Biomedical subjects

C Bernabeu

Publications and source records attributed to C Bernabeu.

At least 37 records · Page 2Linked to original sources

PubMed1992

Decreased tubulin synthesis in synoviocytes from adjuvant-induced arthritic rats.

The first microscopical alterations along adjuvant arthritis induction in rats seem to appear in the synovium. We have studied the protein synthesis pattern of the cells constitutively present in synovial membrane (synoviocytes) and have found an impairment of synthesis of some proteins when synoviocytes are derived from adjuvant arthritic rats. One of these polypeptides was identified as beta tubulin by two-dimensional gel electrophoresis, a membrane transfer assay using a specific monoclonal antibody and peptide mapping. We postulate that a repressed synthesis of tubulin may be an initial step in the triggering of the disease, since the effect was evident at pre-arthritic stages, when infiltration by inflammatory cells had not yet occurred.

Animals↗

PubMed1992

Camptothecin induces differentiation and stimulates the expression of differentiation-related genes in U-937 human promonocytic leukemia cells.

We have studied the effect of the DNA topoisomerase I inhibitor camptothecin on growth, differentiation, and gene expression in U-937 human promonocytic leukemia cells. At a concentration of 20 nM, camptothecin caused significant DNA strand breakage and decreased the growth activity by accumulating cells preferentially at the G2 phase of the cycle. The growth arrest occurred concomitantly with an increase in cell size. Under those conditions, camptothecin induced differentiation, as demonstrated by (a) the capacity of the cells to generate reactive oxygen species, (b) the increase in the surface expression of the leukocyte integrins CD11b/CD18 and CD11c/CD18, (c) the increase in the cellular content of the intermediate filament protein vimentin, and (d) the decrease in the surface expression of the transferrin receptor. Camptothecin also induced the expression of differentiation markers in other human myeloid cells, namely, the promonocytic THP-1 and the myelomonocytic HL-60 cell lines. Northern blot assays revealed that camptothecin stimulated the expression of CD11b, CD11c, and vimentin at the mRNA level. Moreover, the drug increased the transcription rate of the vimentin gene, as shown by "run-on" transcription assays.

Camptothecin↗

PubMed1992

Regulated expression on human macrophages of endoglin, an Arg-Gly-Asp-containing surface antigen.

Endoglin is an endothelial homodimeric membrane antigen containing the tripeptide arginine-glycine-aspartic acid (RGD), which is a recognition motif for adhesion receptors of the integrin family. We have investigated the expression of endoglin by monocyte/macrophage cells from different tissue compartments and at different stages of cell differentiation. Although endoglin is absent from peripheral blood monocytes, it is expressed by in vitro differentiated monocytes as determined by flow cytometry using the endoglin-specific monoclonal antibody 44G4 and 8E11. Furthermore, Northern blot analyses revealed a correlation between the presence of endoglin mRNA and the surface expression of the antigen by in vitro differentiated monocytes. Immunostaining of frozen tissue sections with the 8E11 monoclonal antibody demonstrated the presence of endoglin not only in the endothelium of all the tissues studied, but also on the interstitial macrophages present in the red pulp of the spleen. Using as a model of macrophage differentiation monocytic cell lines treated with phorbol esters, we found that the reactivity of the 8E11 monoclonal antibody is greatly increased on U-937 and HL-60 cells during their PMA-induced differentiation. These findings clearly demonstrate for the first time the regulated expression of the putative adhesion molecule endoglin by macrophages.

Antibodies, Monoclonal↗

PubMed1992

Identification of distinct epitopes of endoglin, an RGD-containing glycoprotein of endothelial cells, leukemic cells, and syncytiotrophoblasts.

