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Free Cholesterol Alters Lipid Raft Structure and Function Regulating Neutrophil Ca²⁺ Entry and Respiratory Burst: Correlations with Calcium Channel Raft Trafficking¹

Kolenkode B. Kannan, Dimitrios Barlos and Carl J. Hauser^2,*,

^* Department of Surgery, Beth Israel-Deaconess Medical Center and Harvard Medical School, Boston, MA 02115; and Department of Surgery, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07101

	Abstract

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Recent studies associate cholesterol excess and atherosclerosis with inflammation. The link between these processes is not understood, but cholesterol is an important component of lipid rafts. Rafts are thought to concentrate membrane signaling molecules and thus regulate cell signaling through G protein-coupled pathways. We used methyl -cyclodextrin to deplete cholesterol from polymorphonuclear neutrophil (PMN) rafts and thus study the effects of raft disruption on G protein-coupled Ca²⁺ mobilization. Methyl -cyclodextrin had no effect on Ca²⁺ store depletion by the G protein-coupled agonists platelet-activating factor or fMLP, but abolished agonist-stimulated Ca²⁺ entry. Free cholesterol at very low concentrations regulated Ca²⁺ entry into PMN via nonspecific Ca²⁺ channels in a biphasic fashion. The specificity of cholesterol regulation for Ca²⁺ entry was confirmed using thapsigargin studies. Responses to cholesterol appear physiologic because they regulate respiratory burst in a proportional biphasic fashion. Investigating further, we found that free cholesterol accumulated in PMN lipid raft fractions, promoting formation and polarization of membrane rafts. Finally, the transient receptor potential calcium channel protein TRPC1 redistributed to raft fractions in response to cholesterol. The uniformly biphasic relationships between cholesterol availability, Ca²⁺ signaling and respiratory burst suggest that Ca²⁺ influx and PMN activation are regulated by the quantitative relationships between cholesterol and other environmental lipid raft components. The association between symptomatic cholesterol excess and inflammation may therefore in part reflect free cholesterol- dependent changes in lipid raft structure that regulate immune cell Ca²⁺ entry. Ca²⁺ entry-dependent responses in other cell types may also reflect cholesterol bioavailability and lipid incorporation into rafts.

	Introduction

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Current evidence suggests that the progression of cholesterol excess to atherosclerosis is linked to the presence of systemic inflammation (1, 2). Lipid rafts are cholesterol- and sphingolipid-enriched domains of cell membranes (3). Interactions of cholesterol with the phospholipids and sphingolipids present in rafts in specific amounts create highly "lipid-ordered" microdomains (4, 5, 6) that recruit signaling proteins and act as staging areas for molecular signaling complexes.

Regulation of cytosolic intracellular free calcium ion concentration ([Ca²⁺]_i)³ is a basic, ubiquitous cell signaling mechanism. In "nonexcitable" eukaryotic cells, agonist-initiated Ca²⁺ mobilization is a biphasic process in which brief bursts of Ca²⁺ release from endoplasmic reticulum (ER) stores are often linked to long-lasting, nonvoltage operated plasma membrane Ca²⁺ entry conductances. These conductances can be referred to as "store-operated", "capacitative," or "calcium release-activated" calcium entry (SOCE/CCE/I_CRAC) depending upon the channel characteristics noted in specific experimental systems. But the increases in [Ca²⁺]_i that activate native cells in vivo are often due to activation of multiple related conductances that contribute to "stimulated calcium entry" (7, 8, 9). Transient receptor potential channel (TRPC) proteins can form cation channels that are activated when G protein-coupled receptors (GPCR) linked to phospholipases release diacylglycerol and inositol 1,4,5 trisphosphate and deplete cell calcium stores (10, 11, 12). Thus TRPC-based channels can mediate agonist-induced Ca²⁺ entry but the mechanisms linking phospholipase activation and store depletion to Ca²⁺ entry through TRPC-based channels remain complex and controversial. Last, rafts are known to play a role in GPCR calcium signaling and disruption of rafts by cholesterol sequestration has been noted to block cell calcium entry (13, 14, 15).

Polymorphonuclear neutrophils (PMN) are primary effector cells of human innate immunity and many facets of PMN activation depend directly on Ca²⁺ entry into the cell (8, 16, 17, 18). PMN lack voltage-gated calcium channels (19) and we have shown that PMN mobilize external Ca²⁺ via mechanisms that mobilize multiple TRPC proteins to the cell membrane (8, 9). Cytoskeletal alterations that prevent TRPC membrane localization block entry of calcium into PMN (9) and TRPC are known to localize to rafts in PMN and other cell types (20, 21).

