Biomedical subjects

M O de Landázuri

Publications and source records attributed to M O de Landázuri.

At least 19 recordsLinked to original sources

PubMed2000

Phosphodiesterases 4D and 7A splice variants in the response of HUVEC cells to TNF-alpha(1).

The mRNA accumulation of phosphodiesterases PDE4D and PDE7A was studied by RNA blot analysis in human umbilical vein endothelial cells (HUVEC) incubated with TNFalpha for different periods. A contrasting behaviour was observed in the mRNA accumulation of the two genes. Further analysis by RT-PCR of the PDE4D and PDE7A splice variants gave different accumulation patterns which may indicate that differential splicing has a role in the regulation of these enzymes. Three previously undescribed PDE4D isoforms, with different accumulation patterns, were also detected. They code for truncated PDE4D isoforms, which could participate in the regulation of PDE4D activity.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2000

Up-regulation of vascular endothelial growth factor receptor Flt-1 after endothelial denudation: role of transcription factor Egr-1.

Vascular endothelial growth factor (VEGF) is highly expressed in vascular remodeling processes and accelerates reendothelialization after mechanical denudation. Two VEGF tyrosine kinase receptors have been reported-fms-like-tyrosine kinase-1 (Flt-1) and kinase domain region (KDR). Little is known about the regulation of the expression of these receptors after vascular injury. Herein, we have analyzed the expression of Flt-1 after mechanical denudation of primary cultures of endothelial cells, which has been considered a useful in vitro model to study endothelium responses to vascular injury. After denudation, the Flt-1 protein and mRNA levels are clearly up-regulated, and transient transfection experiments showed a strong induction of the flt-1 promoter-dependent transcription. Analysis of the flt-1 promoter sequence revealed the presence of a putative binding site for the early growth response factor-1 (Egr-1) at positions -24 to -16. Electrophoretic mobility shift and supershift assays showed that Egr-1 was able to bind to this DNA sequence, and cotransfection of the flt-1 promoter reporter plasmid with an Egr-1 expression vector resulted in enhancement of its transcriptional activity. Furthermore, the mutation of the Egr-1 binding site markedly reduced the denudation-induced flt-1 promoter activity. These data demonstrate that Flt-1 is up-regulated after endothelial denudation and that Egr-1 plays a relevant role in this process.

Binding Sites↗

PubMed1999

Memory B lymphocytes from secondary lymphoid organs interact with E-selectin through a novel glycoprotein ligand.

Recirculation of B lymphocytes through the secondary lymphoid organs is key for recognition and response to foreign antigen. B lymphocytes within secondary lymphoid organs comprise a heterogeneous population of cells at distinct differentiation stages. To ascribe a particular adhesive behavior to discrete B-cell subsets within secondary lymphoid organs, we investigated their functional interaction with endothelial selectins under flow. We describe herein the characterization of a subset of human tonsillar B cells that interact with E-selectin but not P-selectin. E-selectin-interacting B cells had a phenotype of non-germinal center (CD10(-), CD38(-), CD44(+)), memory (IgD-) cells. Furthermore, FucT-VII was expressed selectively in CD44(+) E-selectin-adherent B lymphocytes. B-cell rolling on E-selectin required sialic acid but was independent of previously described selectin ligands. A novel glycoprotein ligand of 240 kDa carrying N-linked glycans was isolated from B-cell membranes by an E-selectin immunoadhesin. Binding of this protein was strictly Ca2+ dependent, was inhibited by a cell adhesion-blocking mAb against E-selectin, and required the presence of sialic acid but not N-linked carbohydrates. Our results enable us to assign to resident memory B lymphocytes a novel adhesion function, the rolling on E-selectin, that provides insights on the adhesion pathways involved in homing of memory B cells to tertiary sites.

Animals↗

PubMed1998

Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions.

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell-cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell-cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with alpha3 beta1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for alpha3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.