Endoglin is a glycoprotein expressed predominantly on human endothelial cells. It was first identified with mAb 44G4, produced against the pre-B acute lymphoblastic HOON cell line. We now report that four mAbs independently produced against human umbilical vein endothelial cells (HUVECs), chronic myelogenous leukemia in blast crisis, or U-937 pro-monocytic cells stimulated with phorbol myristate acetate also react with endoglin. High levels of reactivity of all mAbs were observed with HUVEC, while intermediate levels were seen with HOON and U-937 cells. By sequential immunoprecipitation from HUVEC and U-937 cell extracts, it was established that RMAC8, HEC-19, 8E11, and 1G2 mAbs react with the same protein as 44G4. Three distinct epitopes recognized by 44G4, RMAC8, and 1G2 mAbs were identified by competitive radioimmunoassay and flow cytometry. The HEC-19 epitope is spatially related to the 44G4 epitope, whereas the 8E11 epitope is most closely related to the 1G2 epitope. Western blot analysis showed that all antibodies react with the endoglin dimer (Mr = 170,000) purified from placenta. Immunostaining of sections of full-term placenta revealed reactivity not only with fetal vessels but also with the syncytiotrophoblast, the fetal cell layer which interfaces with maternal blood. When HUVEC monolayers were treated with the different mAbs to endoglin, prior to incubation with U-937 cells, a 5- to 10-fold stimulation of adhesion was observed. A fibronectin hexapeptide containing RGD, but not the corresponding RGE peptide, was capable of inhibiting the increased adhesion, when tested with mAb 44G4 and RMAC8. However, the same peptides had no effect on the binding of any of the five anti-endoglin mAbs to cells. Since 44G4 and RMAC8 recognize two distinct epitopes of endoglin, and since all five mAbs stimulated adhesion, the results suggest that a signal has been triggered through endoglin on HUVECs. Endoglin might be implicated either directly, by binding to a specific integrin-like ligand, or indirectly, by regulating the level of adhesion between certain integrins and their receptors.

Amino Acid Sequence↗

PubMed1992

Arthritis transferred by cells derived from pre-inflammatory rat synovium.

Different cell populations isolated from rats during the period of latency of adjuvant arthritis were injected into the bloodstream of naive rats to test their ability to transfer articular disorders. Synovium-derived cells (synoviocytes) were able to induce arthritis in 3 out of 4 recipient animals, whereas peripheral blood leukocytes, peritoneal exudate macrophages, lymph node cells, synoviocyte lysates and synoviocytes from control animals were not able to do so. This model of cellular transferred arthritis is associated with antibody titres to hsp65 in rat sera. Our findings suggest a crucial role for synovial cells in the pathogenesis of adjuvant disease, which might be linked to their function as accessory cells.

Animals↗

PubMed1991

Antibodies to dietary antigens in rheumatoid arthritis--possible molecular mimicry mechanism.

Antibodies in serum from some patients with rheumatoid arthritis, recognize bovine albumin present in the milk, as determined by immunoprecipitation analysis from 125I-milk extracts. This antigen was also immunoprecipitated from bovine sera. These and ELISA studies showed that BSA is preferentially recognized over other proteins present in the milk. Panel studies demonstrated that although the average reactivity for BSA was high, only one third of the sera tested displayed a reactivity above the mean. The possibility of a molecular mimicry mechanism in RA between this food antigen and other human antigens was investigated. A sequence alignment analysis showed that the residues 141-157 of bovine albumin significantly differed from the corresponding fragment of human albumin, but were highly homologous with human collagen type I, C1q and vitamin D binding protein. In support of the immunogenicity of this fragment, we found that representative RA sera displayed a specific reactivity for a synthetic peptide containing the BSA residues responsible for the homology. Furthermore, most of the epitopes recognized on BSA by the RA sera seem to be conformationally dependent as heat denaturation or reduction followed by alkylation lead to a diminished recognition.

Amino Acid Sequence↗

PubMed1991

Induction of LFA-1-mediated homotypic adhesions in promonocytic U-937 cells occurs independently of cell differentiation.

The differentiation of monocytes into macrophages occurs along with a marked increase in LFA-1-dependent intercellular adhesions. Similarly, the phorbol ester-induced differentiation of U-937 promonocytic cells into macrophage-like cells is morphologically characterized by an important increase in LFA-1/ICAM-1-dependent intercellular homotypic adhesions. Since an important functional role in activation of human T cells has been demonstrated for LFA-1-dependent adherence, we have analyzed whether the induction of LFA-1-dependent intercellular adhesion of human monocytic cells is necessarily accompanied by differentiation of these cells. We found that treatment of the promonocytic U-937 cells with the anti-LFA-1 mAb NKI-L16 induces formation of intercellular clusters, but does not induce cell differentiation as determined by several differentiation markers. These markers include the arrest of cell proliferation, production of reactive oxygen species, changes in the cell surface expression of differentiation-associated antigens such as the transferrin receptor, CD11b and CD11c and changes in the levels of several specific gene transcripts such as CD18 antigen, c-myc, ornithine decarboxylase and vimentin. These findings suggest that LFA-1-dependent adhesion and differentiation of monocytic cells are independent processes.