Because immune cell function depends heavily on Ca²⁺ entry and lipid raft disruption alters calcium signaling (15), we hypothesized that modifications of lipid raft structure or function by cholesterol played a role in PMN Ca²⁺ entry and subsequent activation, potentially linking inflammation mechanistically to the pathogenesis of cholesterol-dependent clinical disease states.

	Materials and Methods

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Research compliance

All studies were performed under the supervision of the Institutional Review Board of the University of Medicine and Dentistry of New Jersey, New Jersey Medical School. Consent was obtained for PMN sampling by phlebotomy.

Materials

Cholesterol assay kits and fura 2-AM were purchased from Molecular Probes. Polyclonal rabbit anti-human TRPC1 Abs as well as HRP-conjugated secondary Abs were obtained from Santa Cruz Biotechnology. The ECL Western blotting detection reagents were purchased from Amersham Biosciences. Protease Inhibitor Cocktail, calpain inhibitor I, Optiprep (60% iodixanol in water; Nycomed), p-nitrophenyl phosphate, cholera toxin B (CTB)-peroxidase conjugates, water-soluble cholesterol, sphingosine-1-phosphate (S1P), platelet-activating factor (PAF), N-fMLP and methyl--cyclodextrin (MCD), and thapsigargin were purchased from Sigma-Aldrich.

PMN isolation

PMN were isolated from volunteers by venipuncture into heparinized (20 U/ml) tubes. Blood was spun (150 x g for 10 min) to remove platelet-rich plasma. Cells were then layered onto equal volumes of Polymorphoprep medium (Robbins Scientific) and separated as we have described (8, 17, 22). PMN layers were removed and mixed with an equal volume of 0.45% sodium chloride solution to restore osmolarity for 5 min. Cells were washed, pelleted, and resuspended in the indicated buffers.

Calcium spectrofluorometry

PMN were suspended in HEPES buffer with 1 mM CaCl₂ and 0.1% BSA and incubated with 2 µg/ml fura 2-AM for 30 min in the dark at 37°C. Cells were divided into aliquots (2 x 10⁶ PMN) and placed on ice in the dark. Before study, samples were warmed to 37°C and centrifuged (4,500 rpm, 5 s), and supernatants were removed. Pellets were resuspended in 200 µl of BSA-free and "nominally calcium-free" HEPES buffer (with 0.3 mM EGTA) and finally injected into cuvettes containing 2.8 ml of the same buffer for study. [Ca²⁺]_i was determined from the fluorescence at 505 nm using 340/380 nm dual wavelength excitation (FluoroMax-2; Jobin Yvon Horiba) as previously described (23, 24).

Respiratory burst

Respiratory burst was assessed in PMN suspensions as previously reported (25). Briefly, 2 x 10⁶ PMN were suspended in 3 ml of HEPES buffer with 0.1% BSA and 15 ng/ml 1,2,3-dihydrorhodamine (DHR) added. Cells were studied at 37°C with constant stirring using excitation at 488 nm and emission at 530 nm. Respiratory burst was allowed to become linear and achieve maximal reaction rate (Vmax). The fluorescent intensity curve of oxidized DHR was compiled for 60 s during Vmax. Curve-fitting analysis was performed over the linear portion (Sigma-Plot 4.0; SPSS). Vmax was assessed as the first derivative of the fluorescent intensity in counts per second.

Lipid raft isolation

Rafts were prepared by an adaptation of the detergent-free methods of Macdonald and Pike (26). After isolation from two to three normal volunteer donors, 5 x 10⁷ PMN were divided into aliquots. Aliquots were treated at 37°C with 10 µg/ml water-soluble cholesterol for 10 min, MCD (10 µM for 10 min), or with vehicle. All steps thereafter were performed on ice. Cells were spun and pellets resuspended in 1 ml of base buffer (20 mM Tris-HCl (pH 7.8), 250 mM sucrose, 1 mM CaCl₂, and 1 mM MgCl₂) containing protease inhibitor and 50 µg/ml calpain inhibitor I. Cells were disrupted by passage through a 22-gauge 3-inch needle 10 times. Fragmented cells were sonicated five times for 20 s, cooling the cells on ice between sonications. Lysates were centrifuged 1000 x g for 10 min, and supernatants were reserved. The cell pellets were resuspended in 1 ml of base buffer with inhibitors and the sonication repeated. The two postnuclear supernatants were combined and an equal volume of 50% Optiprep added. Finally, 4 ml of each lysate (1–2 mg of protein) in 25% Optiprep was placed in the bottom of the centrifuge tubes, and 8 ml of a 0–20% Optiprep gradient was layered over it. Tubes were then centrifuged (52,000 x g; 3 h) and serial 670-µl fractions were collected from the top of the tube using careful pipetting.

