Mycobacterial lipoarabinomannans modulate cytokine production in human T helper cells by interfering with raft/microdomain signalling

Abstract.: Lipoarabinomannans (LAMs) are major lipoglycans of the mycobacterial envelope and constitute immunodominant epitopes of mycobacteria. In this paper, we show that mannose-capped (ManLAM) and non-mannose- capped (PILAM) mycobacterial lipoglycans insert into T helper cell rafts without apparent binding to known receptors. T helper cells modified by the insertion of PILAM responded to CD3 cross-linking by decreasing type 1 (IL-2 and IFN-γ) and increasing type 2 (IL-4 and IL-5) cytokine production. Modification by the mannose-capped ManLAMs had similar, but more limited effects on T helper cell cytokine production. When incorporated into isolated rafts, PILAMs modulated membrane-associated kinases in a dose-dependent manner, inducing increased phosphorylation of Src kinases and Cbp/PAG in Th1 rafts, while decreasing phosphorylation of the same proteins in Th2 rafts. Mycobacterial lipoglycans thus modify the signalling machineries of rafts/microdomains in T helper cells, a modification of the membrane organization that eventually leads to an overall enhancement of type 2 and inhibition of type 1 cytokine productio

High molecular weight surface glycoproteins of murine lymphocytes.

High m.w. glycoproteins in the range of 170 and 210K represent major cell surface components of murine lymphocytes. The expression of such glycoproteins has been characterized for thymocytes and peripheral T and B lymphocytes in BALB/c mice using surface and biosynthetic labeling, nonionic detergent extraction, and immunoprecipitation with antibodies directed against antigenic determinants expressed on the cell surface of a murine T cell lymphoma. Immunoprecipitates were then resolved by 1-dimensional and 2-dimensional gel electrophoresis. Two groups of antigenically distinct glycoproteins have been defined: 1) a 170K component present on thymocytes and peripheral T lymphocytes but undetectable on B lymphocytes; this glycoprotein has been termed T 170 to suggest its potential use for distinguishing T from B lymphocytes; 2) a higher m.w. glycoprotein bearing antigenic determinants common to T and B cells but expressed as one 180K glycoprotein on thymocytes, 2 glycoproteins of 180 and 190K on peripheral T lymphocytes, and as one 210K glycoprotein on B lymphocytes. The relationships of these glycoproteins with the other known lymphocyte surface markers of high m.w. is discussed

Isolation of plasma membrane domains from murine T lymphocytes.

Murine T-lymphoma cells have been homogenized in dense sucrose solution and centrifuged under isopycnic conditions for membrane components. Floating fractions banding between 10% and 22.5% sucrose ("light" membranes) and between 22.5% and 35% sucrose ("heavy" membranes) were shown to consist of smooth membrane vesicles. The amounts of cholesterol and phospholipids recovered after chloroform/methanol extraction were similar in both fractions, but heavy membranes contained two to three times more protein than light membranes. The most striking difference between the two membrane fractions was revealed by their labeled surface glycoprotein patterns on polyacrylamide gels, suggesting that (i) the smooth membrane vesicles originated from the plasma membrane and (ii) two distinct segments of the plasma membrane can be recovered in fractions characterized by specific surface glycoproteins. Light membranes were enriched in Thy-1 antigen, whereas Ly-5 antigen and a 170,000-dalton surface glycoprotein were recovered almost exclusively from heavy membranes, as were metabolically labeled protein spots comigrating with the H-2k/d antigen in two-dimensional electrophoresis. The patterns of the unlabeled proteins in light and heavy membranes appeared similar, except for polypeptides of 180,000 and 85,000 daltons that were found preferentially in heavy membranes. These results support the concept of plasma membrane domains by showing that two distinct populations of plasma membrane vesicles can be isolated and that these populations contain different sets of cell surface glycoproteins