Animals↗

PubMed1997

Expression of the leukocyte early activation antigen CD69 is regulated by the transcription factor AP-1.

The leukocyte Ag CD69, one of the earliest cell surface activation Ags, is up-regulated at the transcriptional level by proinflammatory stimuli involving the NF-kappaB/Rel family of transcription factors. However, promoter fragments lacking a critical kappaB motif respond to other stimuli such as phorbol esters and triggering Abs against TCR/CD3. Since the 5' promoter flanking region of the CD69 gene contains several putative binding sequences for transcription factor activating protein-1 (AP-1), we explored its role in the inducible expression of CD69. Stimuli that induce AP-1, but not NF-kappaB, such as pyrrolidine dithiocarbamate, augmented the cell surface expression of CD69 as well as its mRNA levels, and the promoter activity of the CD69 gene. This up-regulation is accompanied by an increased binding of jun and fos family members to a consensus AP-1 binding site of the proximal (-16) CD69 promoter region, which seems to be functionally responsive to different activation signals and is trans activated by c-jun expression vectors. Furthermore, cotransfection of a dominant negative version of c-jun, but not IkappaB, abolished the inducible transcriptional activity of the CD69 promoter. In conclusion, the inducible expression of the CD69 gene by mitogenic signals is regulated by the transcription factor AP-1.

Animals↗

PubMed1997

Reduced intracellular oxidative metabolism promotes firm adhesion of human polymorphonuclear leukocytes to vascular endothelium under flow conditions.

The interaction of polymorphonuclear leukocytes (PMN) with the vascular endothelium and their subsequent extravasation to the tissues is a key step during different physiological and pathological processes. In certain of these pathologies the oxygen tension becomes very low, leading to reduced cellular oxidative status. To evaluate the effect of lowering the intracellular redox status in the interaction of PMN with the endothelium, exposure to hypoxic conditions as well as treatment with different antioxidant agents was carried out. PMN exposure to hypoxia enhanced beta2 integrin-dependent adhesion to intercellular adhesion molecule-1-coated surfaces, concomitant with a decrease in the intracellular redox status of the cell. As occurs with hypoxia, treatment with antioxidants produced a decrease in the oxidation state of PMN. These agents enhanced adhesion of PMN to human umbilical vein endothelial cells stimulated with tumor necrosis factor-alpha (TNF-alpha), and this effect was also mediated by beta2 integrins LFA-1 and Mac-1. Adhesion studies under defined laminar flow conditions showed that the antioxidant treatment induced an enhanced adhesion mediated by beta2 integrins with a decrease in the fraction of PMN rolling on TNF-alpha-activated endothelial cells. The up-regulated PMN adhesion was correlated to an increase in the expression and activation of integrin Mac-1, without loss of L-selectin surface expression. Altogether, these results demonstrate that a reduction in the intracellular oxidative state produces an enhanced beta2 integrin-dependent adhesion of PMN to stimulated endothelial cells under conditions of flow.

Antioxidants↗

PubMed1996

Pyrrolidine dithiocarbamate inhibits the production of interleukin-6, interleukin-8, and granulocyte-macrophage colony-stimulating factor by human endothelial cells in response to inflammatory mediators: modulation of NF-kappa B and AP-1 transcription factors activity.

