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G Bou-Gharios

Publications and source records attributed to G Bou-Gharios.

33 records · Page 2Linked to original sources
PubMed1992

Expression and function of heterotypic adhesion molecules during differentiation of human skeletal muscle in culture.

The infiltration of skeletal muscle by leukocytes occurs in a variety of myopathies and frequently accompanies muscle degeneration and regeneration. The latter involves development of new myofibers from precursor myoblasts, and so infiltrating cells may interact with muscle at all stages of differentiation. The authors have investigated the surface expression of ligands for T-cell adhesion during the differentiation of human skeletal muscle in vitro. Myoblasts expressed low levels of ICAM-1 (CD54), which remained constant during muscle cell differentiation and could be induced by cytokines such as gamma-interferon. It is therefore likely that ICAM-1 is involved in the invasive accumulation of lymphocytes during skeletal muscle inflammation. In contrast, LFA-3 (CD58) was expressed at higher levels than ICAM-1 on myoblasts, decreased significantly during myogenesis, and was unaffected by immune mediators. Both ICAM-1 and LFA-3 were able to mediate T cell binding to myoblasts, whereas adhesion to myotubes was independent of the LFA-3 ligand. Although expressed throughout myogenesis, human leukocyte antigen class I and CD44 did not appear to mediate T cell binding. The expression of ligands that facilitate interaction of myogenic cells with lymphocytes may have important implications for myoblast transplantation.

Antibodies, Monoclonal↗
PubMed1991

Simultaneous measurement of cell surface and intracellular antigens by multiple flow cytometry.

We have employed a method for permeabilizing lymphocytes with the detergent saponin in order to detect an intracellular protein simultaneously with surface antigens by flow cytometry (FCM). Using monoclonal antibodies specific for the murine CD2 receptor and for the lysosomal enzyme, beta-glucuronidase (Gus), we found that the expression of both of these antigens increased markedly when T cells were activated. Two sensitive methods were used to show that FCM provided an accurate measure of the actual number of CD2 and Gus molecules present in the lymphocytes. Immunogold electron microscopy revealed the precise ultrastructural localization of these different components and corroborated the specificity of the multiple labelling procedure for the simultaneous detection of surface and intracellular antigens. We also developed a three-colour FCM technique which we used to examine the changes in Gus expression in the CD4 and CD8 T cell sub-sets during activation.

Animals↗
PubMed1991

Function and regulation of the murine lymphocyte CD2 receptor.

The CD2 receptor on T-lymphocytes plays a major part in mediating adhesive interactions via the LFA-3 ligand and in transducing signals for lymphocyte activation. In this study the expression, function, and internalization of the CD2 receptor was investigated in resting and activated murine T-cells. Surface iodination of intact lymphocytes showed that both types of cell expressed this antigen as a single polypeptide of 63 KDa, and flow cytometry analysis demonstrated that there was four times as much CD2 on lymphoblasts as on resting cells. Moreover, the CD2 receptor had a more prominent role in the adhesion of the activated lymphocytes to extravascular cells than in the binding of resting cells. Only activated lymphocytes internalized CD2, in the presence or absence of the anti-CD2 monoclonal antibody (mAb) 12-15, more than 80% of the 12-15/CD2 complex being removed from the cell surface within 24 hr. Application of 125I-labelled mAb 12-15 followed by subcellular fractionation on Percoll gradients showed that the complex was internalized initially into a low-density compartment and subsequently transported to heavy-density organelles, in which it was degraded. Immunogold electron microscopy revealed that immediately after the initial binding of mAb 12-15 to the lymphoblasts, the gold particles were localized in clusters exclusively at the plasma membrane. After a short period of culture, the mAb 12-15/CD2 complex was detected in small vesicles near the cell surface. Immunogold staining for a lysosomal enzyme beta-glucuronidase (Gus), for the lysosomal membrane protein LAMP-1, and for the mannose 6-phosphate targetting receptor (MPR) showed that the complex was transported from the endosomal compartment to lysosomal organelles in the activated T-cell. Although mAb 12-15 bound to CD2 in resting T-lymphocytes, in these cells the complex remained associated with the plasma membrane compartment only, even after prolonged culture. These data show that activated but not resting lymphocytes endocytosed the receptor, thereby regulating the expression of this antigen at the plasma membrane. This suggests that the endocytic and lysosomal compartments of lymphocytes have major roles in immune functions, by controlling the level of receptors at the lymphocytes cell surface and thus their response to cytokines and inflammatory mediators as well as their direct interaction with other cells.

