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A Rebollo

Publications and source records attributed to A Rebollo.

At least 37 records · Page 2Linked to original sources
PubMed1997

IL-2 signaling controls actin organization through Rho-like protein family, phosphatidylinositol 3-kinase, and protein kinase C-zeta.

IL-2 and IL-4 induce proliferation of TS1 alpha beta cells. Activation of the zeta isoform of protein kinase C is an important step in IL-2-, but not IL-4-mediated proliferation. In addition, protein kinase C-zeta is implicated in IL-2-mediated actin organization. Given the established involvement of the Rho family of small guanine nucleotide-binding proteins in organization of actin structures, we analyze the possible relationships between Rho and protein kinase C-zeta. Using the Rho-like protein family-specific toxin B from Clostridium difficile, we report in this work that IL-2, but not IL-4, induces a Rho-dependent activation of protein kinase C-zeta. This signaling event is mediated by the activation of phosphatidylinositol 3-kinase. In contrast, IL-4 induces a Rho-independent, phosphatidylinositol 3-kinase-mediated activation of protein kinase C-zeta, but this pathway has no implications in cytoskeleton organization.

Actins↗
PubMed1997

Ras activation leads to cell proliferation or apoptotic cell death upon interleukin-2 stimulation or lymphokine deprivation, respectively.

Lymphokine-dependent cells undergo apoptosis upon lymphokine withdrawal. We describe that lymphokine deprivation of the interleukin (IL)-2- or IL-4-dependent mouse T cell line TS1 alpha beta induces Ras activation which plays a role in programmed cell death, since blocking Ras activity reduces the induction of apoptosis. Induction of apoptosis by lymphokine deprivation can be prevented by expression of the Bcl-2 protein. Rescue from cell death by IL-2 also promotes Ras activation, but, in contrast to lymphokine withdrawal, stimulates Bcl-2 expression. IL-4-induced cell survival is Ras- and Bcl-2 independent. These results are compatible with a model in which cell proliferation requires the simultaneous induction of at least two pathways which act in combination to prevent cell death.

Animals↗
PubMed1997

Rho prevents apoptosis through Bcl-2 expression: implications for interleukin-2 receptor signal transduction.

Here we describe a Rho-mediated apoptosis suppression pathway driven by Bcl-2 expression in the interleukin (IL)-4- or IL-2-dependent murine T cell line TS1 alpha beta. IL-2, but not IL-4, induces Bcl-2 expression through RhoA activation which is inhibited by the specific Rho family inhibitor, Clostridium difficile Toxin B, as well as by a dominant negative RhoA mutant. Using transient transfections of RhoA mutants tagged with the vesicular stomatitis virus glycoprotein, we show that a constitutively active RhoA mutant induces Bcl-2 expression and prevents apoptosis upon IL-4 withdrawal. Finally, we have identified the signaling pathway involved together with RhoA in Bcl-2 induction and show compelling evidence for the implication of phosphatidylinositol 3 kinase and protein kinase C.

Animals↗
PubMed1996

Critical role of Ras in the proliferation and prevention of apoptosis mediated by IL-2.

Activation of the Ras small guanine nucleotide-binding proteins upon IL-2 stimulation of T lymphocytes has been described previously. However, the relevance of the Ras molecules in the cellular events triggered by IL-2 still remained to be clarified. To analyze the effects of Ras inhibition in early proliferative responses to IL-2, we have transiently expressed a Ras dominant negative mutant carrying a point mutation in codon 17 (Ser17Asn or N17) under a tetracycline-controlled expression system. We have found that Ras has a crucial role in both proliferation and prevention of apoptosis through IL-2R, while IL-4 promotes proliferation and inhibits apoptosis by Ras-independent signals. The use of flow cytometry has allowed us to correlate the effects in proliferation and apoptosis with the expression level of the Ras mutant among the transfected population. Furthermore, the availability of a murine T cell line that can be maintained independently in the presence of IL-2 or IL-4 has provided specificity controls, since IL-4 does not trigger Ras activation.

Animals↗
PubMed1996

Association of phosphatidylinositol 3 kinase to protein kinase C zeta during interleukin-2 stimulation.

