**4.2. The influence of cholesterol on the plasma membrane dynamics**

In the lipid bilayer, cholesterol orients with its polar hydroxyl group encountering the aqueous phase and the hydrophobic steroid ring parallel to and buried in the hydrocarbon chains of the phospholipids[50]. This unique orientation allows cholesterol to interact with membrane phospholipids and sphingolipids and thus influence their physicochemistry. Along these lines, cholesterol influences the physical and biological properties of the lipid bilayer and, in doing so, impacts the functions of membrane signaling molecules.

#### *4.2.1. Effects of cholesterol on the physical properties of cellular membranes*

A key function of cholesterol is to modulate the fluidity (i.e., the inverse of microviscosity) of the lipid bilayer. The close inter-positioning of sterols (i.e., cholesterol) between neighboring membrane phospholipids imposes a degree of immobility on the carbon atoms nearest the membrane surface, while increasing the freedom of motion deep within the hydrophobic core of the membrane[51]. In this regard, membrane fluidity reflects the temperature-dependent influence of cholesterol on the gel to liquid-crystal (i.e., solid-like to fluid-like) phase transition of the lipid bilayer[51]. Under physiological conditions (i.e., 37oC), biological membranes adopt a liquid crystalline state whereby increases and decreases in cholesterol content reduce and enhance membrane fluidity, respectively[42, 52, 53].

Operationally, membrane fluidity refers to the ensemble of physical properties that govern the motion of the phospholipid molecules in a membrane, including segmental, rotational, lateral, and translational motions[54]. In this fashion, lipid bilayer fluidity can physically influence the dynamics of membrane-associated molecules including proteins that drive downstream cell functions. Two mechanisms have been proposed to explain how membrane fluidity alters the activities and functions of membrane proteins. One mechanism occurs through effects on protein mobility, particularly lateral diffusion which impacts collisional encounters[54]. This effect appears to be of physiological significance particularly for diffusion-controlled processes that are mediated by large membrane proteins. For example, modulation of membrane fluidity influences Ca++-dependent cAMP signaling through changes in protein mobility that governs the coupling between hormone receptors and the adenylate cyclase catalytic unit[55, 56]. Alternatively, membrane fluidity can also influence the structural flexibility of membrane proteins and, thus, their ability to adopt an optimal conformation for activity[57]. Membrane fluidity has, in fact, been reported to impact the conformation-dependent activation of G-protein coupled receptors (GPCRs) on endothelial cells by affecting changes in the protein tertiary structure[58].

#### *4.2.2. Role of cholesterol in lipid raft structure and function*

230 Lipoproteins – Role in Health and Diseases

52, 53].

extracellular carriers (e.g., lipoproteins). The direction of net flux of cholesterol is governed by its concentration gradient between the lipid bilayer and the carrier molecules. Two mechanisms for this surface transfer have been proposed: 1) formation of transient collision complexes with/without membrane fusion and 2) direct diffusion across the aqueous phase. In principle, these transport modes follow similar kinetics with transfer rates depending on the concentrations and structures of both donor (e.g., extracellular LDL) and acceptor (e.g., cells) particles (for more details, see review[48]). This level of complexity contributes, in part, to the diverse half-times ranging from seconds to hours measured for the uptake of cholesterol by human erythrocytes[49]. Finally, this pathway is not under feedback control.

In the lipid bilayer, cholesterol orients with its polar hydroxyl group encountering the aqueous phase and the hydrophobic steroid ring parallel to and buried in the hydrocarbon chains of the phospholipids[50]. This unique orientation allows cholesterol to interact with membrane phospholipids and sphingolipids and thus influence their physicochemistry. Along these lines, cholesterol influences the physical and biological properties of the lipid

A key function of cholesterol is to modulate the fluidity (i.e., the inverse of microviscosity) of the lipid bilayer. The close inter-positioning of sterols (i.e., cholesterol) between neighboring membrane phospholipids imposes a degree of immobility on the carbon atoms nearest the membrane surface, while increasing the freedom of motion deep within the hydrophobic core of the membrane[51]. In this regard, membrane fluidity reflects the temperature-dependent influence of cholesterol on the gel to liquid-crystal (i.e., solid-like to fluid-like) phase transition of the lipid bilayer[51]. Under physiological conditions (i.e., 37oC), biological membranes adopt a liquid crystalline state whereby increases and decreases in cholesterol content reduce and enhance membrane fluidity, respectively[42,

Operationally, membrane fluidity refers to the ensemble of physical properties that govern the motion of the phospholipid molecules in a membrane, including segmental, rotational, lateral, and translational motions[54]. In this fashion, lipid bilayer fluidity can physically influence the dynamics of membrane-associated molecules including proteins that drive downstream cell functions. Two mechanisms have been proposed to explain how membrane fluidity alters the activities and functions of membrane proteins. One mechanism occurs through effects on protein mobility, particularly lateral diffusion which impacts collisional encounters[54]. This effect appears to be of physiological significance particularly for diffusion-controlled processes that are mediated by large membrane proteins. For example, modulation of membrane fluidity influences Ca++-dependent cAMP signaling through changes in protein mobility that governs the coupling between hormone receptors and the adenylate cyclase catalytic unit[55, 56]. Alternatively, membrane fluidity can also

**4.2. The influence of cholesterol on the plasma membrane dynamics** 

bilayer and, in doing so, impacts the functions of membrane signaling molecules.