Protein in each fraction was assayed by Micro BCA assay method (Pierce). Cholesterol was assayed using an Amplex Red cholesterol assay kit (Molecular Probes). Alkaline phosphatase was assayed using p-nitrophenyl phosphate as substrate (27).

Raft fraction localization with GM-1

Lipid rafts are rich in the ganglioside GM-1, and CTB is a specific GM-1 ligand. We therefore confirmed the distribution of lipid rafts within the PMN fractions isolated as previously described using GM-1 assays (28). Briefly, after treatments, PMN were spun (450 x g; 5 s). Cell pellets were suspended in 100 µl of PBS and incubated with 3 µl of CTB bound to HRP (CTB-HRP: 0.45 mg/ml CTB and 1 mg/ml HRP) for 30 min on ice. Cells were then washed to remove unbound CTB-HRP and processed to obtain raft fractions as described. Dot immunoassays were used to demonstrate the differential distribution of GM-1 in density fractions (29). Briefly, 5 µl of each gradient fraction was applied to slot blots. Standard curves were created using known amounts of CTB bound to HRP. CTB bound to HRP conjugates were then visualized with ECL reagent and quantified on an Alpha Imager 3400 (Alpha Innotech).

Immunohistochemistry for GM-1

As noted, GM-1 ganglioside is widely used as a marker in studies of lipid raft density and distribution. We studied the density and localization of GM-1 by immunohistochemistry to determine whether altered cholesterol content directly induced such changes in raft structure. Freshly isolated PMN were divided into aliquots of 1.5 x 10⁵ cells and allowed to adhere for 20 min to chamber slides coated with 0.01% polylysine (Sigma-Aldrich). Slides were washed with buffer to remove nonadherent cells. Individual chambers were then flushed with 10 µg/ml cholesterol or with vehicle, and cells were allowed to incubate for 5 min at room temperature. Slides were again washed with buffer. Slides were then stained with CTB-FITC (1/50 dilution in 1x PBS; Sigma-Aldrich) for 30 min in the dark on ice. After labeling, cells were washed and then fixed using 2% paraformaldehyde in PBS for 10 min and washed three times with PBS. Negative controls were created by pretreating cells with nonfluorescent CTB before being stained with CTB-FITC. Cells were imaged on a Zeiss Axiovision 200 fluorescent microscope using a x63 objective and Axiovert 4.5 software.

Western blotting to detect TRPC1 in raft fractions

TRPC1 is a prototypic calcium channel protein of the transient receptor potential protein family. PMN express multiple TRPC proteins that are known to traffic to the cell membrane during Ca²⁺ entry (9). We therefore studied TRPC1 raft trafficking in response to cholesterol as an initial proof of principle that raft cholesterol concentration might regulate the distribution of various Ca²⁺ influx-related proteins within the cell membrane. We analyzed 20 µl of each gradient fraction by electrophoresis on a SDS gel followed by Western blotting for TRPC1 protein. The primary rabbit anti-human TRPC1 Ab (Santa Cruz Biotechnology) was used at 1/1600 dilution. Secondary goat anti-rabbit Ab was used at 1/16,000 dilution. An ECL Western blotting detection kit was used to develop the blots that were imaged as described.

	Results

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Cholesterol in lipid rafts regulates G protein-coupled Ca²⁺ entry

Ligation of neutrophil GPCR by PAF depletes ER Ca²⁺ stores. Store depletion then activates cellular Ca²⁺ entry in a sphingosine kinase-dependent fashion (17, 30). We studied whether one or both of these phases of calcium mobilization could be blocked by lipid raft disruption (Fig. 1A). When we used MCD to remove cholesterol from and thus disrupt rafts (21, 31, 32), we found that Ca²⁺ entry was blocked by cholesterol sequestration and small amounts of cholesterol rescued Ca²⁺ entry. Cholesterol depletion had no effect whatsoever on release of Ca²⁺ from cell stores, demonstrating that the afferent arm of GPCR signaling was not affected by raft disruption.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. Cholesterol bioavailability, lipid raft disruption and the modulation of Ca2+ flux. A, PAF (100 nM) in Ca2+-free buffer causes brisk Ca2+ store release from the ER of human PMN. Prolonged Ca2+ entry is seen on CaCl2 readdition. 10 µM MCD has no effect on store release but prevents Ca2+ entry. Cholesterol (3 µg/ml) does not cause calcium entry (see Fig. 6), but rescues Ca2+ entry. Data are traces of the mean ± SE of three independent experiments. B, PMN were stimulated with PAF in increasing cholesterol concentrations. Cholesterol augmented Ca2+ entry without effects on release. Experiments are done in 0.1% BSA. Data are traces of the mean ± SEM of six experiments per trace. PMN are from multiple donors.