Interleukin-2 induces a serine-phosphorylated phosphatidylinositol 3 kinase activity in the mouse T cell line TS1 alpha beta. Moreover, protein kinase C (PKC) zeta directly or indirectly associates with the phosphatidylinositol 3 kinase and the association appears to be necessary for the serine-phosphorylated phosphatidylinositol 3 kinase activity, since release of zeta PKC by competition of binding with peptides spanning the p110 sequence from amino acids 907 to 925 abolishes the serine-phosphorylated phosphatidylinositol 3 kinase activity. This kinase activity is also blocked when zeta PKC expression is inhibited by antisense oligonucleotide. Inhibition of phosphatidylinositol 3 kinase activity by wortmannin does not abolish zeta PKC association.

Amino Acid Sequence↗
PubMed1995

Physical association and functional relationship between protein kinase C zeta and the actin cytoskeleton.

Protein kinase C (PKC) was initially identified as a serine/threonine protein kinase dependent on calcium and phospholipids and shown to be involved in intracellular signaling pathways. PKC isoforms have been classified into four groups: Ca(2+)-dependent conventional PKC alpha, beta I, beta II, gamma; Ca(2+)-independent, novel PKC delta, epsilon, eta, phi; atypical PKC zeta, lambda, iota which are not activated by Ca2+ or diacylglycerol, and the recently discovered PKCmu. We reported that activation of the zeta PKC isoform is an important step in interleukin-2 (IL-2)-mediated proliferation (Gómez, J., Pitton, C., García, A., Martínez, A., Silva, A. and Rebollo, A., Exp. Cell Res. 1995. 218: 105.). zeta PKC is also required for mitogenic activation of fibroblasts and for the maturation pathway activated by insulin and Ras. Contradictory results have been reported regarding the subcellular redistribution of zeta PKC upon activation. We report here, using confocal microscopy, that IL-2 induces expression, translocation and association of zeta PKC to a structure coincident with the actin cytoskeleton. Furthermore, we show that zeta PKC has a role in maintaining the integrity of the actin cytoskeletal structure in IL-2-stimulated cells. On the contrary, zeta PKC is not involved in the actin cytoskeleton organization when cells are maintained in IL-4, confirming our previous results showing that IL-4-induced signal transduction is PKC independent.

Actins↗
PubMed1995

Differential effect of rapamycin and cyclosporin A in proliferation in a murine T cell line expressing either intermediate or high affinity receptor for IL-2.

It has been reported that rapamycin (rap), cyclosporin A (CsA) and FK506 have immunosuppressive effect during the activation process of murine T cells. These drugs were investigated for their suppressive effect on a murine T cell line expressing intermediate (TS1 beta) or high (TS1 alpha beta) affinity IL-2R. Rap and CsA strongly inhibit the IL-2-dependent proliferation of TS1 alpha beta cells while they minimally affect the IL-2-mediated proliferation of TS1 beta cells. FK506 does not have any effect on the IL-2-driven proliferation of either TS1 beta or TS1 alpha beta cells. Simultaneous addition of Rap and CsA or Rap and FK506 inhibit the IL-2-mediated proliferation of TS1 beta and TS1 alpha beta cells and therefore FK506 does not revert the inhibition mediated by Rap in TS1 alpha beta cells. Neither Rap nor CsA affect IL-2R expression and internalization in TS1 alpha beta cells. CsA and Rap strongly inhibit the appearance of DNA binding activity of NF-AT and to a lesser extent NF-kappa B. Rap inhibits IL-2-stimulated phosphatidylinositol 3 (PI3) kinase activity in TS1 alpha beta cells. In TS1 beta cells, Rap activates PI3 kinase on its own, inhibiting the IL-2-stimulated PI3 kinase to a lesser extent. These results suggest that PI3 kinase is a target for Rap action. Our results strongly suggest that we have Rap and CsA sensitive and resistant activation pathways operating in TS1 beta and TS1 alpha beta cells.

Base Sequence↗
PubMed1995

The zeta isoform of protein kinase C controls interleukin-2-mediated proliferation in a murine T cell line: evidence for an additional role of protein kinase C epsilon and beta.

