show that both the p55 and the p75 are involved in the increase in TNF binding sites on LGL and the subsequent acquisition of LAK activity. We also demonstrate that the failure of low doses of IL2 to induce LAK activity is related to their incapacity to induce TNF production. Moreover, when specific antibodies against TNF were added to the culture, the differentiation of LGL into LAK effectors by optimal concentrations of IL2 in our system (2.5-5.0 ng/ml) was partially inhibited. This suggests that TNF may be a physiologic mediator in the sequential activation stages of LGL into LAK effectors.

Cell Differentiation↗
PubMed1989

Expression and regulation of interleukin-1 mRNA and interleukin-1 receptors in human B-cell lines.

Long-term human lymphoid B-cell lines have been described to produce a number of growth factors, including interleukin-1 (IL-1) which may be of importance in the autocrine growth regulation of these cells and may participate in their antigen presenting function. In this report, we have analyzed the production of IL-1 by 12 established B-cell lines at the level of mRNA expression. Among the 5 cell lines containing an IL-1 message, three expressed exclusively IL-1 alpha, one contained traces of IL-1 beta, and only one line contained both. The steady-state level of IL-1 alpha mRNA in these cells could be drastically increased by a short culture of the cells with the protein synthesis inhibitor cycloheximide or with PMA. PMA, however, induced a transient increase in mRNA which could be stabilized by Ca2+ ionophore. Furthermore, only constitutively produced IL-1 mRNA are increased by these compounds which do not induce de novo transcription of silent IL-1 genes in these lines. These data provide the basis for further investigations on the regulation of IL-1 mRNA expression in human B cells. In addition, we studied expression of IL-1 receptors in these lines and observed that only cells which produce IL-1, displayed IL-1 receptors at their surface, as detected by radiolabeled IL-1 binding experiments. These data strongly suggest an autocrine role for IL-1 in these lines. IL-1 mRNA and IL-1 receptors were reciprocally regulated by PMA, which increased IL-1 mRNA and lowered the number of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocytes↗
PubMed1989

Identification of interleukin 1 alpha produced by the 3B6 human EBV-B cell line.

We have previously described a novel protein, derived from the 3B6 EBV B-cell line which apparently displayed IL-1 bioactivity. We report here that the protein previously characterized is not a new species of IL-1. Indeed, we have now been able to clone and express a cDNA which identifies this protein as human thioredoxin, an intracellular dithiol oxydo-reductase enzyme. Thioredoxin however, is not a lymphokine and in particular does not possess IL-1 activity. Our earlier data can be explained in the light of recent findings, reported herein, indicating that IL-1 alpha is produced by the 3B6 line as assessed by antibody inhibition studies and IL-1 alpha mRNA expression. Thus our original protein preparation actually contained traces of IL-1 alpha, which was responsible for the biologic activities and thioredoxin.

Amino Acid Sequence↗
PubMed1989

Functional and molecular characterization of B cell line derived interleukin-1 alpha.

The cytokine secreted by a human hybrid B cell line (STS 25) obtained by fusion of the B lymphoblastoid cell line WI-L2-729-HF2 with neoplastic B cells from a patient with B cell non-Hodgkin's lymphoma (B-NHL) was characterized as IL-1 alpha. STS 25 cells express the idiotypic (Id+) immunoglobulin (Ig) specific for the neoplastic B cells of the B-NHL patient. STS 25 cells are weakly positive for surface mu delta kappa and in addition express the surface markers CD19, CD20, CD23, HLA class I and II, and the 4F2 activation antigen. STS 25 cells are also Epstein-Barr nuclear antigen positive but do not secrete viral particles. Serum-free culture supernatant from STS 25 cells (STS 25 SUP) does not show activity in assays for interleukin-2 (IL-2), -4 (IL-4), -6 (IL-6), interferon or tumor necrosis factor, but is active in the thymocyte costimulation assay and the D10.G4.1 T helper clone proliferation assay for interleukin-1 (IL-1). The IL-1 character of the STS 25 SUP activity was confirmed in inhibition studies with three different poly- or monoclonal anti-IL-1 antibodies (31, 88, and 94% inhibition in thymocyte costimulation assay, respectively). Furthermore, complete blocking of D10.G4.1 cell proliferation mediated by STS 25 SUP was observed by including anti-IL-1 alpha specific antibody in the assay, whereas anti-IL-1 beta antibody had no effect. These results indicate that this STS 25 SUP activity can be attributed to the presence of IL-1 alpha in the supernatant. Northern blot analysis of total STS 25 cellular RNA using IL-1 alpha or IL-1 beta specific probes revealed the constitutive expression of IL-1 alpha messenger RNA by STS 25 cells. In contrast, no IL-1 beta message was detectable, not even after treatment of the cells with phorbol ester or cycloheximide, which resulted in approximately 5-fold enhancement of IL-1 alpha mRNA expression. Binding studies with radiolabeled recombinant (r) IL-1 alpha indicated the presence of high numbers of IL-1 receptors on STS 25 cells (1,170 per cell, Kd = 392 pM). Although both IL-1 alpha and IL-1 beta bound to these IL-1 receptors, no indication was found for IL-1 mediated regulation of STS 25 cell growth.(ABSTRACT TRUNCATED AT 400 WORDS)

B-Lymphocytes↗