Animals↗
PubMed1991

Localization of lysosomal antigens in activated T-lymphocytes.

The lysosomal compartment has been examined in activated T-lymphocytes by immunogold electron microscopy and subcellular fractionation. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of radiolabelled extracts of the T-cells showed that they contained three antigens which are fundamental to normal lysosomal function: a representative lysosomal enzyme beta-glucuronidase, a lysosomal associated membrane protein (LAMP-1), and the cation-independent mannose 6-phosphate lysosomal enzyme targeting receptor (MPR). Immunogold labelling showed that beta-glucuronidase was present in the rough endoplasmic reticulum, the Golgi complex and Golgi-associated vesicles. The enzyme was also found to accumulate in distinct, non-Golgi organelles in which LAMP-1 was co-localized, probably lysosomes. LAMP-1 was also found in tubular elements of the Golgi and in a complex of vesicles clustered near the nucleus where MPR was also present at high density. Fractionation of homogenates from lymphocytes on Percoll gradients revealed that beta-glucuronidase was distributed throughout the low density region containing rough endoplasmic reticulum, Golgi and plasma membrane components, and the high density region which contained only lysosomal activity. Multiple immunogold electron microscopy of the latter fraction showed the presence of homogenous vesicles which had large amounts of beta-glucuronidase within the lumen, LAMP-1 at the periphery and no MPR. These vesicles were probably mature lysosomes, arising from pre-lysosomal organelles enriched for LAMP-1 and MPR.

Animals↗
PubMed1991

Contact-dependent transfer of a lysosomal enzyme from lymphocytes to fibroblasts.

In this study we have examined the mechanism underlying the contact-mediated transfer of a lysosomal enzyme from lymphocytes to fibroblasts in culture. We found that although antibody against the mannose 6-phosphate lysosomal targetting receptor (MPR) completely inhibited fibroblast uptake of the lysosomal enzyme beta-glucuronidase (Gus) from the culture medium, it had no effect on the transfer of the enzyme from normal lymphocytes. In contrast, the presence of antibody that prevented the adhesion of the lymphocytes to the fibroblasts inhibited Gus acquisition but had no effect on endocytosis. Immunogold electron microscopy of the contact site between the two types of cell showed that the transfer of Gus involved uncoated vesicles localized near the cell surface of the fibroblast at sites of contact with the lymphocytes. The acquired lymphocyte enzyme was shown to be transported to the fibroblast lysosomes.

Animals↗
PubMed1990

The activation of resting lymphocytes is accompanied by the biogenesis of lysosomal organelles.

The degradative activity of lymphocytes plays by important role in a number of essential immune functions. In the present study we have examined how the activation of resting lymphocytes, by the mitogen concanavalin A (Con A), affects three major components of the lysosomal compartment: the lysosomal enzyme beta-glucuronidase (Gus); an integral lysosomal membrane protein (LAMP-1); and the mannose 6-phosphate receptor (MPR) which directs lymphocyte enzyme transport. Resting T cells were found to contain only very low levels of these proteins, but they were actively synthesized by, and far more abundant in, stimulated lymphoblasts. Although the lysosomal antigens did not have a distinct cytoplasmic localization in the resting lymphocytes, in the activated T lymphoblasts they were present in several highly developed intracellular structures, including the rough endoplasmic reticulum and the Golgi complex. Furthermore, in these latter cells Gus was also found to be accumulated within the lumen of large vesicles which we characterized as lysosomes by the presence of LAMP-1 at the periphery and by the absence of MPR. Subcellular fractionation confirmed that these organelles were present in the activated lymphocytes only, and not in the resting T cells. Our results demonstrate that lymphocyte activation is accompanied by the synthesis of the enzymic and structural components of the lysosomal compartment which are sorted and assembled into distinct organelles in the activated cell.

