Membrane cholesterol modulates the hyaluronan-binding ability of CD44 in T lymphocytes and controls rolling under shear flow

Abstract

SummaryThe adhesion of circulating lymphocytes to the surface of vascular endothelial cells is important for their recruitment from blood to secondary lymphoid organs and to inflammatory sites. CD44 is a key adhesion molecule for this interaction and its ligand-binding ability is tightly regulated. Here we show that the hyaluronan-binding ability of CD44 in T cells is upregulated by the depletion of membrane cholesterol with methyl-b-cyclodextrin (MbCD), which disintegrates lipid rafts, i.e. cholesterol-and sphingolipid-enriched membrane microdomains. Increasing concentrations of MbCD led to a dose-dependent decrease in cellular cholesterol content and to upregulation of hyaluronan binding. Additionally, a cholesterol-binding agent filipin also increased hyaluronan binding. Cholesterol depletion caused CD44 to be dispersed from cholesterol-enriched membrane microdomains. Cholesterol depletion also increased the number of cells undergoing rolling adhesion under physiological flow conditions. Our results suggest that the ligand-binding ability of CD44 is governed by its cholesterol-dependent allocation to membrane microdomains at the cell surface. These findings provide novel insight into the regulation of T cell adhesion under blood flow.

Recombinant Human Hyaluronidase PH20 Does Not Stimulate an Acute Inflammatory Response and Inhibits Lipopolysaccharide-Induced Neutrophil Recruitment in the Air Pouch Model of Inflammation

“…The interaction between HA on the luminal endothelium and the HA receptor CD44 (present on neutrophils) mediates neutrophilendothelial cell interactions, causes neutrophil adhesion and migration, and contributes to optimal leukocyte recruitment into tissues in response to proinflammatory stimulation (32,33). CD44 on lymphocytes also binds HA on endothelium to mediate the primary adhesion/rolling of lymphocytes on vascular endothelial cells under conditions of physiologic shear stress, and this interaction is important for activated T cell extravasation into an inflamed site in vivo (34)(35)(36). It is therefore hypothesized that Hyal, by degrading and removing HA from tissue, can interfere with the HA-CD44 interaction and consequently reduce neutrophil recruitment into tissues during inflammation, provided that the Hyal used is free of proinflammatory contaminants.…”

Impact of lipid rafts on theT-cell-receptor and peptide-major-histocompatibility-complex interactions under different measurement conditions

“…This, however, may involve other potential underlying mechanisms accounting for the discrepancy. Alternatively, as speculated by Murai et al [28], disruption of lipid rafts may trigger the conformational change that alters the hyaluronan-binding ability of CD44, which needs to be further studied.…”

“…12(d)], because of the increased encounter and rebinding probabilities which weaken the negative impact of protein aggregation. Using this kind of cell-substrate model, Murai et al [28] experimentally examined the case that T cells were perfused across hyaluronan-coated microtiter plates and conversely suggested that the presence of lipid rafts in the T-cell membrane suppresses cell adhesion and migration. Combined with the modeling results, we provide an explanation for this discrepancy regarding the role of lipid rafts in the receptor-ligand interactions.…”

“…Combined with the modeling results, we provide an explanation for this discrepancy regarding the role of lipid rafts in the receptor-ligand interactions. One should notice that, to investigate the effect of lipid rafts on the receptor-ligand interactions, Anderson et al [27] and Murai et al [28] used two different systems in which the interacting partners are TCR and pMHC, and CD44 and hyaluronan, respectively. This, however, may involve other potential underlying mechanisms accounting for the discrepancy.…”

“…They showed that disruption of lipid rafts on APCs by the treatment of cells with raft-disrupting-agent, methyl-β-cyclodextrin (MβCD), inhibits the MHC class II-restricted antigen presentation [27]. They presumed that the increased local concentration of MHC class II molecules in lipid rafts is crucial for the initiation of immune responses, which provides important insights, whereas experimental data of cell-mimetic systems obtained by Murai et al conversely suggest that lipid rafts exert negative control on the receptor-ligand interactions [28], and the corresponding regulatory mechanism is not yet resolved. Taken together, the fundamental mechanism of how lipid rafts might drive TCR-pMHC interactions and the reason for the different experimental results remain unclear and quantitative analysis is lacking.…”