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Biomedical subjects

S Chouaib

Publications and source records attributed to S Chouaib.

At least 19 recordsLinked to original sources
PubMed1995

In vitro processing of human tumor necrosis factor-alpha.

Tumor necrosis factor (TNF)-alpha is initially synthesized as a membrane-bound, cell-associated 26-kDa protein that is further cleaved to yield the soluble 17-kDa form. By using a radiolabeled in vitro translated TNF-alpha precursor we detected a serine proteinase processing activity present in crude membrane preparations of monocytic cells able to generate a 17-kDa active protein. A similar processing pattern was obtained using purified neutral serine proteinase proteinase-3 (PR-3). Moreover, while a secretory leukocyte proteinase inhibitor (a natural serine anti-proteinase) did not affect the in vitro TNF-alpha processing, IgG preparations containing high titers of anti-PR-3 autoantibodies completely blocked this activity. The NH2-terminal sequencing of the reaction products obtained with either membrane preparations or PR-3 showed that cleavage occurs in both cases between Val77 and Arg78. These results together with cellular expression and localization of PR-3 suggest a potential role for this enzyme as an accessory TNF-alpha processing enzyme.

Amino Acid Sequence↗
PubMed1995

Evidence for the binding of a biologically active interleukin-2 to human alpha 2-macroglobulin.

Human alpha 2-macroglobulin (alpha 2M), which irreversibly entraps proteinases through a drastic conformational change, has also been reported to bind various cytokines. The meaning of cytokine binding to native and/or transformed alpha 2M molecules is, however, not understood. In an attempt to elucidate this question, we have studied the interaction of radioiodinated recombinant human interleukin-2 (125I-rhIL-2) with native and chymotrypsin (alpha 2M-C)- or methylamine-transformed (alpha 2M-MA) alpha 2M. Our results show that native and alpha 2M-MA are able to bind 125I-rhIL-2, with binding occurring only with the latter in a covalent manner, whereas the labeled cytokine is proteolyzed when incubated with alpha 2M-entrapped chymotrypsin. The degradation of uncomplexed 125I-rhIL-2 has also been observed in the presence of trypsin, whereas 125I-rhIL-2 bound to alpha 2M-MA is protected. Moreover, the proliferative activity of this cytokine on responsive cells is still maintained either with native alpha 2M- or alpha 2M-MA-complexed rhIL-2 in comparison with that observed with the cytokine alone. Our results, which lead us to consider alpha 2M molecules as IL-2-binding proteins, emphasize the possible role of these molecules as immune response regulators.

Cells, Cultured↗
PubMed1994

Interleukin 12 induces the differentiation of major histocompatibility complex class I-primed cytotoxic T-lymphocyte precursors into allospecific cytotoxic effectors.

The production of interleukin 12 (IL-12) following allogeneic stimulation and its involvement in the differentiation of allospecific cytotoxic T lymphocytes (CTLs) have been investigated. Supernatants of mixed lymphocyte cultures had detectable levels of IL-12 p40 which were completely abrogated after depletion of responder cells from monocytes. While addition to the culture of anti-IL-12 neutralizing antibodies partially inhibited the allogeneic proliferative response and the subsequent CTL activity, addition of IL-12 stimulated both responses, suggesting that endogenously produced IL-12 plays a role in the development of alloreactivity. Furthermore, using primary mixed cultures of lymphocytes from major histocompatibility complex-recombinant siblings identical for class II antigens and displaying class I disparity, we demonstrated that addition of recombinant IL-12 at the sensitizing phase of the primary mixed lymphocyte culture induced CTL activity. Under these stimulation conditions, addition of recombinant IL-12 also triggered cell proliferation, indicating that IL-12 provides both growth and differentiation signals. The mechanism underlying this process does not appear to require IL-2, since IL-12-mediated CTL generation was not abrogated by anti-IL-2 alpha-chain antibodies. IL-12 increased granzyme B and perforin mRNA accumulation in major histocompatibility complex class I-primed lymphocytes, suggesting that this cytokine activates these two genes in CTL precursors. We conclude that IL-12 can stimulate the generation of alloreactive CTLs. We suggest that IL-12 may play a role in helper cell-independent CTL generation.