Animals↗
PubMed1990

Interactions between lymphocytes and dermal fibroblasts: an in vitro model of cutaneous lymphocyte trafficking.

Cultures of dermal fibroblasts were established from skin biopsies of CBA mice and used to study the interactions with murine T-lymphocytes. Electron microscopy showed that zones of contact developed between the fibroblasts and the T-cells, particularly after mitogenic activation. The adhesion of the lymphocytes was temperature-dependent, and many more lymphoblasts than resting cells attached to the fibroblast monolayers. Flow cytometry analysis of the adherent population showed that the most prominent type of resting lymphocyte was of the CD4 phenotype, which was also observed using a T-helper lymphoid cell line. However, neither the CD4 nor the CD8 (T-cytotoxic) antigens were involved in the binding process, and while the fibroblasts expressed Class I MHC molecules (but not Class II), these also had no role in mediating lymphocyte adhesion. Although the fibroblasts did not express the ligand Mala-2, the murine homologue of human ICAM-1, a monoclonal antibody against LFA-1, its cognate receptor on the lymphocytes, nevertheless effectively inhibited binding. T-cell attachment was also partially prevented by antibody against the lymphocyte CD2 antigen and by RGDS, a protein epitope known to mediate a number of receptor-integrin interactions. Moreover, this peptide also rapidly and preferentially detached T-lymphocytes which had previously adhered to the fibroblast monolayers. Lymphocyte binding was substantially elevated following treatment of the fibroblasts with cytokines such as tumor necrosis factor-alpha and interferon-gamma, but not interleukin-1 alpha. This increase in adhesiveness was, however, almost completely abolished by monoclonal antibodies specific for LFA-1 or for Mala-2. The results of this study show that while lymphocytes recognize fibroblasts normally via a number of constitutively expressed receptor-integrin interactions, their adhesion can also be modulated by cytokine-induced changes in the expression of other surface ligands.

Animals↗
PubMed1989

Adhesion of lymphoid cells to fibroblasts in tissue culture.

In this study we have examined the cellular and molecular specificity of lymphocyte interaction with fibroblasts. Using mitogen-activated T-cells, we found that attachment to fibroblasts was highly sensitive to protease treatment, and to an antibody raised against the purified lymphocyte plasma membrane, but it was not mediated by the MEL-14 surface antigen or phosphomannosyl receptors. Lymphocyte interaction with fibroblasts was also unaffected by monoclonal antibodies against the LFA-1, Mac-1, and Class II MHC antigen complexes. In contrast, adhesion of both T- and B-lymphocytes was strongly inhibited by fucoidan, a polymer of sulphated fucose, whereas fucose, mannan, and mannose 6-phosphate had no effect. Both B- and T-lymphoid cell lines were able to recognise and adhere to fibroblasts, although the marked differences between the attachment of the different types of cell did not appear to be related to their immunological function. The attachment of most of the cell lines was prevented by the presence of fucoidan, whereas the inhibition of binding of each of the lymphoid lines in the presence of the anti-T-lymphocyte plasma membrane antibody varied widely. These findings suggest that lymphocyte attachment to fibroblasts involves multiple cell surface receptors, and that these are expressed at different levels on specific T- and B-cells.

Animals↗
PubMed1988

Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts.

The activity of a lysosomal enzyme, alpha-D-mannosidase (EC 3.2.1.24), increased markedly in normal lymphocytes when they were cultured together with fibroblasts from a patient with an inherited deficiency of this enzyme. Cell-to-cell contact was obligatory for this increase in activity, which also required new protein synthesis. The enzyme induced in the co-cultured lymphocytes was a high molecular weight form of alpha-D-mannosidase that was not detected in lymphocytes cultured alone, which had only the low molecular weight mature enzyme. It was this precursor form alone that was directly transferred to the mannosidosis fibroblasts, where it was present initially in organelles of low density. When the culture period was extended the lymphocyte precursor enzyme was transported to the heavy lysosomes in the recipient cells, and correctly processed to the functionally effective mature enzyme.

