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Biomedical subjects

R Moraca

Publications and source records attributed to R Moraca.

15 recordsLinked to original sources
PubMed1998

Functional expression of Fas on mouse bone marrow stromal cells: upregulation by tumor necrosis factor-alpha and interferon-gamma.

In this study we describe the expression and function of Fas in mouse bone marrow (BM) stromal cells (SCs) and cell lines derived from long-term BM cultures. Flow cytometry analysis showed that Fas was expressed on adherent cells from freshly isolated BM and on all cloned SC lines tested. The SC line ME-25 was Fas+ but negative for FasL as detected by reverse transcriptase-polymerase chain reaction. Furthermore, ME-25 was CD44+, VCAM-1+, Mac-3-, Gr-1-, and type IV collagen-. ME-25 treatment with interferon-gamma or tumor necrosis factor-alpha significantly induced upregulation of Fas expression as detected by both flow cytometry and Western blot immunoassay. The same treatment with interleukin (IL)-1, IL-2, or IL-13 had no effect. Functional studies demonstrated that Fas induced a strong increase in apoptosis when engaged with an anti-Fas monoclonal antibody (MoAb). Activated BM T cells induced Fas-dependent cytotoxicity of ME-25 insofar as blocking anti-FasL MoAb inhibited the killing of ME-25 induced by activated BM T cells. These data suggest a possible involvement of Fas-expressing SCs in negative regulatory functions in the BM and provide a starting point for further studies on the role of Fas+ SCs.

Animals↗
PubMed1997

Short-term dexamethasone treatment modulates the expression of the murine TCR zeta gene locus.

Glucocorticoids (GCH) are highly effective agents in controlling inflammation and immune response. We studied the effect of the synthetic GCH dexamethasone (DEX) on the expression of TCR zeta gene splicings that code for some chains belonging to the T-cell receptor (TCR)/CD3 complex. In the DEX-treated hybridoma T-cell line 3DO, TCR zeta gene splicings increase within the first 24 hr (about fourfold increase), as demonstrated by reverse transcriptase-polymerase chain reaction and RNase protection assay. This increase is due to the stimulation of TCR zeta gene locus transcription, as demonstrated by the "run-on" assay. A similar upregulation was observed in murine thymocytes following in vivo DEX treatment. As a consequence of TCR zeta gene locus modulation, the expression of the spliced mRNAs coding for TCR zeta and TCR eta subunits is increased, whereas their relative ratio is only slightly changed. Indeed, the amount of TCR zeta protein in 24-hr DEX-treated cells is fivefold more than that in the untreated cells. A similar effect was seen in 3DO cells treated with hydrocortisone but not in those treated with testosterone. TCR zeta protein increase was confined to the cytoplasm and therefore TCR/CD3 complex expression did not increase. This newly described effect of DEX may constitute an additional molecular mechanism that contributes to its immunomodulating activity.

Animals↗
PubMed1997

A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis.

By comparing untreated and dexamethasone-treated murine T cell hybridoma (3DO) cells by the differential display technique, we have cloned a new gene, GITR (glucocorticoid-induced tumor necrosis factor receptor family-related gene) encoding a new member of the tumor necrosis factor/nerve growth factor receptor family. GITR is a 228-amino acids type I transmembrane protein characterized by three cysteine pseudorepeats in the extracellular domain and similar to CD27 and 4-1BB in the intracellular domain. GITR resulted to be expressed in normal T lymphocytes from thymus, spleen, and lymph nodes, although no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. Furthermore, GITR expression was induced in T lymphocytes upon activation by anti-CD3 mAb, Con A, or phorbol 12-myristate 13-acetate plus Ca-ionophore treatment. The constitutive expression of a transfected GITR gene induced resistance to anti-CD3 mAb-induced apoptosis, whereas antisense GITR mRNA expression lead to increased sensitivity. The protection toward T cell receptor-induced apoptosis was specific, because other apoptotic signals (Fas triggering, dexamethasone treatment, or UV irradiation) were not modulated by GITR transfection. Thus, GITR is a new member of tumor necrosis factor/nerve growth factor receptor family involved in the regulation of T cell receptor-mediated cell death.

Amino Acid Sequence↗
PubMed1997

CD2 rescues T cells from T-cell receptor/CD3 apoptosis: a role for the Fas/Fas-L system.

