Alteration of CD44 expression in HIV type 1-infected T cell lines.

CD44 is known to interfere in HIV replication and to participate in many physiological processes such as lymphocyte binding to high endothelial venules of lymphoid tissue, lymph nodes, and mucosal endothelium. The T cell lines MOLT-4 and CEM, and CEM subclones were infected with the HIV-1 LAI strain and monitored for the expression of CD44 during the course of chronic virus production until the infected cells were at the stage of latent infection. The levels of CD44 protein expression were quantified using cell surface immunostaining and biotinylation. The maturation of CD44 molecules was evaluated by metabolic sulforadiolabeling and CD44 mRNA was visualized by Northern blot analysis. We show a downmodulation of CD44 expression in infected T cell lines and subclones. This phenomenon was most evident at the stage of latent infection. Then, CD44 molecules were undetectable at both the protein and mRNA levels in latently infected CEM cells and CEM subclones. In addition, the 97-kDa standard CD44 isoform showed a shift upward, while detectable during the stage of chronic virus production. In latently infected MOLT-4 cells, the CD44 protein levels were dramatically decreased; CD44 mRNA was detected, but the sizes differed from the mRNA in uninfected cells. Since CD44 is known to regulate in part lymphocyte homing and HIV replication, the alterations that were observed in the expression of this molecule could interfere with the particular homing of HIV-infected cells and/or viral latency.

Autoantibodies directed against CD43 molecules with an altered glycosylation status on human immunodeficiency virus type 1 (HIV-1)-infected CEM cells are found in all HIV-1+ individuals.

Autoantibodies to lymphocytes have been detected in sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals, and several autoantigens have been described. Among them, hyposialylated CD43 has been shown to be a target for autoantibodies in up to 47% of HIV+ individuals. However, the corresponding autoantigen (ie, the incompletely sialylated CD43) has not been isolated from blood cells of HIV-1-infected individuals. Recently, we have observed in vitro that HIV-1 productively or latently infected CEM cells (CEMLAI/NP) express CD43 molecules with modified glycosylation (mogly CD43). Using CEMLAI/NP cells, which do not express any structural viral antigen, we show now that all of the tested HIV+ sera from asymptomatic individuals, and up to 86% of those from subjects at the acquired immunodeficiency syndrome stage contain antibodies (mainly IgM and, to a lesser degree, IgG) that recognize the surface of CEMLAI/NP cells, and precipitate mogly CD43 molecules from the cells lysates. Taken together with our previous demonstration of altered glycosylation of CD43 from HIV-1-infected CEM cells in vitro, the constant antimogly CD43 autoimmune response observed from asymptomatic HIV-1+ subjects is likely to illustrate the occurrence of an altered glycosylation in vivo of the major lymphocyte surface CD43 glycoprotein, associated with HIV-1 infection.

Lymphocytic CD43 and CD45 bear sulfate residues potentially implicated in cell to cell interactions.

CD43 is a major heavily glycosylated lymphocyte surface molecule. It has been shown to play an important role in lymphocyte activation and cell-cell interactions. Here we demonstrate that in human activated lymphocytes and CEM T cells, CD43 is a sulfated molecule. We also observed that CD45, another lymphocyte surface glycoprotein, is a sulfated molecule. 35SO4(2-) incorporation would thus appear to be an appropriate labeling method for CD43 and CD45 visualization. Moreover, we show that the level of cell surface protein sulfation can modulate CD43-mediated homotypic aggregation induced by CD43 monoclonal antibodies. It is well known that glycoprotein sulfation is required for various recognition phenomena. Since there are numerous potential sulfation sites on CD43 and CD45, these residues could play an important role in regulating cell-cell interactions.

Altered glycosylation of leukosialin, CD43, in HIV-1-infected cells of the CEM line.

CD43 (leukosialin, gpL115, sialophorin) is a major sialoglycoprotein widely expressed on hematopoietic cells that is defective in the congenital immunodeficiency Wiskott-Aldrich syndrome. It is thought to play an important role in cell-cell interactions and to be a costimulatory molecule for T lymphocyte activation. Using a metabolic 35SO4(2-) radiolabeling assay or biotinylation of cell surface proteins, we describe here that CD43 are sulfated molecules the glycosylation of which is altered in human immunodeficiency virus type 1 (HIV-1)-infected leukemic T cells of the CEM line. Hyposialylation of O-glycans and changed substitution on N-acetylgalactosamine residues are observed. The glycosylation defect is associated with an impairment of CD43-mediated homotypic aggregation which can be restored by resialylation. The hyposialylation of CD43 on HIV-1+ cells may explain the high prevalence of autoantibodies directed against nonsialylated CD43 that have been detected in HIV-1-infected individuals. A defect in glycosylation of important molecules such as CD43 or, as we recently described, CD45 may explain alterations of T cell functions and viability in HIV-1-infected individuals. In addition, a possible implication of hyposialylation in the HIV-1-infected cells entrapment in lymph nodes could be envisioned.