In order to address a role of protein kinase C in signal transduction through interleukin-2, interleukin-4, and interleukin-9 receptors, we took advantage of the availability of a selective protein kinase C inhibitor, GF109203X, and the availability of TS1 beta and TS1 alpha beta cell lines which can be maintained in interleukin-2, interleukin-4, or interleukin-9 independently. In this report we report that inhibition of protein kinase C activity by GF109203X does not block interleukin-4- or interleukin-9-dependent proliferation and, on the contrary, does block interleukin-2-dependent proliferation, suggesting that interleukin-4 and interleukin-9 do not use signal transduction pathways mediated by protein kinase C and that the common gamma chain of interleukin-2, interleukin-4, and interleukin-9 receptors is not responsible per se for the activation of protein kinase C through interleukin-2 receptor. Moreover, GF109203X induces apoptosis in cells cultured in interleukin-2 but not in interleukin-4 or interleukin-9. Using antisense oligonucleotides, we report that the zeta and epsilon protein kinase C isoforms are involved in signaling through high-affinity interleukin-2 receptor and beta and zeta are involved in signaling through intermediate-affinity interleukin-2 receptor. Taken together, our data indicate that activation of the zeta, beta, and epsilon protein kinase C isoforms is an important step in interleukin-2-mediated proliferation.

Animals↗
PubMed1995

Apoptosis induced by IL-2 withdrawal is associated with an intracellular acidification.

It is known that phorbol esters can protect IL-2-dependent lymphocytes against apoptosis induced by IL-2 withdrawal. However, the mechanism of this effect remains unclear. In this article we show that apoptosis induced by IL-2 withdrawal in the CTLL-2 cell line correlates with a decrease in intracellular pH (pHi). Supplementing the incubation medium with phorbol esters during IL-2 deprivation protects CTLL-2 cells against both apoptosis and intracellular acidification. Interestingly, IL-4 also supports short-term cell survival and maintenance of normal pHi. The protein kinase inhibitor staurosporine prevents the protective effects of IL-2, PMA, and IL-4 on apoptosis and intracellular acidification. In contrast, inhibition of the Na+/H+ antiporter by 5-N-ethyl-N-isopropyl amiloride reverts the protective effects of PMA and IL-4, but only weakly affects IL-2-mediated suppression of apoptosis. Taken together, these results indicate that intracellular acidification may be an important event during apoptosis induced by IL-2 deprivation in the CTLL-2 cell line. Moreover, they suggest a key role for protein kinase C activation both in the maintenance of pHi and in the suppression of apoptosis, through mechanisms which rely on the activation of the Na+/H+ antiporter to a different extent, depending on the rescuing factor employed.

Apoptosis↗
PubMed1995

Action of CCK on CDE diet-induced acute pancreatitis in rats treated with hydrocortisone.

The present work studies the effect of previous hydrocortisone administration (10 mg/kg/day) over 7 days on the later development of diet-induced acute pancreatitis in the rat. Acute pancreatitis was induced by feeding a diet deficient in choline and supplemented with 0.5% ethionine (CDE diet) over 10 days. Hydrocortisone pretreatment exacerbated CDE-induced acute pancreatitis. There was a significant increase in serum amylase, pancreatic edema, and haematocrit levels and an insignificant decrease in pancreatic mass in rats pretreated with hydrocortisone. Pancreatic enzyme secretion was strongly reduced in the rats subjected to acute pancreatitis, and although the drop in enzyme levels did not reach statistical significance, the values of secretion were even further reduced in the animals treated with hydrocortisone, pointing to the absence of pancreatic functionality. This effect can be attributed to enzyme storage elicited by previous hydrocortisone administration; activated intracellularly, these enzymes could aggravate the pathology. Administration of the cholecystokinin octapeptide (CCK-8) (10 micrograms/kg/day) during the development of acute pancreatitis in animals pretreated with hydrocortisone substantially improved the general state of the animals' pancreases. There was a significant decrease in serum amylase, pancreatic edema and haematocrit levels in rats injected with CCK, which was accompanied by an increase in pancreatic functionality. Conversely, the administration of L-364,718 (0.1 mg/kg/day), a CCK antagonist, did not improve pancreatic functionality and did not appreciably affect the general state of the organ. It is concluded that in rats with storage levels increased by hydrocortisone administration that are subjected to acute pancreatitis, the secretagogue effect of CCK is more beneficial than the repose of the gland induced by L-364,718.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗
PubMed1995

A role for the intermediate affinity IL-2R in the protection against glucocorticoid-induced apoptosis.