*4.2.1. Effects of cholesterol on the physical properties of cellular membranes* 

Lipid rafts are nano-scale microdomains that are abundant in the plasma membrane. Structurally, lipid rafts consist of dynamic assemblies of cholesterol and sphingolipids in the outer leaflet of the phospholipid bilayer[59]. The preponderance of saturated hydrocarbon chains in raft sphingolipids renders lipid rafts with a distinct liquid-ordered (i.e., solid-like) phase that is dispersed in the liquid-disordered matrix of the lipid bilayer[60]. One important property of lipid rafts is that they include or exclude proteins to variable extents depending on the raft affinity of proteins[59]. Once individual rafts cluster, they spatially facilitate interactions between raft proteins and expose them to a new membrane environment that is enriched in accessory enzymes and/or second-messenger molecules. In doing so, lipid rafts serve to efficiently initiate and/or amplify signaling cascades. As such, lipid rafts act as signaling platforms that orchestrate outside-in and inside-out signal transduction. Interestingly, these cholesterol-rich microdomains have also been implicated as mechanotransduction centers such as caveolae, a subtype of lipid rafts that reportedly play a role in endothelial mechanotransduction of shear stress and pressure[61-63].

Cholesterol is required to support the formation of lipid rafts and maintain their functionality. It condenses the packing of sphingolipids in the exoplasmic leaflet by occupying the spaces between their saturated hydrocarbon chains near the hydrophilic polar head groups. In this way, cholesterol content and organization influence the stability of lipid rafts with an impact on their capacity to interact with target proteins. Removal or depletion of cholesterol from the plasma membrane using MCD has been widely used to disrupt rafts and disperse raft proteins into the liquid-disordered matrix of the cell membrane[59]. Treatment of cells with cholesterol-sequestering agents (e.g., filipin or nystatin) or inhibition of cholesterol biosynthesis (e.g., lovastatin) as well as addition of exogenous cholesterol into cell membranes also disrupts raft structure leading to an impact on the functions of raft proteins[59]. As a consequence of these lipid raft-related perturbations, neutrophil functions (e.g., chemokine-induced calcium signaling, extracellular regulated kinase activity, cell polarization, shape change, adhesion, migration, integrin expression, and actin polymerization) are altered[64-68].

#### **4.3. Effects of elevated cholesterol environments on neutrophil activity**

Up to this point, we have described how perturbations in extracellular cholesterol levels modify the membrane physicochemistry and the mode by which these modifications may influence membrane protein-related signaling in the neutrophil. The altered cell signaling capacity of membrane-bound proteins is followed by changes in cell behavior that contribute to the principal role of the neutrophil as the first responder to tissue damage and infection. In this way, the influence of membrane cholesterol on the ability of the neutrophil to sense its environment extends to basic cell functions including cell adhesion and migration, phagocytosis, ROS production, and degranulation. We will discuss the effects of cholesterol on these cell functions in order to illustrate the link between the lipid bilayer properties and the control of neutrophil activation.

### *4.3.1. Expression of membrane adhesion molecules*

Upon agonist stimulation, neutrophils exhibit upregulated expression of adhesion molecules that facilitate their recruitment to sites of inflammation by enabling their binding to other cells (e.g., leukocytes, platelets, endothelium)[33]. Two classes of adhesion molecules govern leukocyte interactions with other cells: selectins and integrins. In addition to the ligands for platelet (P)- or endothelial (E)- selectins, neutrophils constitutively express leukocyte (L)-selectins and 2 (i.e., CD18) integrins, which participate in their initial capture and firm adhesion to other cells, respectively[33]. Currently, the impact of hypercholesterolemia on expression of the selectin family of adhesion molecules is unclear since neutrophils in a cholesterol-rich environment have been reported to exhibit both elevated surface expression[69] and cleavage of L-selectins[70]. In the case of the integrins, surface levels of CD18, particularly Mac-1 (CD11b/CD18), are elevated on neutrophils exposed to a hypercholesterolemic environment both in vitro and in vivo[29, 69, 70]. Notably, surface expression of Mac-1 by neutrophils in hypercholesterolemic patients positively correlates with serum cholesterol levels[29]. But, cholesterol enrichment does not appear to alter the expression of LFA-1 (CD11a/CD18)[69], another CD18 subtype. Thus, the influence of extracellular cholesterol levels on neutrophil adhesion molecule expression is receptor-specific.