Anti-CD3 monoclonal antibodies (MoAbs) and glucocorticoid hormones induce apoptosis in immature thymocytes and peripheral T lymphocytes. This process is inhibited by a number of growth factors, including interleukin-2 (IL-2), IL-3, and IL-4, as well as by triggering of the adhesion molecule CD44, which would indicate that signals generated by membrane receptors can modulate the survival of lymphoid cells. To investigate whether triggering of CD2 may also affect apoptosis in lymphoid cells, we analyzed the effect of stimulation with anti-CD2 MoAbs on T-cell apoptosis induced by two stimuli, anti-CD3 MoAbs and dexamethasone (DEX), using a hybridoma T-cell line and a T-helper cell clone. The results show that CD2 engagement decreased anti-CD3 MoAb-induced apoptosis, but did not influence DEX-induced cell death. Furthermore, the decrease appeared to be related to the expression of Fas/APO-1 (CD95) and Fas-ligand (Fas-L). In fact, we show that CD2 stimulation inhibits apoptosis by preventing the CD3-induced upregulation of Fas and Fas-L in a Fas-dependent experimental system. These data suggest that a costimulatory molecule may control a deletion pathway and may therefore contribute to the regulation of peripheral tolerance.

Animals↗
PubMed1997

Deoxycholic acid and SCFA-induced apoptosis in the human tumor cell-line HT-29 and possible mechanisms.

Short chain fatty acids (propionate and butyrate) and deoxycholic acid (DCA) are able to induce apoptosis in HT-29 colonic tumor cell line, but DCA induces a much higher level of apoptosis than butyrate and propionate. Mixtures of DCA with butyrate or propionate enhance the effect of the single components. Apoptosis is not affected by the PKC, PTK or de novo mRNA and protein synthesis inhibitors, so that the involvement of these enzymes and processes is ruled out. In contrast, DCA-induced apoptosis is directly related to [Ca2+]i concentration as demonstrated by the apoptosis inhibition caused by [Ca2+]i chelator BAPTA/AM.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

A new dexamethasone-induced gene of the leucine zipper family protects T lymphocytes from TCR/CD3-activated cell death.

By comparing mRNA species expressed in dexamethasone (DEX)-treated and untreated murine thymocytes, we have identified a gene, glucocorticoid-induced leucine zipper (GILZ), encoding a new member of the leucine zipper family. GILZ was found expressed in normal lymphocytes from thymus, spleen, and lymph nodes, whereas low or no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. In thymocytes and peripheral T cells, GILZ gene expression is induced by DEX. Furthermore, GILZ expression selectively protects T cells from apoptosis induced by treatment with anti-CD3 monoclonal antibody but not by treatment with other apoptotic stimuli. This antiapoptotic effect correlates with inhibition of Fas and Fas ligand expression. Thus, GILZ is a candidate transcription factor involved in the regulation of apoptosis of T cells.

Amino Acid Sequence↗
PubMed1997

Effect of dexamethasone on T-cell receptor/CD3 expression.

Glucocorticoid hormones (GCH) are anti-inflammatory and immunosuppressive agents that inhibit T-cell growth and activation. Since the T-cell receptor (TCR)/CD3 complex mediates T-lymphocyte activation, we studied the effect of in vitro dexamethasone (DEX), a synthetic GCH, on TCR/CD3 expression. DEX-treatment of a hybridoma T-cell line and normal un-transformed T-cell clones induced a decrease of the TCR/ CD3 membrane expression after 4 days. After 4 weeks, TCR/CD3 was undetectable. However, the amount of mRNAs coding TCR/CD3 chains, including TCR alpha, TCR beta, CD3 gamma, CD3 theta and CD3 epsilon, as well as the amount of CD3 epsilon protein, a major component of the complex, were unaltered. By contrast, a decrease of the mRNAs deriving from the TCR zeta gene locus, as well as of the TCR zeta protein which is responsible for the membrane expression of the TCR/CD3 complex, was induced. These data suggest that the down-modulation of TCR expression is due to the diminution of TCR zeta gene products in DEX-treated cells.

Animals↗
PubMed1997

Possible mechanisms involved in apoptosis of colon tumor cell lines induced by deoxycholic acid, short-chain fatty acids, and their mixtures.