Recent work has shown that T lymphocytes undergo apoptosis upon treatment with the glucocorticoid analogue dexamethasone. These cells can be protected from the effect of dexamethasone by interleukin-2 (IL-2) or IL-4. We were interested in analysing whether a transfected cell dependent on three different lymphokines could be protected by them from the effect of dexamethasone. In addition, we took advantage of our cellular system, in which we expressed intermediate- or high-affinity IL-2R independently, to analyse the role of these receptors in the protection from glucocorticoid-induced apoptosis. In this report we show that IL-2 rescues murine T cells expressing exogenous intermediate- (TS1 beta) or high-affinity (TS1 alpha beta) IL-2 receptor (IL-2R) from dexamethasone-induced apoptosis. This result suggests that intermediate-affinity IL-2R alone can replace high-affinity IL-2R for the protection from the effect of dexamethasone. In addition, IL-4 and IL-9 are rescue-factors, as well as IL-2, of glucocorticoid-treated TS1 beta and TS1 alpha beta cells. Our data suggest that the presence of the alpha-chain of the IL-2R is not required for rescue by IL-2 from the effect of dexamethasone. In addition, we show that proliferation is not required for preventing glucocorticoid-induced apoptosis. This result implies a new role for the intermediate-affinity IL-2R.

Animals↗
PubMed1994

Detection of inflammation/infection with human polyclonal immunoglobulin G labelled with 99Tcm.

Radiolabelled human polyclonal immunoglobulin G (IgG) has been successful in identifying inflammatory/infectious processes in human and animal models. We studied 71 patients with suspicion of inflammation of varied origin and location (44 of musculoskeletal location and 27 other) using 99Tcm. IgG images correctly identified 21/22 inflammatory sites of musculoskeletal origin and only 4/14 sites of soft-tissue location. Five false negative studies corresponded to granulomatous processes, three of them tuberculosis. Four false positive studies were obtained in the musculoskeletal group corresponding to three synovial tumours and a Charcot joint. No false positive results were seen in the soft tissue group. 99Tcm-IgG performs well in the identification of bone-joint lesions and rules out non-inflammatory conditions (with the important exception of tumours). The role of 99Tcm-IgG in soft-tissue inflammatory sites, especially in highly vascular organs is inferior, with a high yield of false negative studies. Granulomatous lesions probably represent situations of low or absent IgG uptake.

Adolescent↗
PubMed1993

High-affinity and intermediate-affinity forms of the human interleukin 2 receptor, expressed in an interleukin 9-dependent murine T cell line, deliver proliferative signals via differences in their transduction pathways.

Although several reports have claimed that the p70 IL-2R per se transduces a growth signal in lymphoid and non-lymphoid systems, there is no convincing evidence for this in lymphoid T cell lines. In order to investigate the mechanism of IL-2R-dependent signal transduction pathways via the p70 IL-2R in lymphoid T cells, we have established two IL-9-dependent murine (TS1) cell lines stably expressing the human p70 IL-2R subunit or the human p55-p70 IL-2R complex. Whereas the parental T cell line, TS1, proliferated in response to IL-9 or IL-4 but not IL-2, cell lines stably expressing human p70 IL-2R or p55-p70 IL-2R complex proliferated in response to IL-2. This implies that the murine T cell, TS1, contains all the intracellular components necessary for directing IL-2 signalling. In human p55-p70 IL-2R-transfected cells, the expression of a functional murine p55 IL-2R seemed to be induced and regulated by IL-2. In human p70 IL-2R-transfected cells, in the presence of IL-2 an interaction requiring only intermediate-affinity was sufficient for transduction of a proliferative signal. In addition human p70 IL-2R per se, is biologically functional via a transduction pathway not requiring induction of murine p55 IL-2R and consecutive high-affinity complex reconstitution. Thus, although both transfected cell lines can transduce a proliferative signal in the presence of IL-2, a difference in their transduction pathway is probably involved for the induction of p55 IL-2R.

Animals↗
PubMed1993

Intermediate- and high-affinity interleukin-2 receptors expressed in an IL-4-dependent T-cell line induce different signals.