Animals↗
PubMed1988

Ultrastructural localization of a lysosomal enzyme in resin-embedded lymphocytes.

The intracellular distribution of lysosomal enzymes in lymphocytes has previously been only poorly defined, mainly by cytochemical procedures of low resolution. In the present study we have used a post-embedding immunogold technique to identify the precise ultrastructural localization of a lysosomal enzyme, beta-glucuronidase, in activated lymphocytes embedded in Lowicryl K4M resin. We show that this enzyme is present in the rough endoplasmic reticulum, in the Golgi complex, and in vesicular organelles which probably include lysosomes.

Animals↗
PubMed1988

Forms and intracellular distribution of alpha-D-mannosidases in murine liver and spleen.

1. The intracellular distribution of alpha-D-mannosidase in homogenates of murine liver and spleen was investigated by differential and gradient density centrifugation. 2. In both tissues an enzyme with a neutral pH optimum was found in the cytosol together with an alpha-D-mannosidase with optimal activity between pH 5.5 and 6.0 which was also partially membrane-bound. 3. In liver the acidic alpha-D-mannosidase was obtained almost entirely in a particulate form distributed equally between a heterogeneous low density region and heavy density lysosomes. 4. The lysosomal form of the liver enzyme was purified to electrophoretic homogeneity and shown to be a glycoprotein composed of four identical subunits of molecular weight 65 kDa. 5. Antibody raised against the purified liver alpha-D-mannosidase immunoprecipitated a polypeptide from spleen which had the same molecular size. This acidic enzyme was the predominant type of alpha-D-mannosidase in spleen, but in contrast to liver, it was obtained mainly in a cytosoluble form, the remaining activity being present in the heterogeneous light density compartment. 6. Although both tissues contain the same molecular form of the acidic alpha-D-mannosidase, in murine spleen this enzyme does not appear to be associated with stable heavy density lysosomes.

Animals↗
PubMed1988

Ultrastructural studies of a lysosomal enzyme during lymphocyte activation.

A post-embedding immunogold technique has been used for the ultrastructural localization of a lysosomal enzyme, beta-glucuronidase, in resting and activated T- and B-lymphocytes. The results presented here show that mitogen-induced stimulation of T- and B-cells was associated with an increase in the amount of enzyme in the Golgi complex and rough endoplasmic reticulum, organelles which were rarely present in the resting lymphocytes.

Animals↗
PubMed1986

Effects of mitogenic stimulation on lymphocyte alpha-D-mannosidases.

Three types of alpha-D-mannosidase are present in human and murine lymphocytes. Their levels increased substantially when the cells were activated by T-cell mitogens, concanavalin A (Con A) and phytohaemagglutinin (PHA), and in the murine cells also by lipopolysaccharide (LPS), a B-cell mitogen. The intracellular localization of the alpha-D-mannosidases in the non-stimulated and activated murine cells was investigated by fractionation of lymphocyte lysates on colloidal silica (Percoll) and discontinuous sucrose gradients. In both types of cell, an enzyme having optimal activity at neutral pH was obtained in the cytosolic fraction and another alpha-D-mannosidase most active at an intermediate pH was obtained partly in membrane-bound form. In contrast, an acidic alpha-D-mannosidase, which was particularly elevated in the activated murine spleen cells, had a distribution in these lymphoblasts which was markedly different from that in non-stimulated lymphocytes. In the latter, the major proportion of the activity was obtained in a cytosolic fraction and the remainder in a particulate fraction of light density, whereas the enzyme in activated lymphocytes was distributed between vesicles of light and heavy density comparable with lysosomal organelles. Moreover, the acidic alpha-D-mannosidase still remained membrane bound even when cell lysates were prepared under hypotonic conditions which disrupt lysosome integrity. These results suggest that lymphocyte activation involves either stabilization of fragile lysosomes present in resting cells or de novo synthesis of lysosome-like structures. The acidic alpha-D-mannosidase present within isolated, intact lysosomes was found to be in a form, A, whereas a different form, B, was most prominent in whole-cell extracts of both types of lymphocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Compartmentation↗