Exogenous cholesterol regulates raft cholesterol content

Because cholesterol availability modulated Ca²⁺ entry, we sought to determine whether this effect was specifically related to cholesterol incorporation into rafts. We therefore isolated PMN rafts after treatment with cholesterol or MCD to raise or lower cholesterol bioavailability. No detergents were used in raft preparation. Raft fractions were identified by their high cholesterol and low protein content, their high alkaline phosphatase activity (Fig. 2A), and the presence of GM-1 ganglioside (Fig. 2B). These experiments show that bioavailable cholesterol is incorporated preferentially into the low density fractions displaying raft markers (Fig. 2C). Conversely, treatment of PMN with MCD removed cholesterol from raft fractions with little effect on higher density fractions (Fig. 2C). The degree of cholesterol depletion by MCD was similar to that seen in studies using whole cells (35), also suggesting MCD specifically depletes raft cholesterol.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Fractionation for raft markers and cholesterol. Of note, data are all representative traces and that peaks in individual separations may move slightly left or right. Also, individual studies may focus on subsets of the fractions to optimize results. Thus peaks do not line up exactly. A, Alkaline phosphatase demonstrates a typical concentration peak in the light membrane raft fractions. B, The raft marker GM-1 ganglioside was probed using CTB in slot blots of raft fractions prepared from PMN undergoing no treatment or 10 min incubation in 10 µg/ml cholesterol. GM-1 peaked in fractions 2–6. C, Cholesterol concentration in raft fractions from PMN incubated with 10 µg/ml cholesterol, 10 mM MCD, or 10 min in BSA-free buffer only (No Tx). Rafts were isolated by centrifugation on 0–20% Optiprep gradients (52,000 x g for 3 h). Cholesterol is reported as micrograms per milligram of protein in the individual fractions. Data are a representative study (n = 4 experiments, each using pooled cells from two to four healthy donors).

Because cholesterol uptake into rafts augmented agonist-initiated Ca²⁺ entry, we studied whether cholesterol could stimulate Ca²⁺ uptake directly. We found that cholesterol caused Ca²⁺ entry without any antecedent release of calcium stores. This finding was best seen in albumin-free medium in which immediate dose-dependent responses were seen (Fig. 3A). We used a standard battery of channel inhibitors to investigate this phenomenon further. Ca²⁺ entry was inhibited by the inorganic channel blocker lanthanum (La³⁺, 1 mM) showing that it is cation channel-dependent. Similar Ca²⁺ entry was seen in albumin-containing medium (0.1% BSA), but these results required longer incubation, suggesting albumin-bound cholesterol desorbs onto cells gradually. To further delineate the type of cation channels involved we then studied cholesterol mediated Ca²⁺ uptake in the presence of L-type (verapamil) and nonspecific (SKF96365) calcium channel inhibitors (Fig. 3B). Dose-response curves were created (data not shown) but maximal activity was found at or near the expected concentrations based on prior publications. Nonspecific Ca²⁺ channel blockade (with SKF96365) quantitatively inhibited cholesterol-mediated Ca²⁺ influx. As expected, L-type channel blockade (verapamil) had no effect at any concentration because PMN lack voltage-gated calcium channels (36).

View larger version (40K): [in this window] [in a new window]   FIGURE 3. Direct cholesterol regulation of calcium channels. A, Cholesterol stimulates dose-dependent Ca2+ influx. Entry is blocked by 1 mM lanthanum (La). Nickel (Ni2+) but not gadolinium (Gd3+) act similarly (data not shown). The inorganic ions lanthanum and nickel block all cationic channels. Gadolinium at submicromolar concentration is believed specific for "store-operated" channels. Taken together, these observations show Ca2+ entry after cholesterol incorporation into rafts reflects the activation of nonspecific cation transporters and not a loss of membrane function. B, Cholesterol incorporation-mediated Ca2+ entry was sensitive to the nonspecific dihydropyridine SKF96365 (SKF, 80 µM) shown at IC50 30 µM. L-type channel blockade with verapamil (VER) up to 100 µM shown, had no effect at all. Data in all traces are mean ± SE of three to four experiments per condition. PMN isolates are from multiple donors.