In order to understand the respective roles of IL-2R alpha and IL-2R beta subunits in transmission of the interleukin-2 (IL-2)-mediated growth signals, we have established two IL-4-dependent murine T-cell clones stably expressing the human IL-2R beta chain and three clones stably expressing the human IL-2R alpha chain. Whereas parental LD8 cells (which express only the murine IL-2R beta chain) do not proliferate in response to IL-2, cell lines stably expressing human IL-2R beta or the chimeric IL-2R alpha beta complex proliferate in response to IL-2. Stably transfected cells expressing the chimeric high-affinity receptor (human IL-2R alpha and murine IL-2R beta) expressed de novo endogenous murine IL-2R alpha when cultured in the presence of IL-2 but not IL-4. Both chimeric and endogenous receptors are functional in response to IL-2, since only addition of both anti-human and anti-murine IL-2R alpha monoclonal antibodies (mAb) inhibited IL-2-induced proliferation. Taken together, our results strongly suggest that human and murine IL-2R beta molecules are different since interaction of IL-2 with human p70 IL-2R is sufficient for transduction of proliferative signals in the absence of p55 IL-2R or, alternatively, that over-expression of the IL-2R beta chain renders cells responsive to IL-2. In addition, IL-2 stimulation of T cells through different forms of IL-2R results in the induction of distinct cellular responses.

Blotting, Northern↗
PubMed1992

Immunochemical characterization of antigenic domains on human IL-2: spatially distinct epitopes are associated with binding to the p55 and p70 subunits of IL-2 receptor.

We have isolated and characterized 8 mAb against human rIL-2. All recognize nonglycosylated rIL-2 in liquid phase with similar affinities (Kd approximately 1 nM). Based on the epitopes of the IL-2 molecule that they recognize and their pattern of reactivity against glycosylated and non-glycosylated IL-2, they have been classified into four groups. The first group of anti-IL-2 mAb (2C4, 19B11 and 12C2) inhibits IL-2 binding to p70 IL-2R, while the second one (16F11, 18E1 and 2A4) prevents its binding to p55 IL-2R. These two groups neutralize IL-2 activity in a T cell proliferation assay equally well, due to their similar inhibition of IL-2 binding to high affinity IL-2R. Two mAb, 3H9 and 17F4, recognize separate epitopes on IL-2 molecule, are poor inhibitors of IL-2 binding, and they are inefficient in the neutralization of its biological activity; they have been assigned to the third and fourth groups, respectively. These results show that mAb from the first and second group recognize two epitopes of the human IL-2 molecule which probably overlap the p70 IL-2R and p55 IL-2R binding sites, respectively. In addition, these areas together form the high affinity IL-2R binding site. The two mAb from the third and fourth group recognized epitopes of IL-2 not directly involved in IL-2 binding to its receptor. All eight mAb anti-human IL-2 recognize murine IL-2 and with the exception of one, 17F4 mAb are also able to neutralize it in a T cell proliferation assay. The relationship between the structure and the function of the IL-2 molecule is discussed.

Animals↗
PubMed1990

IL-2-dependent proliferation of murine T cells requires expression of both the p55 and p70 subunits of the IL-2 receptor.

An IL-4-dependent T cell clone (LD8) was isolated from the murine IL-2-dependent cytotoxic T cell line C30.1. This clone has lost the capacity to proliferate in response to IL-2 after long-term culture in IL-4. LD8 cells express the p70, but not the p55, subunit of the IL-2R on their cell surface. The number of p70 IL-2R molecules on LD8 cells is comparable with the number of high-affinity IL-2R on the parental C30.1 cell line. LD8 cells can efficiently internalize IL-2 through the p70 IL-2R subunit. Following stimulation by IL-2, LD8 cells up-regulate p70 IL-2R mRNA, but do not express p55 IL-2R mRNA. IL-2-dependent proliferation of LD8 cells was reconstituted after introduction and expression of a human p55 IL-2R cDNA. To further investigate the role of p70 IL-2R, we have measured IL-2-induced proliferation of C30.1 cells in the presence of three anti-p55 IL-2R mAb (5A2, PC61, and 7D4) that recognize different epitopes. Under the experimental conditions used, the combination of anti-p55 IL-2R mAb prevents the formation of high-affinity IL-2R, but does not affect the binding of IL-2 to p70 IL-2R or IL-2 internalization. However, these three mAb inhibit proliferation of C30.1 cells even in the presence of IL-2 concentrations sufficient to saturate p70 IL-2R. Together these results demonstrate that p70 IL-2R alone is not sufficient to transmit IL-2-induced growth signals and that formation of p55-p70 IL-2R complex is required for IL-2-dependent proliferation of murine T cells.

Animals↗
PubMed1990

OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor.

The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation.

Animals↗