Endothelial cells (EC) play a key role in the inflammatory response, both by the production of proinflammatory cytokines and by their interaction with leukocytes. Molecular genetic analysis has demonstrated that functional NF-kappa B sites are involved in the transcription of interleukin-6 (IL-6), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes in response to inflammatory mediators. Thus, we have explored the effect of two inhibitors of the NF-kappa B activation, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), on the production of these cytokines by EC. Both PDTC and NAC inhibited, in a dose-dependent manner, the synthesis of IL-6, IL-8, and GM-CSF induced by tumor necrosis factor (TNF)-alpha or bacterial lipopolysaccharides (LPS) in human umbilical vein endothelial cells (HUVEC). PDTC appeared to prevent IL-6, IL-8, and GM-CSF gene transcription, as it blocked the induction of specific mRNA by TNF-alpha or LPS. The TNF-alpha mediated transcriptional activation of a chloramphenicol acetyltransferase (CAT) plasmid containing three copies of the -72 kappa B binding site from the IL-6 promoter was abrogated by PDTC. According to transfection experiments, electrophoretic mobility shift assays (EMSA) demonstrated that the antioxidant prevented the induction of NF-kappa B DNA-binding activity by TNF-alpha. Under the same conditions, PDTC by itself or in combination with TNF-alpha, enhanced the DNA-binding activity of AP-1, as well as c-fos and c-jun mRNA levels. Altogether, these results indicate that the antioxidant PDTC specifically inhibits the transcription of IL-6, IL-8, and GM-CSF genes through the inhibition of the NF-kappa B activation, while increasing the expression of AP-1. Our data make evident the antiinflammatory and immunoregulatory potential of the pharmacological inhibition of the NF-kappa B activation. In addition, PDTC and related molecules may be a useful tool to explore the expression of genes involved in the inflammatory response.

Acetylcysteine↗

PubMed1996

Transcriptional up-regulation of intracellular adhesion molecule-1 in human endothelial cells by the antioxidant pyrrolidine dithiocarbamate involves the activation of activating protein-1.

The redox status of the cell plays an essential role in regulating signal transduction, transcription factor activity, and expression of cell surface molecules. In this study, we show that pyrrolidine dithiocarbamate (PDTC), a potent antioxidant agent, upregulated the cell surface expression of intercellular adhesion molecule-1 (ICAM-1) in human endothelial cells (EC). Further analysis of PDTC-mediated ICAM-1 up-regulation revealed that PDTC increased ICAM-1 mRNA levels and augmented its gene promoter activity. Transfection experiments in EC with reporter constructs harboring nested deletion fragments of the ICAM-1 promoter indicated the presence of a functional PDTC-responsive region located between positions -136 to -353 of the promoter. Gel retardation assays together with supershift analysis revealed that PDTC induced the binding of c-fos and c-jun to a consensus activating protein-1 (AP-1) binding site located at position -284. PDTC alone or in combination with TNF-alpha enhanced AP-1-dependent transactivation in HUVEC, as determined by DNA binding assays. The functional implication of AP-1 in the transcription of the ICAM-1 gene was further demonstrated by cotransfection experiments in which a c-jun expression vector induced the promoter activity of the PDTC-responsive element of the ICAM-1 promoter. Taken together, these results indicate that the antioxidant PDTC induces transcriptional activation of ICAM-1 and that this induction is mediated at least in part by the transcription factor AP-1. This mechanism might be operative in pathologic conditions in which a redox imbalance plays a key role, such as ischemia/reperfusion injury or arteriosclerosis.

Antioxidants↗

PubMed1996

Dithiocarbamates trigger differentiation and induction of CD11c gene through AP-1 in the myeloid lineage.

It has recently been shown that the alteration of the cell-redox status affects the transcription factor expression and activity. Dithiocarbamates (DTCs) are potent antioxidant agents that can switch the expression of genes dependent on the activation of the transcription factors AP-1 and NF kappa B. In this study, we show that these agents triggered the expression of genes involved in myeloid differentiation of the promonocytic U-937 cell line. DTCs promoted differentiation-associated changes that included the surface up-regulation of beta 2-integrins (CD11a-c/CD18), cell growth arrest concomitant with transferrin receptor (CD71) down-modulation, induction of the nonspecific esterase enzyme, and a rapid drop in the mRNA levels of c-myc. A further analysis, focused on the molecular mechanisms leading to the activation of CD11c expression, revealed that the pyrrolidine derivative of DTC (PDTC) increased CD11c mRNA levels and augmented its gene promoter activity. Transfection experiments with reporter constructs harboring different promoter regions of CD11c gene, indicated the presence of a functional DTC-responsive region located between positions -160 and +40 of the promoter. Gel retardation assays revealed that the PDTC-induced DNA-protein complexes were restricted to members of the Fos and Jun families that bound to an AP-1 site located at position -60 from the transcription start site. A role for this site was confirmed by in vitro mutagenesis experiments that indicated the functional importance of this site for the CD11c gene transcriptional activation in response to PDTC. The effect of DTCs on myeloid cell differentiation supports a possible role for these agents in the therapy of some bone marrow-derived malignancies.