Because cholesterol had no discernible effect on ER Ca²⁺ store release in response to PAF, we examined the possibility that it could play a direct role in the regulation of store-operated Ca²⁺ entry, a Ca²⁺ entry mechanism that is completely independent of GPCR activation. To study this possibility, we used thapsigargin, which blocks ER Ca²⁺ ATPase pumps, thus depleting ER Ca²⁺ stores directly and causing calcium entry independent of GPCR activation. As with PAF-mediated entry, we found that MCD markedly inhibited thapsigargin-initiated Ca²⁺ entry (Fig. 4, top). This inhibition was specific for MCD’s effect on cholesterol in that that the inhibition was immediately reversed and even overcome by cholesterol readdition (Fig. 4, bottom).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Cholesterol regulates Ca2+ entry responses to thapsigargin (TG). Top, Cholesterol depletion by MCD pretreatment (10 µM, 10 min) has no effect on thapsigargin-initiated store depletion but blocks calcium influx responses to thapsigargin on recalcification. Bottom, Specificity the effect of MCD for cholesterol. Suppression of thapsigargin-induced Ca2+ entry by MCD (10 µM in the cuvette) on recalcification (at t = 200 s) is immediately reversed by readdition of 10 µM cholesterol to the cuvette (at t = 250 s).

Next we evaluated the functional relevance of cholesterol-mediated Ca²⁺ influx. We studied a PMN model in which respiratory burst depends entirely on extracellular calcium entry (37, 38) to see whether respiratory burst would vary with the incorporation of cholesterol into rafts. First, we showed that cholesterol sequestration by MCD inhibited respiratory burst (Fig. 5A, histogram bar 4) and next that cholesterol replacement rescued respiratory burst from MCD inhibition (Fig. 5A, histogram bar 5). Thus we showed respiratory burst in this model depends on the presence of cholesterol in intact rafts.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. PMN respiratory burst varies with the incorporation of cholesterol into rafts in a model dependent on extracellular calcium entry. A, A total of 2 x 106 PMN was pretreated for 5 min in Ca2+ and BSA-free conditions with 10 µM cholesterol, 10 µM MCD, or vehicle as noted. Cells were then transferred to a cuvette containing DHR and stimulated with N fMLP (100 nM) at t = 30 s and then Tg (1 µM) at t = 100 s (fMLP/Tg). Cuvettes were followed 2–3 min until the linear Vmax phase for respiratory burst (RB) was achieved. This protocol causes respiratory burst that has been reported to depend entirely on uptake of external Ca2+ (37 38 ). In preliminary studies, we confirmed that respiratory burst was inhibited >85% by adding EGTA to the medium. Control studies using either cholesterol preincubation without fMLP or thapsigargin or with cholesterol (5–60 µg/ml) added directly to the cuvette do not initiate respiratory burst (30 µg/ml, histogram bar 2). BSA (0.1%) is used in all experiments because respiratory burst proceeds poorly without it. Basal fMLP/Tg induced respiratory burst (histogram bar 3) was markedly inhibited by MCD (histogram bar 4) and rescued by 10 µM cholesterol (histogram bar 5). Data are mean ± SE of Vmax (n = 6 for experimental conditions and n = 2–3 for controls). ANOVA/Dunn pairwise comparison shows fMLP/Tg (histogram bar 3) greater than others (*, p < 0.05). Cholesterol repletion (histogram bar 5) rescued respiratory burst from MCD (histogram bar 4) (*, p < 0.05). B, PMN were previously cholesterol-depleted (10 µM MCD) or preincubated with increasing concentrations of cholesterol for 5 min. Cells were then transferred to cuvettes with Ca+/BSA+ medium, and respiratory burst was induced by treatment with fMLP/Tg exactly as described. Vmax for the respiratory burst was assessed 2–3 min later. Pretreatment with MCD decreased (histogram bar 1) and pretreatment with 10 µg/ml cholesterol increased (histogram bar 5) the respiratory burst response to fMLP/Tg significantly (*, p < 0.05) vs medium-only control cells (histogram bar 2). At higher cholesterol pretreatment concentrations this augmentation was lost: pretreatment with 30 or 60 µg/ml cholesterol (histogram bars 6 and 7) actually decreased respiratory burst as compared with pretreatment with 10 µg/ml (histogram bar 5) (#, p < 0.05). Data are mean ± SE of Vmax for DHR oxidation (n = 6 isolates/group). Statistics are by ANOVA/Dunn comparisons.