Antibodies, Monoclonal↗

PubMed1991

Effects of poliovirus replication on undifferentiated and differentiated monocytic U937 cells: comparative studies with human macrophages.

Poliovirus infection of either undifferentiated or differentiated U937 cells produced a decrease in the percentage of cells positive for the surface expression of the CD4, CD11c, CD14, or 8E11 antigens. The number of 4F2 surface molecules per cell increased in infected normal U937 cells, but was unaffected in differentiated cells. The level of O2- production in infected differentiated U937 cells was approximately 50% of that found when not infected. Finally, poliovirus RNA levels and infectious particle production were similar in either cell type.

Antigens, CD↗

PubMed1991

Cellular and humoral reactivity pattern to the mycobacterial heat shock protein HSP65 in adjuvant arthritis susceptible and resistant Wistar rats.

We have analyzed the cellular and humoral immunity to the mycobacterial 65 KDa heat shock protein (hsp65) in a group of Freund's Adjuvant-immunized rats with a limited susceptibility to Adjuvant arthritis. According to the arthritis indices during the period of study (35 days), two different groups of rats could be distinguished; a) autoimmune Adjuvant arthritic rats (AA), and b) Non-arthritic animals (NA), including both rats which did not display any disease symptoms and rats suffering mild transient inflammation. The cellular response to the immunizing agent (Mycobacterium tuberculosis) or the mitogen Concanavalin A was comparable between both groups of rats. However, we detected an impaired cellular response to the individual hsp65 antigen in the animals that did not develop the disease. On the contrary, the level of hsp65-specific antibodies was much higher in NA animals than in AA rats suggesting a protective role for the hsp65 specific antibodies.

Animals↗

PubMed1991

Differentiation of human promonocytic leukemia U-937 cells with DNA topoisomerase II inhibitors: induction of vimentin gene expression.

The administration of the DNA topoisomerase II inhibitors 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) (10(-7) M), VP-16 (2 x 10(-7) M), or novobiocin (1.5 x 10(-4) M) reduces the growth activity of human promonocytic leukemia U-937 cells, by arresting them preferentially at the G2 (m-AMSA and VP-16) or at the G1 and G2 (novobiocin) phases of the cell cycle. Under these conditions, m-AMSA and VP-16 induce the differentiation of the cells efficiently, as proved both by an increase in the production of reactive oxygen species and by the activation of the surface expression of CD11b and CD11c, two differentiation-specific antigens. Novobiocin also induces the expression of those differentiation markers, but to a lesser extent. Analyses by Northern blot indicate that the topoisomerase II inhibitors reduce the levels of c-myc and beta-actin mRNA and increase the levels of vimentin mRNA. The expression of vimentin is also stimulated at the protein level, as indicated by immunofluorescence assays. This represents one of the few known instances in which topoisomerase inhibitors stimulate gene expression in eukaryotic cells.

Amsacrine↗

PubMed1990

Phorbol esters induce differentiation of U-937 human promonocytic cells in the absence of LFA-1/ICAM-1-mediated intercellular adhesion.

Intercellular adhesions which occur during the mononuclear phagocyte differentiation are predominantly mediated by the lymphocyte-function-associated antigen-1 (LFA-1) family and the intercellular-adhesion molecule-1 (ICAM-1) which is a ligand for LFA-1. Thus, differentiation of U-937 promonocytic cells induced by phorbol esters occurs concomitantly with intercellular LFA-1/ICAM-1-dependent cluster formation. Since these homotypic adhesions can be inhibited by monoclonal antibodies (mAb) directed to either LFA-1 or ICAM-1, we have analyzed whether the lack of cell-cell adhesions impairs the differentiation process. Treatment of U-937 cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in the presence of mAb to LFA-1 or ICAM-1 antigens yielded cells free from homotypic adhesions but differentiated as evidenced by their decreased proliferation and enhanced capacity for generation of superoxide anion. In addition, expression of the CD11c antigen was increased, whereas the transferrin receptor disappeared from the cell surface. Vimentin gene transcription was also greatly augmented as opposed to a clear diminution in the levels of c-myc and ornithine decarboxylase transcripts. These results clearly demonstrate that phorbol esters can induce differentiation of monocytic cells independently of cell-cell adhesion.