Adult↗
PubMed1994

Thioredoxin increases the proliferation of human B-cell lines through a protein kinase C-dependent mechanism.

Thioredoxin (Trx) catalyzes thiol-disulfide oxidoreductions. We and others recently showed that human Trx could function as an autocrine growth factor for human lymphoid cells immortalized by the human T-lymphotrophic virus type I or the Epstein-Barr virus. Here we report that reduced Trx from Escherichia coli generated by NADPH and thioredoxin reductase increases the proliferation of an Epstein-barr virus(+)-B cell line 1G8, which constitutively produces low amounts of human Trx. This proliferative effect involved the activation of protein kinase C through its translocation to the membrane. Staurosporin and calphostin C, two inhibitors of protein kinase C, but not of H8, a protein kinase A inhibitor, were able to block Trx-dependent proliferation. The addition of Trx to 1G8 cells resulted in the formation of inositol 1,4,5-triphosphate and sn-1,2-diacylglycerol by a phosphoinositide-specific phospholipase C, as well as increased free calcium concentration. Diacylglycerol showed a biphasic increase; the first phase, corresponding to an early peak (30 s) of inositol 1,4,5-triphosphate and a second larger, prolonged phase. The second phase was inhibited by propranolol, a specific inhibitor of phosphohydrolase, indicating that it is most likely derived from phosphatidylcholine hydrolysis by the sequential action of phospholipase D and phosphatidic acid phosphohydrolase. Our data suggest that enhanced phosphoinositide-specific phospholipase C activity induced by the dithiol form of Trx in 1G8 cells is associated to protein kinase C activation, and thus plays a role in the permanent growth of Epstein-Barr virus-infected B cells.

B-Lymphocytes↗
PubMed1994

Resistance to TNF-alpha and adriamycin in the human breast cancer MCF-7 cell line: relationship to MDR1, MnSOD, and TNF gene expression.

The molecular basis of cross-resistance to tumor necrosis factor (TNF) and Adriamycin has been investigated using the breast adenocarcinoma cell line MCF7/p, its Adriamycin-resistant counterpart MCF7AdrR, and the MDR1 gene-transduced MCF-7 cells (MCF7/MDR1). While the parental cell line MCF7/p was TNF-sensitive, MCF7AdrR was TNF-resistant. The TNF resistance exhibited by MCF7AdrR cells was not due to a lack of TNF receptor expression because both cell lines express comparable levels of p75 and p55 receptors as revealed by immunofluorescence analysis. NF-kappa B translocation, which is an essential transducing signal of the TNF-induced lysis pathway, does not appear to be involved in this resistance as assessed by gel shift experiments. In order to determine the role of MDR1 gene expression in the development of this cross-resistance, MCF7/p cells transfected by the MDR1 gene were examined. Our data showed that the expression of the MDR1 gene in these cells resulted in a relative resistance of these cells to Adriamycin without affecting their susceptibility to TNF killing. The implication of the manganese superoxide dismutase and endogenous TNF expression in the cross-resistance by MCF7AdrR cells to Adriamycin and TNF has also been investigated. Northern blot analysis indicated that following TNF stimulation, the expression of 4-kilobase and 1-kilobase manganese superoxide dismutase mRNAs were 9- to 10-fold induced in MCF7AdrR cells as compared to MCF7/p and MCF7/MDR1 cells. This suggests a possible involvement of this enzyme in the Adriamycin-induced resistance to TNF. Although TNF-treatment of MCF7/p and MDR-cells induced endogenous TNF expression in these cells, the level of mRNA induction was selectively enhanced in MCF7AdrR cells (7- to 8-fold greater in MCF7AdrR cells as compared to MCF7/p and MCF7/MDR1 cells). Collectively, these data indicate that the expression of the MDR1 gene in MCF7/p cells following gene transfection is not sufficient for the acquisition of TNF resistance by MCF7/MDR1 cells. Furthermore, our data provide the first evidence that Adriamycin-induced resistance to TNF in MCF7AdrR cells may, in part, involve an overexpression of endogenous TNF and manganese superoxide dismutase genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

IL2-R alpha chain inhibitory factor (p29) produced by HIV-infected macrophages: cell target and mode of action.

