Altered lipid raft–associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus
Elizabeth C. Jury, Panagiotis S. Kabouridis, Fabian Flores-Borja, Rizgar A. Mageed, David A. Isenberg
Elizabeth C. Jury, Panagiotis S. Kabouridis, Fabian Flores-Borja, Rizgar A. Mageed, David A. Isenberg
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Article Autoimmunity

Altered lipid raft–associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus

Abstract

Systemic lupus erythematosus (SLE) is characterized by abnormalities in T lymphocyte receptor–mediated signal transduction pathways. Our previous studies have established that lymphocyte-specific protein tyrosine kinase (LCK) is reduced in T lymphocytes from patients with SLE and that this reduction is associated with disease activity and parallels an increase in LCK ubiquitination independent of T cell activation. This study investigated the expression of molecules that regulate LCK homeostasis, such as CD45, C-terminal Src kinase (CSK), and c-Cbl, in lipid raft domains from SLE T cells and investigated the localization of these proteins during T cell receptor (TCR) triggering. Our results indicate that the expression of raft-associated ganglioside, GM1, is increased in T cells from SLE patients and LCK may be differentially regulated due to an alteration in the association of CD45 with lipid raft domains. CD45 tyrosine phosphatase, which regulates LCK activity, was differentially expressed and its localization into lipid rafts was increased in T cells from patients with SLE. Furthermore, T cells allowed to “rest” in vitro showed a reversal of the changes in LCK, CD45, and GM1 expression. The results also revealed that alterations in the level of GM1 expression and lipid raft occupancy cannot be induced by serum factors from patients with SLE but indicated that cell-cell contact, activating aberrant proximal signaling pathways, may be important in influencing abnormalities in T cell signaling and, therefore, function in patients with SLE.

Authors

Elizabeth C. Jury, Panagiotis S. Kabouridis, Fabian Flores-Borja, Rizgar A. Mageed, David A. Isenberg

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CD45 has an altered pattern of expression and membrane localization in T...
CD45 has an altered pattern of expression and membrane localization in T cells from patients with SLE. Lipid raft and non-raft fractions were prepared from T cells from patients with SLE and controls (Normal). Fractions were separated by 8% SDS-PAGE and analyzed by Western blotting. (A) A representative experiment showing T cell non-raft fractions from patients with SLE and healthy controls analyzed by Western blot for CD45, LCK, c-Cbl, and CSK. Actin was used as a control for equal protein loading. (B) Graph displaying cumulative semiquantitative results for CD45, CSK, c-Cbl, and LCK expression in T cell non-raft fractions from ten SLE patients and five healthy controls. The relative levels of LCK, CSK, CD45, and c-Cbl were estimated by comparison of the intensity of each band to that of the actin control. (C) Western blot representative of three experiments showing expression of CD45, active LCK (Src pY414), and inactive LCK (LCK pY505) in T cell lipid rafts. LAT expression is not altered in SLE T cells (26) and is used as a marker of equal protein loading. (D) LCK was immunoprecipitated from 50-μg fractions of whole–T cell lysates for patients with SLE and controls (Normal and RA). Immunoprecipitates (IP) were analyzed by Western blot and probed for CD45 and LCK.