Antibodies, Monoclonal↗

PubMed1990

The CD11c antigen couples concanavalin A binding to generation of superoxide anion in human phagocytes.

We have found that an anti-CD11c monoclonal antibody (MAb) inhibits the respiratory burst induced in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells as well as in human peripheral blood monocytes and neutrophils upon cell stimulation with concanavalin A. The MAb had no effect, however, when the added stimulus was fMet-Leu-Phe or PMA. Flow cytometry analyses indicated that concanavalin A was able to interact with CD11c. The anti-CD11c MAb inhibited significantly concanavalin A binding to differentiated U937 cells, and concanavalin A blocked binding of anti-CD11c MAb to the cells. Binding of labelled concanavalin A to membrane proteins which were separated by PAGE and transferred to nitrocellulose paper indicated that proteins with apparent molecular masses similar to those of CD11c (150 kDa) and CD18 (95 kDa) molecules were the main concanavalin A-binding proteins in differentiated U937 cells as well as in mature neutrophils. Similar experiments carried out in the presence of the anti-CD11c MAb showed a specific and significant inhibition of concanavalin A binding to the CD11c molecule. These results indicate that concanavalin A binds to the CD11c molecule and this binding is responsible for the concanavalin A-induced respiratory burst in PMA-differentiated U937 cells as well as in human mature monocytes and neutrophils.

Antibodies, Monoclonal↗

PubMed1990

Induction of T cell activation by monoclonal antibodies specific for the transferrin receptor.

The effect of the monoclonal antibodies (mAb), FG 1/5, FG 1/6 and FG 2/12, specific for different epitopes of the transferrin receptor (TfR) on T cell activation was studied. mAb FG 1/6 but not FG 2/12 or FG 1/5 was able to induce T cell proliferation in presence of submitogenic doses of phorbol esters. The costimulatory effect of FG 1/6 was seen only with phorbol esters known to be activators of protein kinase C. This proliferation occurred at low concentration (0.5 micrograms/ml) of antibody, required the simultaneous presence of both stimuli, phorbol esters and FG 1/6, and was independent of the presence of accessory cells. Furthermore, FG 1/6 mAb was able to increase the rate of modulation of CD3 surface expression induced by phorbol esters. FG 1/6 induced interleukin (IL) 2 synthesis by normal and transformed T lymphocytes. In addition, anti-IL2 receptor antibodies inhibited FG 1/6 plus phorbol ester-induced proliferation. Our results indicate that FG 1/6 mAb may provide to the T cells complementary signals to protein kinase C and that this activation is mediated by the IL2/IL 2R pathway.

Animals↗

PubMed1989

S-phase inhibitors induce vimentin expression in human promonocytic U-937 cells.

The administration of hydroxyurea (3 x 10(-4) M) and cytosine arabinoside (10(-7) M) greatly induces the expression of the vimentin gene in human promonocytic leukemia U-937 cells. The induction takes place at both the mRNA and protein levels, as demonstrated by Northern blot, immunoblot and immunofluorescence assays. On the contrary, the drugs inhibit the expression of c-myc and ornithine decarboxylase, and do not modify significantly the expression of beta-actin. Since hydroxyurea and cytosine arabinoside trigger the phenotypic differentiation of U-937 cells, as demonstrated by the induction of the differentiation-specific CD11b and CD11c antigens, it is concluded that vimentin expression might be implicated in the maturation of these cells.

Antigens, Differentiation, Myelomonocytic↗

PubMed1989

Characterization of a novel myeloid antigen regulated during differentiation of monocytic cells.