We recently reported that HIV-infected adherent cells spontaneously produce a 29-kDa protein (p29), most probably of nonviral origin, which inhibits expression of the IL2R alpha chain on activated normal T cells. Studying the mode of action of this molecule, we observed that monocyte depletion of normal PBMC either by plastic adherence or by complement-mediated cytotoxicity using macrophage-specific monoclonal antibodies abrogated the inhibitory effect of p29. The biological activity of p29 in adherent cell-depleted PBMC could be restored either with rIL1 or with as little as 10(5) autologous adherent cells/ml. Moreover, p29 could not inhibit IL1 biologic activity, nor its binding to IL1 receptor. In addition, p29 could not inhibit the production of IL1 and TNF alpha by normal adherent cells. Positive and negative cell sorting of normal T cells as well as two-color immunofluorescence studies revealed that CD8+ subsets are the main cell targets of p29, which also mediated an impaired generation of alloantigen-specific CTL. Conversely, only a slight inhibitory activity could be detected in highly purified CD4+ cells. Our findings suggest that HIV-induced production of p29 inhibitory factor by adherent cells may be involved in mechanisms responsible for some of the impaired cytotoxic functions observed in AIDS patients.

CD8 Antigens↗
PubMed1992

The development of human tumor-cell resistance to TNF-alpha does not confer resistance to cytokine-induced cellular cytotoxic mechanisms.

We have derived a TNF-alpha-resistant clone (RA-I) from the parental TNF-sensitive human breast-adenocarcinoma cell line (MCF-7). The acquisition of TNF-resistance was not associated with endogenous TNF production or with differential levels of TNF receptors since both MCF-7 and RA-I display comparable TNF-receptor expression. We have investigated the relationship between acquisition of resistance to TNF and susceptibility to lysis by cytokine-activated effectors. Experiments were performed using human peripheral-blood monocytes stimulated with IL-2, IFN or GM-CSF, and lymphokine-activated killer cells as effector cytotoxic cells. Our data indicate that both TNF-resistant (RA-I) and TNF-sensitive (MCF-7) cells were killed by IL-2-activated monocytes. Incubation of monocytes with IFN also resulted in the activation of their tumoricidal activity against MCF-7 and RA-I. When stimulated monocytes were pre-incubated in the presence of a TNF-specific neutralizing monoclonal antibody, prior to co-culture with target cells, no effect on their lytic capacity was observed. Thus, the monocyte killing does not appear to involve the membrane form of TNF. These observations suggest that, in our experimental system, IL-2 and IFN are able to induce non-TNF-mediated mechanisms of cytotoxicity by monocytes. Experiments performed using GM-CSF and LPS for monocyte stimulation indicate that, although both reagents were efficient in inducing the membrane form of TNF on monocytes, they did not enhance the cell-killing capacity towards MCF-7 and RA-I targets. Furthermore, using IL-2-stimulated LGL as effector cells, we show in this study that the TNF-resistant clone RA-I was as sensitive as MCF-7 to human LAK cells.

Adenocarcinoma↗
PubMed1992

Heterogeneity among human nasopharyngeal carcinoma cell lines for inflammatory cytokines mRNA expression levels.

Using polymerase chain reaction (PCR), we confirmed the expression of interleukin-1 alpha (IL-1 alpha) by the human nasopharyngeal carcinoma (NPC) cell line C15 without contribution of either human IL-1 beta or mouse IL-1 alpha in the biological activity previously found in C15. However we showed that IL-1 alpha was not expressed in all NPCs. IL-1 beta and/or tumor necrosis factor (TNF)-alpha genes could also be activated, independently from the number of Epstein Barr Virus (EBV) copies harbored by the cells. Interestingly, the primary tumor C15 showed a profile of TNF-sensitive tumor while C17, C18 and C19 which were derived from metastasis have a typical profile of TNF-resistant cells. Furthermore, the inflammatory cytokines whose genes are classically induced by IL-1 and TNF were found expressed only in C17 and C19 suggesting another level of heterogeneity among NPCs.

Animals↗
PubMed1992

Tumor necrosis factor-mediated cell lysis in vitro: relationship to cAMP accumulation and guanine nucleotide-binding proteins.