Moreover, neutrophils exposed to elevated cholesterol levels undergo increased adhesive interactions with other cells. For example, neutrophils with increased membrane cholesterol exhibit enhanced tethering and firm arrest on activated endothelial cell monolayers[41, 71, 72]. Moreover, neutrophils exposed to hypercholesterolemia display increased heterotypic adhesion to platelets[73] as well as increased homotypic aggregation in response to 10 M N-formyl-Met-Leu-Phe (fMLP)[74]. These studies confirm that cholesterol-dependent modulation of adhesion molecule expression has an impact on neutrophil adhesion to other leukocytes, platelets, or the endothelium lining the blood vessel lumen.

#### *4.3.2. ROS production*

Neutrophil-derived ROS includes superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radicals (- OH), and NO-related oxidants. Notably, total production of ROS by neutrophils in a hyperlipidemic environment positively correlates with levels of triglycerides and LDL but not with total amount of cholesterol in the plasma[75]. Recent studies, however, did demonstrate a positive correlation between O2- release rate and plasma cholesterol levels[29]. Interestingly, enhanced O2- release by neutrophils was detected in other clinical states associated with cardiovascular complications, namely hypertension and diabetes[76, 77], which are usually accompanied by hyperlipidemia. In fact, elevations in extracellular cholesterol levels have been shown to enhance neutrophil respiratory burst in response to agonist stimulation. Moreover, plasma activity of superoxide dismutase (SOD), which scavenges ROS, decreases with increases in total cholesterol[75].

#### *4.3.3. Degranulation*

232 Lipoproteins – Role in Health and Diseases

receptor-specific.

*4.3.2. ROS production* 

radicals (-

properties and the control of neutrophil activation.

*4.3.1. Expression of membrane adhesion molecules* 

infection. In this way, the influence of membrane cholesterol on the ability of the neutrophil to sense its environment extends to basic cell functions including cell adhesion and migration, phagocytosis, ROS production, and degranulation. We will discuss the effects of cholesterol on these cell functions in order to illustrate the link between the lipid bilayer

Upon agonist stimulation, neutrophils exhibit upregulated expression of adhesion molecules that facilitate their recruitment to sites of inflammation by enabling their binding to other cells (e.g., leukocytes, platelets, endothelium)[33]. Two classes of adhesion molecules govern leukocyte interactions with other cells: selectins and integrins. In addition to the ligands for platelet (P)- or endothelial (E)- selectins, neutrophils constitutively express leukocyte (L)-selectins and 2 (i.e., CD18) integrins, which participate in their initial capture and firm adhesion to other cells, respectively[33]. Currently, the impact of hypercholesterolemia on expression of the selectin family of adhesion molecules is unclear since neutrophils in a cholesterol-rich environment have been reported to exhibit both elevated surface expression[69] and cleavage of L-selectins[70]. In the case of the integrins, surface levels of CD18, particularly Mac-1 (CD11b/CD18), are elevated on neutrophils exposed to a hypercholesterolemic environment both in vitro and in vivo[29, 69, 70]. Notably, surface expression of Mac-1 by neutrophils in hypercholesterolemic patients positively correlates with serum cholesterol levels[29]. But, cholesterol enrichment does not appear to alter the expression of LFA-1 (CD11a/CD18)[69], another CD18 subtype. Thus, the influence of extracellular cholesterol levels on neutrophil adhesion molecule expression is

Moreover, neutrophils exposed to elevated cholesterol levels undergo increased adhesive interactions with other cells. For example, neutrophils with increased membrane cholesterol exhibit enhanced tethering and firm arrest on activated endothelial cell monolayers[41, 71, 72]. Moreover, neutrophils exposed to hypercholesterolemia display increased heterotypic adhesion to platelets[73] as well as increased homotypic aggregation in response to 10 M N-formyl-Met-Leu-Phe (fMLP)[74]. These studies confirm that cholesterol-dependent modulation of adhesion molecule expression has an impact on neutrophil adhesion to other

Neutrophil-derived ROS includes superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl

a hyperlipidemic environment positively correlates with levels of triglycerides and LDL but not with total amount of cholesterol in the plasma[75]. Recent studies, however, did demonstrate a positive correlation between O2- release rate and plasma cholesterol levels[29]. Interestingly, enhanced O2- release by neutrophils was detected in other clinical states associated with cardiovascular complications, namely hypertension and diabetes[76,

OH), and NO-related oxidants. Notably, total production of ROS by neutrophils in

leukocytes, platelets, or the endothelium lining the blood vessel lumen.

Neutrophils contain four main types of granules: primary, secondary, and tertiary granules as well as secretory vesicles. These granules contain a multitude of cytokines (e.g., interleukins, tumor necrosis factor-, etc.), enzymes (e.g., MPO, etc.), and proteases (e.g., cathepsins, MMPs, etc.). Upon activation, neutrophils degranulate and release these bioactive mediators into the extracellular milieu. Interestingly, although neutrophils from hyperlipidemic patients contain significantly lower levels of intracellular MPO, sera from these patients exhibit significantly higher levels of MPO[29]. These results point to a degranulation process that further links the activation state of neutrophils with the cholesterol levels in the blood environment.