Base Sequence↗

PubMed1995

B-cell homotypic adhesion through exon-A restricted epitopes of CD45 involves LFA-1/ICAM-1, ICAM-3 interactions, and induces coclustering of CD45 and LFA-1.

Lymphocyte interactions with other leukocytes and other cell types, as well as with components of the extracellular matrix, are one of the key steps in the immune response. Three novel monoclonal antibodies (MoAbs) have been produced and selected for their ability to induce intercellular adhesion in B cells. These three MoAbs immunoprecipitated a polypeptide of 220 kD, displaying specific phosphotyrosine phosphatase activity that has been identified as CD45. These MoAbs recognize epitopes located on the alternative spliced exon-A-encoded region of CD45. These epitopes are of polypeptidic nature, but they can be masked by addition of carbohydrate during CD45 biosynthesis. Interestingly enough, CD45 epitopes recognized by these MoAbs appeared to be selectively expressed on both peripheral blood and tonsillar B lymphocytes as well as on peripheral blood natural killer (NK) cells. CD45-mediated intercellular adhesion was abrogated upon incubation with anti-leukocyte function-associated antigen 1 (anti-LFA-1), intercellular cell adhesion molecule 1 (ICAM-1), and ICAM-3 MoAbs, thus indicating that this phenomenon involved both LFA-1/ICAM-1 and LFA-1/ICAM-3 cell adhesion pathways. Moreover, CD45-mediated cell aggregation was also inhibited by preincubation with some conventional anti-CD45 MoAbs. Interestingly, the triggering of cell aggregation through CD45 induced membrane surface relocation of CD45 and LFA-1 molecules, with both of them colocalizing at cell-cell contact areas of B-cell aggregates. Studies with inhibitors of both phosphotyrosine phosphatase and tyrosine kinase activities suggest that CD45 phosphotyrosine phosphatase activity could be involved in CD45-mediated cell aggregation. Taken together, these results support the notion that CD45 is a key molecule in the regulation of LFA-1-mediated cell-cell interactions.

Antibodies, Monoclonal↗

PubMed1995

Expression of vascular adhesion molecules on human endothelia in autoimmune thyroid disorders.

Cellular activation and expression of certain adhesion molecules within vascular endothelium is a critical event in leucocyte recruitment and emigration. A wide array of different adhesion receptors has been identified to mediate the interaction between endothelial cells (EC) and leucocyte subpopulations. In this study, the tissue expression of E-selectin, P-selectin, CD31, and endoglin endothelial cell adhesion molecules was studied on thyroid tissue from patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). We found an up-regulated expression of E-selectin in EC in GD and HT thyroids, specifically in those areas more severely inflamed, with no reactivity in control thyroids. P-selectin was basally expressed in postcapillary venules in control glands, with an increased expression in HT and GD glands. On the other hand, increased CD31 expression was found on perifollicular, small and large venule EC from GD and HT glands, that correlated with the severity of mononuclear infiltration. In addition, CD31 expression was observed in some intrathyroidal macrophages and T cells in close proximity to CD31+ EC. Furthermore, a markedly enhanced expression of endoglin, a transforming growth factor-beta binding protein, was mainly located on perifollicular EC and EC from small venules as well as in adjacent macrophages from GD and HT thyroid glands. This enhanced expression of E- and P-selectins, CD31 and endoglin by thyroid EC in GD and HT may reflect their ability to regulate leucocyte trafficking and activation.