To verify that the biphasic respiratory burst responses to cholesterol see specifically reflected cholesterol regulation of calcium entry, we studied whether exposure to cholesterol concentrations above 10 µg/ml could suppress PMN Ca²⁺ uptake. We found that this suppression was true both for direct cholesterol stimulation of Ca²⁺ entry (Fig. 6A) and for the synergistic combination of an ineffective concentration of S1P with cholesterol (Fig. 6B). This finding is of special significance because S1P synthesis links chemoattractant-induced store depletion to Ca²⁺ influx (30).

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Biphasic cholesterol regulation of calcium entry. A, PMN were incubated 30 min in the cholesterol concentrations shown with 0.1% BSA added. Cells were then placed in BSA/Ca2+-free medium, which was recalcified at t = 50 s. Ca2+ uptake was maximal at 10 µg/ml cholesterol. Thus modulation of Ca2+ uptake by cholesterol is biphasic. B, S1P signals store-operated Ca2+ entry in PMN (30 ). A total of 1 µM S1P (injected into the cuvette at t = 30 s) causes no measurable Ca2+ entry above "leak" in the absence of cholesterol (i.e., the "zero cholesterol" control trace), but is synergistic with cholesterol preincubation, allowing the biphasic response to 10–30 µg/ml cholesterol to be clearly seen. Each trace shown is the mean of two to three studies done using PMN from different donors. Because Ca2+ entry rises and then falls with increasing cholesterol, the scale has been expanded and error bars omitted to allow more accurate inspection of the overlapping traces.

To confirm that the effects of cholesterol on GPCR-mediated Ca²⁺ entry were generalized and not restricted to PAF, we evaluated the effects of cholesterol on fMLP-mediated Ca²⁺ entry. Consistent with PAF, fMLP-linked Ca²⁺ entry responses were suppressed by MCD and augmented by cholesterol (Fig. 7, top). To further demonstrate that in our model GPCR-mediated Ca²⁺ entry and respiratory burst were regulated in a coordinated and linked fashion, we studied the fMLP-mediated Ca²⁺ entry responses of PMN at very high cholesterol concentrations. Again, we found that high cholesterol concentrations suppressed calcium entry (Fig. 7, bottom).

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Cholesterol modulates Ca2+ entry responses to fMLP. Top, Cholesterol depletion by MCD preincubation (10 µg/ml for 10 min) has no effect on ER store depletion but blocks calcium influx responses to fMLP. Similar to the responses seen to PAF, repletion of cholesterol to 10 µM enhances calcium entry. Bottom, Again, high concentrations of cholesterol have no effect on ER store depletion by fMLP but diminish subsequent calcium entry. These findings again demonstrate that free cholesterol causes global changes to the composition/structure of the plasma membrane, which have the capacity to modify the Ca2+ entry signals elicited by multiple G protein-coupled mediators in a similar biphasic fashion.

Calcium entry into PMN depends upon the redistribution of TRPC proteins (9). Calyculin A (CalyA) stabilizes cortical actin, forming a "cortical bar" that prevents TRPC proteins from trafficking to membranes. CalyA also blocks Ca²⁺ influx in response to Ca²⁺ entry-mobilizing signals. Cytochalasin D prevents actin assembly. Used with CalyA, it prevents the formation of the cortical bar which inhibits protein traffic to and from the cell membrane. Cytochalasin D, therefore, serves as a control to demonstrate the specificity of the CalyA effect for actin reorganization (9, 40). We examined cholesterol-mediated Ca²⁺ influx in the presence of CalyA and found that it was abolished (Fig. 8). Consistent with our prior studies of TRPC trafficking in PMN, cytochalasin D reversed the inhibition of cholesterol-induced Ca²⁺ influx by CalyA (9). These experiments show cholesterol incorporation into rafts regulates Ca²⁺ entry by mechanisms that are opposed by stabilizing cortical actin, which presumably prevents redistribution of proteins to and from the cell membrane. These findings are consistent with the concept that incorporation of cholesterol into rafts localizes Ca²⁺ channels to the cell membrane.

View larger version (41K): [in this window] [in a new window]   FIGURE 8. CalyA (50 nM) stabilizes cortical actin forming a "cortical bar" and preventing translocation of proteins to the cell membrane. Thus CalyA abolishes Ca2+ entry responses to G protein-coupled agonists (9 ). CalyA blocks the Ca2+ entry response to cholesterol. Preincubation in Cytochalasin D (CytoD, 2.5 µM) blocks actin assembly and reverses the effects of CalyA. Cytochalasin D rescues the Ca2+ entry response to cholesterol that was inhibited by CalyA. Data are the mean ± SE for n = 3 experiments shown in all traces.

