Membrane cholesterol modulates the fluid shear stress response of polymorphonuclear leukocytes via its effects on membrane fluidity

Abstract

Specifically, fluid flow-derived shear stresses deactivate leukocytes via actions on the conformational activities of proteins on the cell surface. Because membrane properties affect activities of membranebound proteins, we hypothesized that changes in the physical properties of cell membranes influence PMNL sensitivity to fluid shear stress. For this purpose, we modified PMNL membranes and showed that the cellular mechanosensitivity to shear was impaired whether we increased, reduced, or disrupted the organization of cholesterol within the lipid bilayer. Notably, PMNLs with enriched membrane cholesterol exhibited attenuated pseudopod retraction responses to shear that were recovered by select concentrations of benzyl alcohol (a membrane fluidizer). In fact, PMNL responses to shear positively correlated (R 2 ϭ 0.96; P Ͻ 0.0001) with cholesterol-related membrane fluidity. Moreover, in low-density lipoprotein receptor-deficient (LDLr Ϫ/Ϫ ) mice fed a high-fat diet (a hypercholesterolemia model), PMNL shear-responses correlated (R 2 ϭ 0.5; P Ͻ 0.01) with blood concentrations of unesterified (i.e., free) cholesterol. In this regard, the shear-responses of PMNLs gradually diminished and eventually reversed as free cholesterol levels in blood increased during 8 wk of the high-fat diet. Collectively, our results provided evidence that cholesterol is an important component of the PMNL mechanotransducing capacity and elevated membrane cholesterol impairs PMNL shearresponses at least partially through its impact on membrane fluidity. This cholesterol-linked perturbation may contribute to dysregulated PMNL activity (e.g., chronic inflammation) related to hypercholesterolemia and causal for cardiovascular pathologies (e.g., atherosclerosis).flow; cell deactivation; pseudopod retraction; mechanotransduction; hypercholesterolemia WHETHER ADHERED TO A SURFACE or freely suspended in plasma, polymorphonuclear leukocytes (PMNLs; particularly neutrophils) sense and respond to fluid shear stress (force per unit area acting tangential to the cell surface; dyn/cm 2 ) imposed by blood flow. Specifically, fluid shear stress maintains PMNLs in a deactivated state by reducing or preventing formation of pseudopod(s), a morphological hallmark of an activated PMNL, while at the same time, promoting cleavage of cell surface-associated ␤ 2 (CD18) integrins involved in cell adhesion and migration (30,48). The cell-inactivating action of shear stress is depressed upon treatment of cells with threshold concentrations of biochemical agonists, such as formyl peptide (N-formyl-Met-LeuPhe or FMLP; Ͼ 0.01 M) and platelet-activating factor (Ͼ0.1 M) (15), pointing to circulatory fluid flow as a negative mechano-regulator of PMNL activity in the absence of inflammatory agents, i.e., cell stimulation overrides this negative fluid shear stress control. Moreover, impaired leukocyte responses to shear stress, due to agonist stimulation or a pathologically inflamed state, have been linked to increased PMNL entrapment in the microcirculation of normotensive r...

Linking the Pathobiology of Hypercholesterolemia with the Neutrophil Mechanotransduction

“…Interestingly, neutrophils from LDLr-/-mice fed a HFD exhibit a reduced and even reversed shear stress response relative to cells from similar mice maintained on a regular chow (i.e., normal) diet (ND) [40]. These observations were consistent with our in vitro data correlating membrane cholesterol levels with neutrophil mechanosensitivity [40].…”

“…Thus, there is also an optimal membrane fluidity level permissive for shear-induced neutrophil deactivation. This was confirmed by regression analysis [40] which revealed a linear relationship ( Figure 5B) between membrane cholesterol-related fluidity and the degree to which neutrophils within a population are inactivated by fluid flow. Membrane cholesterol enrichment therefore impairs neutrophil mechanosensitivity, at least in part, through its impact on membrane fluidity.…”

“…Moreover, the membrane must be capable of supporting the formation of lipid rafts. But, the critical evidence from this work are our observations [40] that membrane fluidizer, benzyl alcohol, was capable of counteracting the rigidifying effects of membrane cholesterol enhancement (with…”

“…In addition to de novo biosynthesis, mammalian cells can take up cholesterol from the extracellular milieu. Exposure to elevated cholesterol levels both in vivo and in vitro enhances cholesterol abundance within the plasma membrane of neutrophils and other blood cells [39][40][41][42]. These findings, in conjunction with the wealth of evidence demonstrating the influence of the extracellular cholesterol levels on neutrophil activity, point to membrane cholesterol enrichment as a potential link between hypercholesterolemia and chronic neutrophil activity.…”

“…GPCR and CD18 conformational activity, protease release, and/or production of ROS, that must bidirectionally transmit biological activity across the cell membrane ( Figure 4). Recently, we reported that neutrophil deactivation by shear stress depends on the cholesterol-dependent physicochemical properties (i.e., fluidity) of the cell membrane [40]. Fundamentally, we showed that the deactivating actions of fluid shear require a cell membrane containing an optimal level of cholesterol.…”