We have investigated the modulation of tumor necrosis factor (TNF)-mediated tumor cell lysis by cAMP. Among a panel of human breast tumor cell lines, MCF7 and MDA MB 231 were shown to be, respectively, sensitive and resistant to TNF-mediated cell lysis in vitro. 125I-labeled TNF-binding experiments demonstrated that both cell lines bind TNF, indicating that the differential sensitivity to TNF was not related to TNF receptor expression. To study the relationship between TNF-mediated cell lysis and cAMP accumulation, cAMP measurement was performed following TNF treatment. Our data show that TNF alone did not induce an enhancement of intracellular cAMP accumulation either in the TNF-sensitive or in the TNF-resistant cell line. Experiments in which cells were exposed to forskolin revealed that this cAMP elevating drug was efficient in enhancing the sensitivity to TNF of MCF7 cell line. This potentiating effect of forskolin was maximal for suboptimal concentrations of TNF (10 ng/ml), reaching up to 100% when forskolin was added at 100 microM. However, co-stimulating with forskolin of either MDA MB 231 or a TNF-resistant MCF7 clone (MCF7-R-A1) did not induce any reversal of resistance to TNF. We further assessed the interaction of TNF with transmembrane signalling and the possible involvement of guanine nucleotide-binding proteins (G-proteins). Bacterial toxin-catalyzed ADP ribosylation of MCF7 and MDA MB 231 membranes was, therefore, performed. Using cholera toxin, we demonstrate that TNF treatment did not quantitatively alter the activity of stimulatory G-proteins either in MCF7 or MDA MB 231 cell line. In contrast, pertussis toxin-catalyzed ADP ribosylation experiments suggest a functional coupling of TNF receptors to a 40-kDa pertussis toxin-sensitive G-protein in the TNF-sensitive MCF78 cell line but not in the TNF-resistant MDA MB 231 cell line. Taken together, these data indicate that cAMP might play a role in TNF-mediated cell lysis and are in support of the involvement of a pertussis toxin-sensitive G-protein in TNF-mediated MCF7 cells lysis.

Adenosine Diphosphate Ribose↗
PubMed1992

Interleukin-4 differentially regulates interleukin-2-mediated and CD2-mediated induction of human lymphokine-activated killer effectors.

Natural killer (NK) cells can be differentiated into lymphokine-activated killer (LAK) effectors following stimulation with interleukin (IL)-2. This induction can be negatively regulated by IL-4. In this study, we demonstrate that the stimulation of NK cells through the CD2 pathway with (9-1 + 9.6) monoclonal antibodies can also induce these cells to secrete tumor necrosis factor-alpha (TNF-alpha) and to differentiate into LAK effectors. More importantly, our data indicate that, in contrast to the IL-2-induced LAK generation, the anti-CD2-triggered LAK activity was not regulated by IL-4. IL-4 was found to enhance the LAK activity as well as NK cell proliferation following activation with anti-CD2 by a mechanism involving, at least in part, an increased TNF-alpha production. Using immobilized monoclonal antibodies against the Fc receptor (Fc gamma RIII or CD16) for NK stimulation, we also observed that the anti-CD16-induced LAK activity was not inhibited by IL-4. These data further point to a pivotal role of TNF-alpha as a regulatory cytokine in anti-CD2-induced LAK generation, and suggest that IL-4 could serve as a discriminatory factor between two distinct pathways involved in the activation of non-MHC-restricted cytotoxicity.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Serum levels and receptor expression of tumor necrosis factor-alpha following human allogeneic and autologous bone marrow transplantation.