We knew exogenous cholesterol caused changes in raft composition, Ca²⁺ entry and respiratory burst. We therefore questioned whether cholesterol enrichment of rafts might alter functionally important raft structural properties, such as their coalescence into macrodomains or polarization to specific regions of the cell. We studied this question with immunofluorescent microscopy, using CTB-FITC to bind GM-1 (28) and visualize rafts directly. We found that exposure to free cholesterol (10 µg/ml) increased membrane GM-1 available for binding as well as causing brisk coalescence of GM-1 into polarized raft macrodomains (Fig. 9). Thus, altering raft lipid composition by cholesterol addition changes raft structural characteristics known to be associated with function.

View larger version (154K): [in this window] [in a new window]   FIGURE 9. Neutrophil lipid rafts were visualized by staining cells with CTB linked to fluorescein (CTB-FITC). CTB is a specific ligand for the ganglioside raft marker GM-1. Identical fields are shown in phase-contrast (bottom) and fluorescent (top) modes. Adherent cells treated in the chamber slide with cholesterol (10 µg/ml, 5 min) are on the left. Cells similarly treated with vehicle on the same slide and imaged under identical conditions are on the right. Cholesterol is noted to cause aggregation of the lipid raft marker GM-1 into polarized macrodomains. Without cholesterol treatment, cells demonstrated only diffuse staining. Data represent 10–12 fields examined per study slide. Cells are chosen for fluorescent study on the basis of spacing and normal morphology under phase-contrast. Control cells pretreated with unlabeled CTB before CTB-FITC show little fluorescence.

Cholesterol caused changes in raft composition, structure, and function (Figs. 2 and 9). Also, regulation of Ca²⁺ entry by cholesterol was dependent upon protein trafficking to the plasma membrane (Fig. 8). We therefore hypothesized that cholesterol might regulate trafficking of calcium channel proteins to rafts. The regulation of PMN Ca²⁺ entry in vivo relies upon multiple channel proteins (9) and these may show varied responses to changes in raft composition. Based upon earlier work by our group and others, however, we initially asked whether cholesterol might regulate raft trafficking of TRPC1. TRPC1 is a prototypical TRPC protein that can both localize to rafts and transmit nonspecific Ca²⁺ entry currents (9, 20, 41, 42, 43, 44).

Using our fractionation methods, we noted that fractions 2–4 displayed the highest concentration of raft markers and exhibited maximal cholesterol incorporation (Fig. 2). We therefore used Western blots to probe for TRPC1 in fractions 2–4 of pooled normal volunteer PMN fractionated with or without prior incubation in 10 µg/ml cholesterol (Fig. 10). These studies demonstrate that preincubation of neutrophils in cholesterol increases the amount of TRPC1 protein in membrane lipid raft fractions.

View larger version (41K): [in this window] [in a new window]   FIGURE 10. Three different experimental preparations of pooled, isolated normal PMN (each preparation obtained from n = 2–4 volunteers) were probed for raft TRPC1 with and without prior cholesterol treatment. For each experiment, 5 x 107 PMN were fragmented, lysed, and centrifuged on an Optiprep gradient as described in Materials and Methods. For each experiment (a–c) and condition (with (+) or without (–) cholesterol) a mixture of 5 µl each of raft fractions 2–4 (total 15 µl per lane) were run out on a single SDS-PAGE gel alongside 20 µl of reserved crude extract and positive control "(+)" protein. After transfer, blots were probed for TRPC1, developed and visualized by autoradiography as described in Materials and Methods. The total amount of TRPC1 in the crude lysates was fairly consistent across the three experiments. In each of the three experiments, however, there was a clear increase in the amount of TRPC1 protein found in the raft fractions after cholesterol treatment.

View larger version (47K): [in this window] [in a new window]   FIGURE 11. Individual donor PMN samples were divided into two groups and treated with either cholesterol or vehicle. Treated and untreated PMN were fragmented, lysed, and centrifuged on Optiprep gradients simultaneously. Raft fractions were obtained and identical volumes (20 µl) of raft fractions made from the treated and untreated samples were electrophoresed together. Gels were transferred, stained for TRPC1, and developed side by side. A typical redistribution of the TRPC1 signal (right to left) is well seen. Multiple preparations (n = 5) resulted in similar shifts. Fractions 7–9 appeared to lose TRPC1 especially rapidly as the lighter raft fractions gained it. It is unclear whether the movement seen suggests a spatial reorganization of TRPC1 protein across the plasma membranes or from endomembranes of higher density to the surface. MW, molecular weight marker. Positive control proteins were run with fractions 10–18 (data not shown).