Antigens, CD↗

PubMed1994

Human CD45RC specificity. A novel marker for T cells at different maturation and activation stages.

The different isoforms of CD45 seem to play an important role in thymocyte maturation and T cell activation and function. To investigate the particular contribution of CD45-exon C (exon 6) to human T cell development, we first investigated the expression of the CD45-exon C isoforms on different human lymphoid cell populations. A wide panel of mAbs against the different isoforms of CD45 was generated by immunizing mice with purified human CD45. Two of these mAbs (RP1/12 and RP2/19) selectively recognized mouse 300-19 pre-B cells that had been transfected with CD45 cDNA that contained exon C, suggesting that epitopes recognized by these mAbs are dependent on the expression of exon C. Immunofluorescence analysis of PBLs showed that CD45RC is expressed at high levels on B, NK, and CD8+ T cells, although a subset of CD4+ T cells was found to be negative. Activation of PBMCs and purified NK cells resulted in down-regulation of CD45RC, which took place in a coordinate manner involving both down-regulation of the expression of CD45RA and CD45RB and up-regulation of CD45R0. Two-color immunofluorescence analyses showed that thymocytes expressing CD45RC were CD1-to lo, CD3hi and HLA class Ihi, typical markers of mature thymocytes. Accordingly, immunohistochemical analysis of a normal thymus showed that whereas the anti-CD45RA mAb stained only a subset of medullary thymocytes, the anti-CD45RC mAb stained the majority of medullary thymocytes. Thymocytes that bear CD45RA also express CD45RC, but do not express CD45RO; a subset of CD45 RA- RO+ thymocytes was also detected. Immunoprecipitation of CD45 molecules from both PBMC and thymocyte cell lysates with mAbs against the different specificities of this molecule revealed that the RC determinants are found on CD45 polypeptides of 220 and 205 kDa that also bear RB or both RB and RA determinants, respectively. Together these results support both the existence of a tight regulation of the expression of CD45 specificities during lymphocyte activation and thymocyte maturation and also that CD45RC is selectively expressed by mature medullary thymocytes during T cell ontogeny.

Alternative Splicing↗

PubMed1994

Adhesion molecules from the LFA-1/ICAM-1,3 and VLA-4/VCAM-1 pathways on T lymphocytes and vascular endothelium in Graves' and Hashimoto's thyroid glands.

Lymphocytic infiltration of the thyroid gland in autoimmune thyroid disorders requires, as a first step, their attachment to endothelial cells (EC) and subsequently, their interaction with thyrocytes and extracellular matrix proteins. A number of different ligand molecules have been identified to mediate the interaction between EC and leukocyte subpopulations. In this study, we examined by flow cytometry and immunohistochemical techniques, the expression of integrin receptors and their counter-receptors by infiltrating lymphocytes and vascular endothelium in thyroid glands from patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). A high proportion of GD intrathyroidal T lymphocytes expressed the CD69 and gp95/85 (Ea2) activation antigens as well as an increased number of LFA-alpha L, VLA-alpha 1, -alpha 4, -alpha 5, and beta 1 integrin receptors, as compared with peripheral blood T lymphocytes from the same patients. The expression of intercellular adhesion molecule (ICAM)-1 was increased in EC from GD and HT thyroids. In addition, an up-regulated de novo expression of vascular cell adhesion molecule (VCAM)-1 was found in EC in GD and HT thyroids, with no reactivity in control thyroids. Dendritic cells in thyroid lymphoid follicles were also positive for ICAM-1 and VCAM-1. In addition, most of intrathyroidal mononuclear cells expressed the ICAM-3 adhesion molecule. This enhanced expression of ICAM-1 and VCAM-1 by thyroid EC in GD and HT may reflect their ability to regulate leukocyte trafficking and activation by means of the expression of specific ligand molecules. Our data suggest that both the LFA-1/ICAM-1, ICAM-3 and VLA-4/VCAM-1 pathways could play a relevant role in localizing and perpetuating the autoimmune response in the thyroid gland in autoimmune thyroid disorders.