We have investigated tumor necrosis factor-alpha levels in serum samples of patients before and after allogenic (16 patients) or autologous (8 patients) bone marrow transplantation. A sensitive immunoradiometric assay for monitoring levels of endogenous tumor necrosis factor-alpha was used. The serum levels of tumor necrosis factor-alpha were found to be relatively low (ranging from less than 15 to 77 pg/ml). Among 13 patients having graft-versus-host disease following allogeneic bone marrow transplantation 8 patients did not have detectable tumor necrosis factor-alpha (less than 15 pg/ml) while 4 out of 8 patients undergoing autologous bone marrow transplantation had detectable tumor necrosis factor-alpha levels (15 pg/ml), indicating a lack of correlation between tumor necrosis factor-alpha serum levels and the occurrence of graft-versus-host disease. Because the tumor necrosis factor-alpha levels detected in patient sera could be regulated by TNF-receptor expression, the presence of TNF-receptor on patients' peripheral blood mononuclear cells was also studied using fluorescent liposome-conjugated tumor necrosis factor-alpha and immunofluorescence analysis. Our data indicate that peripheral blood mononuclear cells of some patients receiving either autologous or allogeneic bone marrow transplantation expressed significant levels of TNF-receptors, suggesting a lack of correlation between TNF-receptor expression and graft-versus-host disease development.

Bone Marrow Transplantation↗
PubMed1992

[Tumor necrosis factor: pleiotropic cytokine].

Originally described for its capacity to induce hemorrhagic necrosis of transplantable tumors in mice, TNF-alpha also exerts cytotoxic effects against some tumor cell lines in vitro. It is now known that TNF is an essential mediator of cellular immunity and a wide variety of biological activities of TNF in vitro and in vivo has been reported. TNF is an important mediator of inflammation and is involved during the pathogenesis of several auto-immune, infectious or cancer diseases. While some immunomodulatory properties of TNF have at least been partially elucidated, the biochemical basis of TNF cytotoxic action remains largely unknown. Furthermore, the molecular mechanisms of TNF susceptibility have yet to be clarified. Clinical studies with recombinant TNF as an anticancer agent are encouraging. Multiple phase I and phase II trials have been carried out without major therapeutic effect. In fact, TNF resistance and TNF-induced systemic toxicity are two major limitations for the use of TNF as an antineoplastic agent. The clinical application of human TNF remains an area of active research and innovative approaches such as gene therapy need to be elaborated. The elucidation of the process of lysis and the modulation of TNF resistance are crucial to the future development of TNF and its use in antitumor therapy.

Cytokines↗
PubMed1991

More insights into the complex physiology of TNF.

Tumor necrosis factor (TNF), a multifactorial cytokine involved in a plethora of cell regulatory and differentiative processes, was the subject of a recent conference. It is perhaps best known as a major player in the pathophysiology of numerous diseases but TNF clearly has many beneficial effects and is considered to be a promising therapeutic agent.

Animals↗
PubMed1991

Evidence that residual host cells surviving the conditioning regimen to allogeneic bone marrow transplantation inhibit donor hematopoiesis in vitro--the role of TNF-alpha.

Blood cells were obtained from patients selected for allogeneic bone marrow transplantation who had undergone a conditioning regimen (CR) with high-dose chemotherapy and total body irradiation. The majority of residual cells bore CD3 antigen (range: 68-98%), and the CD4/CD8 ratio was normal. The effect of these residual/radioresistant cells on donor bone marrow hematopoiesis was investigated in eleven cases. Growth of donor CFU-GM and BFU-E was inhibited by 22-65% and 29-77%, respectively, when donor marrow was cocultured with residual cells at various ratios. In contrast, blood cells obtained from two patients prior to CR had no inhibitory effect. Supernatants obtained following incubation of residual cells from 9 patients were able to inhibit the growth of CFU-GM and BFU-E from normal unrelated subjects, whereas supernatants obtained before CR and from cultured normal marrow had no inhibitory effect. In addition, in blocking experiments, an anti-TNF-alpha MoAb was able to prevent this inhibition. Thus, TBI might be able to select and/or activate cells responsible for hematopoietic growth inhibition by a mechanism involving, at least in part, TNF-alpha.

Adult↗
PubMed1991

[TNF: antitumoral agent at the border lines of immunity and inflammation].