CD3-stimulated Jurkat T cells mediate IL-1 beta production in monocytic THP-1 cells. Role of LFA-1 molecule and participation of CD69 T cell antigen.

In this study we investigated the T cell signals required for monocyte activation. We used an in vitro co-culture system involving two human cell lines: Jurkat T cells and THP-1 monocytes. Monocyte activation was monitored by measuring IL-1 beta production, whereas IL-2 secretion reflected Jurkat activation. We showed that CD-3 -stimulated Jurkat cells delivered an IL-1-inductive signal to THP-1 cells through a cellular contact which was independent of THP-1 Fc receptors cross-linking. Stimulation of IL-1 beta production did not appear to require lymphokine secretion by T cell since a lymphokine defective mutant of Jurkat cell was able to deliver the stimulatory signal. The LFA-1 molecule was clearly shown to participate in the cooperation process, but its role was likely to be restricted to mediating initial adhesive interaction rather than to transducing the IL-1 -inductive signal. Interestingly, the co-culture stimulated by (Fab')2 fragments of CD3 mAb displayed an enhanced IL-1 beta production without any increase of IL-2 secretion. This result indicated that Jurkat cells could stimulate THP-1 cells even when they were only partially activated. The kinetics and conditions of IL-1 beta production called our attention to the early T cell activation antigen CD69. We then showed that CD69 mAb interfered with transmission of the IL-1 inductive signal (40-50% inhibition of IL-1 production). Our results are suggestive of a new role for CD69 molecule intervening in the T lymphocyte-dependent monocyte activation process.

Inhibition of phorbol ester-induced cell activation in microgravity.

T lymphocytes and monocytes were exposed to microgravity and activated to produce interleukin 2 and interleukin 1, respectively. When Jurkat T cells were triggered with monoclonal antibodies directed against the CD3/T cell receptor complex in the presence of THP-1 monocytes used as accessory cells, cell-to-cell contacts took place in microgravity leading to normal production of interleukin 2 and interleukin 1, as compared to ground controls. In contrast, when cells were individually stimulated by soluble substances including a protein kinase C activating phorbol ester, the production of interleukin 1 and interleukin 2 was dramatically inhibited during microgravity exposure. This result indicates that microgravity may affect the cellular target of phorbol ester.

Isolation and characterization of a T lymphocyte mutant defective in the protein kinase C signal transduction pathway.

The phorbol ester TPA is a potent protein kinase C (PKC) activator and a cofactor in the activation of the human Jurkat leukemic T cell line. We have studied the implication of the PKC signaling pathway in the process of T cell activation by generating TPA resistant mutants of Jurkat. These mutants were obtained by recovery of cells that survived a growth arrest induced by TPA. Several cellular phenomena dependent on TPA were dramatically altered in the mutated cells. The mutants were unable to form homoaggregates upon TPA stimulation. Moreover, they did not produce interleukin-2 after activation through engagement of the T cell receptor, in the presence of TPA. These results suggest that the PKC signaling pathway activated by TPA is defective in these cells. In an attempt to define and locate the defect present in the mutants, we have analysed the biochemical properties of PKC, the cellular receptor of TPA. The increase in kinase activity and the translocation of the enzyme to the plasma membrane after stimulation by TPA appeared to be normal in the mutants. We hypothesize that a metabolic step, critical for the completion of T cell activation, distinct from protein kinase C, is impaired in the mutant cells.

Stimulation of human interleukin 1 production and specific mRNA expression by microtubule-disrupting drugs.

The production of interleukin 1 (IL1), a pleiotropic monocyte-derived interleukin, can be induced in vitro by various stimuli. The present study shows that cytochalasins which inhibit actin filament polymerization in various cell types have no significant effect on IL1 production from human monocytic cells. On the contrary, microtubule disrupters such as colchicine, vinblastine, and vincristine dramatically potentiate (15- to 35-fold), in a dose-dependent fashion, cell-associated IL1 and to a lesser extent (2.5- to 7-fold) released IL1 in the myelomonocytic THP1 cell line and in adherent peripheral blood mononuclear cells. The enhancing effect of the drugs was blocked by actinomycin D and by cycloheximide and was accompanied by an increase of specific IL1 beta mRNA expression as measured by Northern blot analysis, thus indicating that these drugs act at a transcriptional or post-transcriptional IL1 gene expression level.