Antigens, CD↗

PubMed1994

B lymphocyte binding to E- and P-selectins is mediated through the de novo expression of carbohydrates on in vitro and in vivo activated human B cells.

Cell adhesion to endothelium regulates the trafficking and recruitment of leukocytes towards lymphoid organs and sites of inflammation. This phenomenon is mediated by the expression of a number of adhesion molecules on both the endothelium and circulating cells. Activation of endothelial cells (EC) with different stimuli induces the expression of several adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1), involved in their interaction with circulating cells. In this report, we have studied the binding of nonactivated and activated B cells to purified E- and P-selectins. Activated but not resting B cells were able to interact with both selectins. This binding capacity of activated B cells paralleled the induction of different carbohydrate epitopes (Lewisx, sialyl-Lewisx, CD57 and CDw65) as well as other molecules bearing these or related epitopes in myeloid cells (L-selectin, alpha L beta 2 and alpha X beta 2 integrins, and CD35) involved in the interaction of different cell types with selectins. B cells infiltrating inflamed tissues like in Hashimoto's thyroiditis, also expressed these selectin-binding carbohydrates in parallel with the expression of E-selectin by surrounding follicular dendritic cells. Moreover, the crosslinking of these selectin-binding epitopes resulted in an increased binding of B cells to different integrin ligands. Thus, in addition to the involvement of integrins, E- and P-selectins could play an important role in the interaction of B lymphocytes with the endothelium during B cell extravasation into lymphoid tissues and inflammatory foci as well as in their organization into lymphoid organs.

B-Lymphocytes↗

PubMed1993

Alpha 4 beta 7 integrin mediates B cell binding to fibronectin and vascular cell adhesion molecule-1. Expression and function of alpha 4 integrins on human B lymphocytes.

Cell-cell and cell-extracellular matrix interactions are mediated by a wide array of cell surface molecules known as adhesion receptors, including the integrin family that comprises numerous alpha beta heterodimers. A new integrin group, the beta 7 subfamily, has been recently defined. Its two members, alpha 4 beta 7 and alpha H beta 7, are involved in the lymphocyte migration to the Peyer's patches and the intestinal mucosa, respectively. We have analyzed the expression of alpha 4 beta 7 integrin on B cells from different cellular compartments and at different activation states. Resting peripheral blood B lymphocytes constitutively express large amounts of alpha 4 beta 7. By contrast, alpha 4 beta 7 integrin, which is absent on resident B cells from different lymphoid tissues, is induced upon activation. Functional studies indicates that alpha 4 beta 7 is mediating B cell attachment to fibronectin and vascular cell adhesion molecule-1 through distinct epitopes on this integrin. Furthermore, the alpha 4 beta 7 integrin is also implicated in intercellular interactions as deduced by the ability of anti-alpha 4 beta 7 mAb to trigger homotypic B cell aggregation. Finally, alpha 4 beta 7 and alpha 4 beta 1 integrins redistribute at the cell membrane in a similar clustering pattern when B cells attach to fibronectin- and vascular cell adhesion molecule-1-coated surfaces. Our studies demonstrate the differential regulation on the expression and function of alpha 4 beta 7 integrin among different human B cell populations.

Antigens, Neoplasm↗

PubMed1993

Costimulation of cAMP and protein kinase C pathways inhibits the CD3-dependent T cell activation and leads to a persistent expression of the AP-1 transcription factor.

