**4. The influence of cholesterol on neutrophil activity**

One way hypercholesterolemia may influence the activation state of neutrophils is to modify the lipid composition of biological membranes. Cholesterol is an essential component of mammalian cell membranes. Approximately 90% of the free (i.e. unesterified) cholesterol in cells resides in the plasma membrane[38]. These sterol molecules not only maintain the integrity of cell membranes, but also play an important role in regulating membrane properties (e.g., microviscosity) and functions (e.g., via their influence on membrane-bound signaling components). 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[39-42]. 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. To better understand this link, we next describe the possible cholesterol uptake pathways, the influence of cholesterol on physicochemical properties of the cell membrane, and lastly, the influence of cholesterol on neutrophil activity.

#### **4.1. Transport of extracellular cholesterol into the plasma membrane**

Due to its insolubility in aqueous media, cholesterol must be transported complexed to carrier molecules, i.e. within the hydrophobic cores of lipoproteins[43]. Lipoproteins (e.g., LDL) in the blood plasma are positioned in close proximity to the circulating blood cells. Conditions that elevate cholesterol-enriched lipoprotein levels may thus favor cholesterol transport into the membranes of these blood cells[39, 42]. In the laboratory, cyclodextrin derivatives (e.g., methyl--cyclodextrin or MCD), synthetic cholesterol carrier molecules, are commonly used to alter membrane cholesterol abundance. Such treatments elicit acute changes in membrane cholesterol levels and downstream cell activity indicating the existence of mechanism(s) that permit rapid transport of cholesterol into nearby cell membranes. Cholesterol uptake may occur by either receptor dependent/independent endocytosis followed by rapid membrane mobilization[44] or direct exchange between the hydrophobic environments of carrier molecules and the lipid bilayer(Figure 2).

#### *4.1.1. Receptor-dependent endocytosis*

228 Lipoproteins – Role in Health and Diseases

perfusion.

perfusion.

state of neutrophil activation is, thus, a critical determinant of tissue blood flow and

In summary, as a result of their arsenal of noxious agents and their effects on microvascular blood flow, it is evident that tight regulation of neutrophil activity is an essential requirement for a healthy circulation. A failure to either prevent or "turn-off" cell activity, e.g., due to hypercholesterolemia, leads to sustained neutrophil activation which has potential impacts not only in terms of the initiation and progression of atherosclerosis in large arteries but also as it relates to microvascular blood flow and downstream tissue

One way hypercholesterolemia may influence the activation state of neutrophils is to modify the lipid composition of biological membranes. Cholesterol is an essential component of mammalian cell membranes. Approximately 90% of the free (i.e. unesterified) cholesterol in cells resides in the plasma membrane[38]. These sterol molecules not only maintain the integrity of cell membranes, but also play an important role in regulating membrane properties (e.g., microviscosity) and functions (e.g., via their influence on membrane-bound signaling components). 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[39-42]. 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. To better understand this link, we next describe the possible cholesterol uptake pathways, the influence of cholesterol on physicochemical properties of

the cell membrane, and lastly, the influence of cholesterol on neutrophil activity.

**4.1. Transport of extracellular cholesterol into the plasma membrane** 

hydrophobic environments of carrier molecules and the lipid bilayer(Figure 2).

Due to its insolubility in aqueous media, cholesterol must be transported complexed to carrier molecules, i.e. within the hydrophobic cores of lipoproteins[43]. Lipoproteins (e.g., LDL) in the blood plasma are positioned in close proximity to the circulating blood cells. Conditions that elevate cholesterol-enriched lipoprotein levels may thus favor cholesterol transport into the membranes of these blood cells[39, 42]. In the laboratory, cyclodextrin derivatives (e.g., methyl--cyclodextrin or MCD), synthetic cholesterol carrier molecules, are commonly used to alter membrane cholesterol abundance. Such treatments elicit acute changes in membrane cholesterol levels and downstream cell activity indicating the existence of mechanism(s) that permit rapid transport of cholesterol into nearby cell membranes. Cholesterol uptake may occur by either receptor dependent/independent endocytosis followed by rapid membrane mobilization[44] or direct exchange between the

**4. The influence of cholesterol on neutrophil activity** 

For a variety of cell types (including leukocytes)[45], LDL-cholesterol is taken up in vivo mainly through LDL receptor (LDLr)-mediated endocytosis. LDLr expression is subject to feedback regulation and, thus, is unlikely to contribute significantly to the overaccumulation of cellular cholesterol[43]. Cellular uptake of cholesterol can also occur via endocytosis mediated by other receptors[43]. Potentially, these pathways can load cholesterol continuously into the cell leading to cholesterol elevations in the plasma membrane[43]. For example, a family of scavenger receptors have been identified on monocytes, macrophages, and SMCs[6] that, by binding to modified LDL (e.g., oxidized and acetylated LDL) with high affinity, account for the majority of cholesterol uptake by these cells[6]. To our knowledge, such scavenger receptors have not yet been identified for neutrophils.

**Figure 2. Schematic representation of three possible modes of cholesterol uptake.** A: Receptormediated endocytosis; B: Direct surface exchange of cholesterol between extracellular carrier molecules and plasma membrane which may occur due to the formation of a transient collision complex without (➀) or with (➁) membrane fusion or resulting from diffusion across the aqueous phase (➂); C: Receptor-independent endocytosis.

#### *4.1.2. Receptor-independent endocytosis*

The entire LDL particle can be internalized as a result of fluid or bulk endocytosis without receptor-mediated LDL binding to the cell surface[46]. It is taken up at a rate strictly proportional to its concentration in the extracellular milieu[43]. Alternatively, some LDL, e.g., cationized LDL, can also be taken up by the cell through a non-specific low affinity adsorptive endocytotic process. In this case, endocytosis occurs after cationized LDL binds to the negatively charged membrane surface[47]. For both of these modes of endocytosis, cholesterol transport is not influenced by intracellular cholesterol levels and thus may lead to progressive cholesterol uptake[43].

#### *4.1.3. Cholesterol surface exchange*

Cholesterol may also directly enter or exit the plasma membrane[48]. In this case, free cholesterol is exchanged between the hydrophobic cores of the plasma membrane and 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.