Apoptosis of tumor cells is an important growth-regulating event in tumor masses. In this study we have confirmed that deoxycholic acid (DCA) and the short-chain fatty acids (SCFA) butyrate and propionate induce a time- and concentration-dependent apoptosis in two human colon tumor cell lines: HT-29 and CaCO2. DCA is more potent, inducing effects at low concentration (50 microM) and after 24 hours of incubation, whereas SCFA (4 mM) requires 72-96 hours of treatment. Combining low concentrations of DCA (12.5-25 microM) with butyrate and propionate (4 mM) produces an additive effect on the percentage of apoptotic cells, as demonstrated by flow cytometry and DNA fragmentation. Protein kinase C, protein tyrosine kinase, and gene transcription/translation inhibitors do not significantly modify the rate of apoptosis, whereas the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) completely abolishes the DCA-induced effect without affecting the SCFA-induced apoptosis. Measurement of intracellular Ca2+ by inverted fluorescence microscopy reveals that DCA induces a rapid increase of cytosolic Ca2+ that is abolished when the cells are preincubated with BAPTA-AM, whereas ethyleneglycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid has a minimal effect. In contrast, SCFA does not modify the intracellular Ca2+ concentration. Thus the DCA-induced apoptosis is a Ca(2+)-dependent process, whereas the intracellular signals responsible for the SCFA-induced effect remain unknown. The ionophore activity of DCA could be responsible for the increased intracellular Ca2+, but other mechanisms, such as activation of phospholipase C and phosphoinositide hydrolysis, have to be considered.

Apoptosis↗
PubMed1996

Dexamethasone modulates CD2 expression.

Glucocorticoid hormones (GCs) are able to modulate leukocyte activity. We studied the effect of dexamethasone (DEX) on the expression of CD2, an adhesion molecule involved in T-lymphocyte homing and activation. Results of flow cytometry analysis and immunoprecipitation with anti-CD2 monoclonal antibodies (mAbs) indicated that in vitro treatment with DEX augments CD2 expression in transformed T-cell lines. This effect correlated with a rapid increase in the mRNA and was inhibited by actinomycin-D (AD). The DEX-induced CD2 augmentation was transient, peaked at days 1-2 and returned to the levels of untreated controls at days 3-4. It was a dose-dependent phenomenon, mediated by the GC receptor (GCR), because it was inhibited by the GCR antagonist RU486, and was not induced by other steroids such as testosterone and progesterone. This CD2 modulation could presumably contribute to GC-induced effects on T-cell activity.

Animals↗
PubMed1995

T lymphocytes bearing the gamma delta T cell receptor are susceptible to steroid-induced programmed cell death.

The mechanisms by which glucocorticoids suppress immune responses have not yet been clearly defined. In steroid-sensitive pathological conditions, an increase in gamma delta T cells can occur in certain untreated systemic autoimmune disorders and seems to be a peristent feature in most cases of systemic lupus erythematosus (SLE). Our previously published data demonstrated that immunosuppressive therapy normalized this expanded SLE T cell subset in parallel with clinical remission of the symptoms. To establish how corticosteroid treatment determines the disappearance of peripheral blood gamma delta T lymphocytes, circulating alpha beta and gamma delta T lymphocytes from seven SLE subjects with active disease and seven healthy individuals were cultured in the presence or absence of 10(-7) M Dexamethasone (DEX). Cell suspensions were then analysed for DNA fragmentation, characteristic of apoptotic cell death, by a new cytofluorimetric method. Conventional agarose-gel electrophoresis on the same T cell populations was carried out for comparison. Regular follow-ups for 6 months revealed in vivo steroid treatment determined a dramatic fall in SLE blood gamma delta T cells, and in vitro experiments seem to indicate that DEX-triggered apoptotic signals are confined to the double negative (CD4-CD8-) gamma delta T cell subpopulation which disappears after in vivo immunosuppressive therapy. Clinical and pathological remission of some autoimmune diseases is often obtained by corticosteroids. Our results offer new insights on the mechanisms through these hormones exert their potent inhibitory activities on immune system cells postulated to play a role in the generation of autoimmune responses.

Adult↗
PubMed1995

IL-4 is able to reverse the CD2-mediated negative apoptotic signal to CD4-CD8- alpha beta and/or gamma delta T lymphocytes.