The effects mediated by a combined stimulation of cAMP- and protein kinase C (PKC)-dependent pathways have been investigated in different cellular systems, and it has been shown that they may complement each other in activating cell proliferation and differentiation. In this report, we show that upon the stimulation of both pathways T lymphocytes became refractory to activation via the CD3/T cell receptor (TcR) complex. T cells preincubated with phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (Bt2cAMP) displayed a deficient proliferative ability in response to anti-CD3 mAb stimulation, whereas lymphocytes treated individually with either Bt2cAMP or PMA responded comparably to untreated samples. We detected an association between the reduced mitogenic response and low expression of both interleukin-2 (IL-2) and the alpha chain (CD25) of the IL-2 receptor (IL-2R). Analysis of intracellular Ca2+ mobilization suggested that the CD3/TcR-dependent signal transduction was impaired in PMA/Bt2cAMP-treated cells. Remarkably, we observed that these samples displayed a persistent expression of the c-fos protooncogene, associated to an increased AP-1 DNA-binding activity, whereas no variations of CREB or NF-kB were detected. Neither Bt2cAMP nor PMA individually mediated these sustained effects, which therefore appear as a consequence of the interplay between both metabolic stimuli. Altogether, the data provide the evidence that both pathways complement each other in regulating gene expression and, conversely, downregulate the TcR transduction mechanisms.

Base Sequence↗

PubMed1992

Human T cell activation through the activation-inducer molecule/CD69 enhances the activity of transcription factor AP-1.

The induction of the AP-1 transcription factor has been ascribed to the early events leading to T cell differentiation and activation. We have studied the regulation of AP-1 activity in human peripheral blood T lymphocytes stimulated through the activation inducer molecule (AIM)/CD69 activation pathway. Phorbol esters are required to induce AIM/CD69 cell-surface expression as well as for triggering the proliferation of T cells in conjunction with anti-AIM mAb. Mobility shift assays showed that addition of anti-AIM mAb to PMA-treated T lymphocytes markedly enhanced the binding activity of AP-1 to its cognate sequence, the phorbol ester response element. In contrast, anti-AIM mAb did not induce any change in the binding activity of NF-kappa B, a transcription factor whose activity is also regulated by protein kinase C. The increase in AP-1-binding activity was accompanied by the marked stimulation of the transcription of c-fos but not that of c-jun. Blockade of the DNA-binding complexes with an anti-Fos mAb demonstrated a direct participation of c-Fos in the AP-1 complexes induced by anti-AIM mAb. Most of the AP-1 activity could be eliminated when the anti-AIM mAb was added to the culture medium in the presence of cycloheximide, suggesting that de novo protein synthesis is crucial for the induction of AP-1-binding activity. These data provide the evidence that activation of human peripheral blood T cells through the AIM activation pathway regulate the activity of AP-1. Therefore, this pathway appears as a crucial step in the initiation of early T cell activation events.

Antibodies, Monoclonal↗

PubMed1992

VLA family in rheumatoid arthritis: evidence for in vivo regulated adhesion of synovial fluid T cells to fibronectin through VLA-5 integrin.

Adhesion of T cells to extracellular matrix (ECM) proteins through VLA integrin receptors is crucial for lymphocyte trafficking, tissue localization and inflammatory function. We have investigated the expression of different VLA integrins (VLA-1-5) on peripheral blood (PB) and synovial fluid (SF) T lymphocytes from patients with rheumatoid arthritis (RA). Their expression on different cell types from synovial membrane (SM) is also reported. The role of VLA-4 fibronectin (FN) receptors in the interaction of activated SF T cells from RA patients with a 38-kD fragment of FN has been previously demonstrated. Here we have focused functional studies on VLA-5 as an alternative FN receptor for RA T cells. A significant higher proportion of SF T cells were able to bind to an 80-kD fragment of FN, containing the Arg-Gly-Asp (RGD) cell binding site, compared with PB T cells. This attachment was almost completely inhibited by anti-VLA-5 MoAbs as well as by RGD peptides. This enhanced capability by SF T cells appears to be independent of the level of the surface expression of the receptor and correlates better with their activation state as determined by the expression of the activation molecule AIM (CD69). The evidence for the expression of VLA heterodimers on both SF and SM cells from RA patients suggests the possible implication of ECM proteins in mediating and perpetuating inflammation in vivo.

Adult↗