Tumor Necrosis Factor-alpha (TNF) is mainly produced by activated monocytes and exerts pleiotropic biological effects on a wide variety of both normal and transformed cells. Originally described for its capacity to induce hemorrhagic necrosis of transplantable tumors in vivo and cytolysis of some tumor cells in vitro, TNF has also been shown to play an essential role during the inflammatory response, exerting dual, both beneficial and deleterious, effects. TNF, via a local production appears to be a key cytokine involved in antiviral, antibacterial and antiparasitic host defense mechanisms. Contreversely, deregulation of the inflammatory and immune reactions can be associated with a systemic TNF production, leading to toxic secondary effects. Recent cloning of the TNF receptors has provided additional insights in the complex physiology of TNF. It is now clearly established that both type I and type II TNF receptors can be cleaved and released as TNF binding proteins. The soluble fragments of TNF receptors can inhibit TNF-mediated tumor cell lysis in vitro and might therefore serve as regulators of TNF action. The antitumor potency of TNF also reflects the pleiotropic aspect of TNF. TNF-induced tumor regression, observed in various preclinical studies, appears to result from at least three distinct biological effects: mainly hemorrhagic necrosis via TNF action on tumor endothelium, TNF immunomodulatory activity on immune effector cells, and presumably a direct TNF-mediated cytotoxic effect against tumor cells. From the clinical trials performed with distinct recombinant materials a consensus has emerged about the disappopinting anticancer efficacy of TNF used in systemic administration. Further studies, aimed at better understanding the complex action of TNF, are required to possibly enhance its therapeutic index and subsequently to assess whether TNF still remains a promising therapeutic agent.

Animals↗
PubMed1991

Role of calcium on interleukin-1 production by monocytes: its relevance during T cell proliferation.

T cell proliferation, in the presence of monocytes, triggered either by an anti-CD3 monoclonal antibody (mAb) or by a mitogenic pair of anti-CD2 mAbs was inhibited either by the calcium chelator EGTA or the calcium channel blocker nifedipin. Antibodies against interleukin-1 (IL-1) inhibited T cell proliferation in both mitogenic systems. However inhibition achieved by anti-IL-1 beta Ab was greater than by anti-IL-1 alpha Ab while the combination of both anti-rIL-1 alpha Ab + anti-rIL-1 beta could completely inhibit the CD3-triggered T cell proliferation. On the other hand, IL-1 production by LPS-stimulated monocytes was strongly decreased both by EGTA and nifedipin. Northern blot analysis showed that this inhibition paralleled a decrease of IL-1 alpha and beta messenger RNA (mRNA) expression only in the presence of EGTA but not in the presence of nifedipin. These results indicate that EGTA acted at the transcriptional level while nifedipin acted at a yet undefined posttranscriptional level. Thus, it is suggested that the impairment of T cell proliferation by calcium inhibitors could result not only from an effect on Ca2+ influx in T cells but also from interfering with the function of accessory cells, such as the production of IL-1.

Antibodies, Monoclonal↗
PubMed1991

Tumor necrosis factor alpha: a costimulator for cytotoxic cell differentiation.

Large granular lymphocytes (LGL) can be activated by interleukin-2 (IL-2) into lymphokine-activated killers (LAK). Tumor Necrosis factor alpha was found to act synergistically with very low concentrations of IL-2 which were ineffective by themselves in inducing LAK activity. We demonstrate that the failure of low doses of IL-2 to induce LAK generation was related to their incapacity to induce TNF production by LGL. When specific antibodies against TNF were added to the culture, the differentiation of LGL into LAK effectors by optimal concentrations of IL-2 in our system was partially inhibited suggesting that TNF may be a physiologic mediator in the sequential activation stages of LGL into LAK effectors. We have also investigated the functional interaction between IL-2 and TNF on the generation of alloreactive CTL. Using primary mixed cultures of lymphocytes from a MHC-recombinant sibling identical for MHC class II antigens and displaying MHC class I disparity, our data indicate that addition of exogenous TNF at the sensitizing phase of the primary mixed lymphocyte reaction did not result in CTL activation. However, when simultaneously added with IL-2, TNF could promote an optimal induction of cytotoxic T cell generation. The TNF property to potentiate the cytotoxic function appears to involve an enhancement of IL-2 induced serine esterase activity suggesting that TNF may affect some components of the cellular lytic machinery. In conclusion the data presented here are most consistent with the suggestion that TNF could increase the IL-2 signaling efficiency or potentiate activation signaling involved in the acquisition of lytic competence by cytotoxic T cells.

Cell Differentiation↗