The monoclonal antibody HC1/6 generated against phorbol 12-myristate 13-acetate-treated U-937 cells recognizes a new cell surface antigen with a broad relative molecular mass ranging from 100 to 150 kDa. This antigen is also present on monocytes, platelets and endothelial cells and is weakly expressed by granulocytes. In contrast, it is absent from T, B and erythroblastoid cells. The antigen HC1/6 is also expressed by normal tissue macrophages in tonsil, lung and kidney, as well as in skin biopsies from pathologies such as sarcoidosis and lepromatous leprosy. The expression of the HC1/6 antigen is increased up to 5-fold when U-937 (promonocytic) and HL-60 (myelomonocytic) cell lines are stimulated with phorbol 12-myristate 13-acetate. Conversely, the expression of the HC1/6 antigen is down-regulated in monocytes upon treatment with interferon-gamma. These findings are discussed in relation with other myeloid cell surface markers.

Antibodies, Monoclonal↗

PubMed1989

A monoclonal antibody to CD11c antigen inhibits the production of superoxide anion induced by concanavalin A in PMA-differentiated U-937 cells.

We have examined the effect of different monoclonal antibodies (MAb) to the CD11/CD18 family of surface antigens on the production of superoxide anion by phorbol myristate acetate (PMA)-differentiated U-937 cells upon stimulation with concanavalin A (Con A). The anti-CD11c MAb HC1/1 displayed a significant inhibition, whereas MAb to the other members of the CD11 family did not show a relevant inhibitory effect. These findings suggest that the CD11c antigen is involved in the triggering of the respiratory burst response by mononuclear phagocytic cells.

Antibodies, Monoclonal↗

PubMed1989

Poliovirus infection interferes with the phorbol ester-induced differentiation of the monocytic U937 cell line.

Monocytic U937 cells can differentiate in vitro into macrophage-like cells by treatment with phorbol esters such as phorbol 12-myristate 13-acetate (PMA). We have analyzed the effect of poliovirus infection in this pathway of differentiation. Poliovirus RNA replication took place in both untreated and PMA-treated U937 cells infected before or after PMA addition, although a slight reduction in poliovirus RNA levels was observed in PMA-treated cells at late times postinfection. Total protein synthesis remained unchanged during the first 5 h of infection both in normal and PMA-treated cells. However, an inhibition on total RNA synthesis was observed early in infection. PMA-induced c-myc mRNA expression was abolished when infection took place 1 h before PMA addition but was just partially inhibited when poliovirus was added 1 h after PMA stimulation. Fluorescence flow cytometry analysis revealed that poliovirus infection induced an increase in the number of 4F2 molecules per cell in normal U937 cells and a slight decrease in the number of positive cells for the antigens CD14, CD4 and CD11c in both untreated or PMA-treated U937 cells. These findings suggest that poliovirus infection of U937 cells interferes at various levels with monocyte maturation yielding cells which are unable to undergo the complete pathway of differentiation to macrophages.

Antigens, Surface↗

PubMed1989

Synoviocytes type A bind exogenous antigens recognized by antibodies present in rheumatoid arthritis.

The presence of antibodies in rheumatoid arthritis (RA) patients to antigens on the synoviocyte surface has recently been reported (Scand. J. Immunol. 27, 295, 1988). Here we have further characterized these antigens and found that they are exogenous proteins acquired from the bovine serum used in the culture medium. By immunoprecipitation and ELISA studies, we have identified bovine albumin and transferrin as the antigens recognized by the RA antibodies. These specificities were found not only in the sera but also in the synovial fluid from RA patients. A comparative study with a large panel of RA sera did not show a correlation in the antibody specificities for bovine albumin, bovine transferrin, or the 65-kDa heat shock protein from Mycobacterium bovis. Similar experiments using rabbit and monkey sera as well as human synovial fluid and serum as a source of antigen did not reveal any reactivity with a highly positive RA serum. By sequence alignment, a high degree of homology between residues 142-156 from bovine albumin and residues 65-78 from human pro-collagen alpha 1 (I) was found. The capacity of the synoviocytes to bind exogenous antigens and the presence of antibodies to bovine proteins, normally present in the diet, suggest a role for these type A synoviocytes as well as a possible involvement of food antigens in the pathogenesis of RA.

Animals↗