Dynamic reorganization of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to sites of CD4 engagement
Introduction
Chemokine receptors belong to the family of seven transmembrane G protein-coupled receptors that are essential for immune cell migration and homing in response to soluble mediators called “chemokines.” We have previously demonstrated that chemokine receptor conformation, more specifically CCR5 and CXCR4, is significantly altered upon cholesterol extraction with β-cyclodextrin (BCD) [1], [2]. Interestingly, these studies also revealed that SDF-1α (CXCL12) and MIP-1β (CCL4) preferentially bind to chemokine receptors associated with cholesterol- and sphingolipid-enriched membrane microdomains termed “lipid rafts,” despite the observation that the majority of cell surface chemokine receptors did not co-localize with lipid rafts on these cells. There is a current debate on chemokine receptor–lipid raft localization where several groups have demonstrated significant levels of chemokine receptors associated with rafts before and/or after stimulation, while others have failed to identify any association [3], [4], [5], [6]. This issue is partly confounded by the differences in raft isolation techniques and the varying cell types examined in such studies as well as possible differences in the levels of receptor expression, which may influence receptor–raft localization. Nonetheless, lipid rafts appear to play an important role in the function of many chemokine receptors and the significance of receptor–raft interplay remains to be fully understood.
Polarization and lipid raft recruitment in immune cells have been demonstrated in response to cross-linking of various receptors, including CD3 and CD28, as well as by cytokine and chemokine stimulation [3], [7]. For example, formation of the immunological synapse (IS) between an antigen presenting cell and a T cell causes significant redistribution of T cell lipids and proteins, such as ganglioside GM1, LFA-1, and TCR, to specific regions of the contact zone [8]. Other distinct immune synapses are also observed with NK cell interactions as well [8]. Signaling through these contact zones is designed to transmit the proper signals from one cell to the other, which can include signals for cell activation, inactivation, or cytolysis. The redistribution of lipid rafts to the IS on T cells is believed to be critical for the transmission of the appropriate signal, especially during T cell activation, by providing stable platforms for the accumulation of intracellular signaling molecules and by providing sites for cytoskeletal assembly [9]. Additionally, polarization of cellular components including chemokine receptors and lipid rafts has been demonstrated in immune cells responding to migratory signals such as chemokine gradients [3], [10], [11]. Furthermore, chemokine signaling is believed to enhance T cell responsiveness during activation [12], [13], [14], [15]. The redistribution of chemokine receptors to the IS in the context of CD4 signaling, which may influence T cell signaling outcomes, has not been determined.
Several groups have recently demonstrated that CD4, chemokine receptors, LFA-1, and cytoskeletal proteins on target cells are recruited to sites of contact with an infected dendritic cell or T cell, dubbing this union the “HIV synapse,” supporting the models for cell-to-cell transmission of HIV and other retroviruses [16], [17], [18], [19], [20], [21], [22]. This process bears amazing resemblance to the formation of the IS where T cell interactions with APCs results in the recruitment of lipid rafts, adhesion molecules, and the T cell receptor (TCR) complex to the point of cell-to-cell contact [23]. The similarity between these immune-associated synapses suggests a commonality between the processes mediating these raft recruitment. CD4 signaling and raft association in the IS has also been demonstrated to be required for TCR/PKCγ raft association and clustering [24]. Here, we sought to examine if solid-phase engagement of the CD4 molecule alone is sufficient to induce human T cells redistribution of chemokine receptors, lipid rafts, and adhesion molecules to the point of cell contact. Our studies revealed that engagement of CD4 alone on human T cells does induce chemokine receptor, cholesterol, GM1, adhesion molecules, and GPI-anchored proteins, but not CD45, recruitment to contact sites. This process requires lck signaling, F-actin polymerization, and the presence of bioactive cholesterol. Moreover, we also demonstrate that both lipid rafts and CXCR4 on target T cells become polarized to sites of contact with HIV-infected cells. The implications of these findings on T cell activation and HIV infectivity will be discussed.
Section snippets
Cells and reagents
Jurkat T cells (clone E6.1), Molt-4, Sup-T1, and CEM-NKR-CCR5 (referred to as CEM-R5) cells were obtained through the AIDS Research Reagents and Reference Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health) from Dr. A. Weiss and Dr. A. Trkola. The JCaM 1.6 variant of E6.1 Jurkat cells was obtained from Dr. Ron Wange at the National Institute on Aging. Cells were grown in RPMI-1640 (Mediatech; Cellgro, Herndon, VA) supplemented with
CD4 engagement by antibody-bound beads induces cell clustering
Some adhesion molecules including LFA-1 require functional lipid rafts for optimal activity [26], [27]. Similarly, SDF-1-induced stimulation of VLA-4, but not LFA-1 avidity, under shear flow, requires lipid rafts [28]. Additionally, previous studies by Woods et al. [29] have demonstrated that CD4 engagement is capable of activating β1 integrin via lck-dependent signaling. To assess if CD4 engagement can induce the recruitment of adhesion molecules and cholesterol to sites of CD4 contact, we
Discussion
The data presented here supports the concept that CD4 co-stimulation may play a critical role in raft and co-stimulatory molecule recruitment during T cell activation or selection in the thymus [30]. During contact with HLA proteins on APCs, CD4 signaling itself may facilitate cellular reorganization, establishing, and/or maintaining the IS, via recruiting adhesion molecules and signaling molecules associated with lipid rafts. This could contribute to some of the differential lipid raft capping
Acknowledgments
We would like to thank Drs. Vishwa Deep-Dixit and Eric Schaffer for their helpful discussions. We also thank Dr. Ron Wange for his generous gift of JCaM 1.6 cells.
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