Activation of immature thymocytes or transformed T lymphocytes via T-cell receptor (TCR)/CD3 signalling can induce programmed cell death (apoptosis). Recent data indicate that anti-CD3/TCR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral blood T lymphocytes. Here we report that triggering of resting CD4-CD8-TCR alpha beta+ and/or TCR gamma delta+ via the alternative CD2-dependent activation pathway is able to induce programmed cell death. A pair of mitogenic anti-CD2 mAb provoked a dramatic rise in [Ca2+]i that was almost entirely sustained by extracellular fluxes, and the inhibition of membrane [Ca2+/Mg2+] ATPase. The resulting endonuclease activation was able to induce DNA fragmentation, as revealed by propidium iodide staining and gel electrophoresis. Induction of apoptosis was prevented by the presence of interleukin-4 (IL-4) as well as by endonuclease inactivation with 100 microM ZnCl2, but enhanced by the contemporary block of protein kinase C. Thus it seems that in resting T lymphocytes the strong calcium signal delivered by the alternative CD2 activation pathway may act as a negative apoptotic signal in both alpha beta and gamma delta T cells with low (non-major histocompatibility complex restricted) antigenic affinity, so limiting the extension of polyclonal T-cell growth.

Antibodies, Monoclonal↗
PubMed1994

The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells.

Genistein, a natural isoflavonoid phytoestrogen, is a strong inhibitor of protein tyrosine kinases. We analyzed the effects of genistein on in vitro growth, cell-cycle progression and chromatin structure of Jurkat cells, a T-cell leukemia line with a constitutively increased tyrosine phosphorylation pattern. Exposure of in vitro cultured Jurkat cells to genistein resulted in a dose-dependent, growth inhibition. Cell-cycle analysis of genistein-treated cells revealed a G2/M arrest at low genistein concentrations (5-10 micrograms/ml), while at higher doses (20-30 micrograms/ml) there was also a perturbation in S-phase progression. The derangements in cell-cycle control were followed by apoptotic death of genistein-treated cells. Immunocytochemical analysis of cells stained with a FITC-conjugated anti-phosphotyrosine monoclonal antibody showed that 30 micrograms/ml genistein effectively inhibit tyrosine kinase activity in cultured Jurkat cells. Our results indicate that the natural isoflavone genistein antagonizes tumor cell growth through both cell-cycle arrest and induction of apoptosis and suggest that it could be a promising new agent in cancer therapy.

Antineoplastic Agents↗
PubMed1992

Interleukins modulate glucocorticoid-induced thymocyte apoptosis.

Glucocorticoid hormones, calcium ionophores and anti-CD3 monoclonal antibodies induce apoptosis in mouse thymocytes. This type of cell death, which is characterized by an extensive DNA fragmentation into oligonucleosomal subunits, occurs in the intrathymic process of negative selection, and is involved in the deletion of autoreactive T-cells during thymic maturation. A number of cytokines are able to modulate apoptosis, and interleukins, including interleukin-1, interleukin-2, and interleukin-4, play a crucial role in thymic maturation and T-cell development. We tested the effects of several cytokines on the glucocorticoid hormone-induced apoptosis of mouse thymocytes in vitro, and demonstrated that interleukin-1 alpha, interleukin-2, and interleukin-4 inhibit the apoptosis induced by dexamethasone, but that interleukin-3 and interleukin-6 exert no noteworthy effect. Dose-response experiments indicated that interleukin-4 is more potent than interleukin-1 alpha and interleukin-2 in inhibiting dexamethasone-induced apoptosis. Furthermore, interleukin-4 fully inhibited the DNA fragmentation induced by the protein kinase-C activator 12-O-tetradecanoylphorbol-13-acetate, but was ineffective against apoptosis induced by the calcium ionophore A23187. These results suggest that interleukins regulate the thymic selection process by acting as modulators of the negative selection process.

Animals↗
PubMed1992

IL-2-dependent generation of natural killer cells from bone marrow: role of MAC-1-, NK1-1- precursors.

We have previously shown that interleukin-2 (IL-2) is able to induce the generation of natural killer (NK) activity in bone marrow (BM) cell cultures from mice pretreated with 5-fluorouracil (5-FU). Cell fractionation experiments to analyze the nature of BM precursors indicate that MAC-1-, NK1-1- noncytotoxic precursors are induced by IL-2 to proliferate and generate cytolytic NK cells. These data demonstrate that the phenotype and functional characteristics of the IL-2-responsive cells in the FUBM are different from those of mature NK cells in that they are MAC-1+, NK1.1+, CD3- and susceptible to boosting by IFN-alpha.

Animals↗