	Discussion

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The current findings have many potential implications. Clinically, activation of immune cells by cholesterol may help explain the association of atherosclerosis with increased C-reactive protein (1). High serum or tissue-free cholesterol availability might also activate immune cells that contribute to the progression of atherosclerotic lesions. Also, some of the reported effects of statin drugs on cell signaling and immunity may reflect altered lipid raft structure and function due to decreased cholesterol availability. Conversely, cholesterol deficiency could limit inflammatory responses, and indeed where decreased serum cholesterol occurs in trauma, burns, and other critical illnesses, it appears to predispose to sepsis and death (45, 46, 47, 48, 49). Practically speaking, these findings suggest cholesterol excess and deficiency states may alter cell signaling in ways that are modifiable by dietary or pharmacologic cholesterol control. Mechanistically, our findings suggest the equilibrium between free cholesterol and raft cholesterol acts as a determinant of raft function. Although it is clear that not all protein-protein interactions require lipid rafts (50), there is wide agreement that rafts play a significant role in cell signaling (5, 6, 51). Moreover, there is no theoretical reason that raft trafficking and protein-protein scaffolding interactions should not coexist and collaborate in the initiation and regulation of signaling events.

Rafts are ternary mixtures of sphingolipids and cholesterol packed between unsaturated phospholipids, and signaling proteins preferentially traffic to rafts because of this lipid-ordered physical state. The mole fraction of cholesterol in rafts has been assessed at 25% (4, 52). Further ordering of rafts can occur at mole fraction of 33%, but at higher concentrations still, cholesterol is incorporated into rafts without complexing to sphingolipids (51). Thus, the physical properties of lipid rafts may vary markedly around an optimal cholesterol mole fraction of 33% with higher or lower cholesterol concentrations having a disordering effect (5, 6, 51). Our studies show that PMN Ca²⁺ entry and respiratory burst appear to be regulated in accordance with this principle, potentially reflecting a novel and basic raft structure-function relationship in which altered lipid ordering and maximal affinity for specific signaling proteins are seen at specific free cholesterol concentrations. This mechanistic concept of raft structure could also explain why cholesterol at very high concentrations can suppress immune cell function (39).

Cholesterol is present in human plasma at concentrations of milligrams per milliliter. The vast majority is complexed in lipoproteins, but clinically measured free cholesterol levels still far exceed the low cholesterol concentrations used to elicit Ca²⁺ entry in this study. Low concentrations of albumin (0.1% BSA) prevent the instantaneous induction of Ca²⁺ entry by cholesterol (e.g., Figs. 1 and 3). But over time cholesterol stimulates typical Ca²⁺ entry in the presence of albumin (e.g., Fig. 6). Thus although clinical free cholesterol is albumin-bound and albumin can delay cholesterol’s effects on signaling in vitro, cells are continuously exposed to cholesterol in vivo in an equilibrium where cholesterol desorbs from albumin to cell membranes. Moreover, albumin concentrations vary markedly from plasma to the interstitium in health, and may vary further as a function of disease states. These relationships suggest great caution in creating scientific models to study the role of cholesterol in cell signaling, but the equilibrium between albumin-bound and raft-incorporated cholesterol may help determine how variations in cholesterol concentration influence cell signaling and function at specific locations, over relevant time periods, and in clinical disease states.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grant 2 R01 GM059179 from the National Institutes of Health (to C.J.H.).

² Address correspondence and reprint requests Dr. Carl J. Hauser, Department of Surgery, Lowry Medical Office Building, Room 2G, Beth Israel-Deaconess Medical Center, 110 Francis Street, Boston, MA 02215. E-mail address: cjhauser{at}bidmc.harvard.edu

³ Abbreviations used in this paper: [Ca²⁺]_i, cytosolic free calcium ion concentration; MCD, methyl--cyclodextrin; GPCR, G protein-coupled receptor; PAF, platelet-activating factor; PMN, polymorphonuclear neutrophil; ER, endoplasmic reticulum; S1P, sphingosine-1-phosphate; TRPC, transient receptor potential channel; CTB, cholera toxin B; DHR, 1,2,3-dihydrorhodamine; CalyA, calyculin A; Vmax, maximal reaction rate; fMLP/Tg, fMLP plus thapsigarin.

Received for publication April 21, 2006. Accepted for publication February 3, 2007.

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