Cholesterol-modulating Agents Selectively Inhibit Calcium Influx Induced by Chemoattractants in Human Neutrophils

Abstract

The effects of cholesterol-perturbing agents on the mobilization of calcium induced upon the stimulation of human neutrophils by chemotactic factors were tested. Methyl-␤-cyclodextrin and filipin did not alter the initial peak of calcium mobilization but shortened the duration of the calcium spike that followed the addition of fMet-Leu-Phe. These agents also inhibited the influx of Mn 2؉ induced by fMet-Leu-Phe or thapsigargin. Methyl-␤-cyclodextrin and filipin completely abrogated the mobilization of calcium induced by 10 ؊10 M platelet-activating factor, which at this concentration depends to a major extent on an influx of calcium as well as the influx of calcium induced by 10 ؊7 M platelet-activating factor. On the other hand, methyl-␤-cyclodextrin and filipin enhanced the mobilization of calcium induced by ligation of Fc␥RIIA, an agonist that did not induce a detectable influx of calcium. Finally, methyl-␤-cyclodextrin and filipin enhanced the stimulation of the profile of tyrosine phosphorylation, the activity of phospholipase D (PLD), and the production of superoxide anions induced by fMet-Leu-Phe. These results suggest that the calcium channels utilized by chemotactic factors in human neutrophils are either located in cholesterol-rich regions of the plasma membrane, or that the mechanisms that lead to their opening depend on the integrity of these microdomains.

Cutting Edge: Optical Microspectrophotometry Supports the Existence of Gel Phase Lipid Rafts at the Lamellipodium of Neutrophils: Apparent Role in Calcium Signaling

“…Cholesterol-rich lipid rafts have recently been suggested to participate in neutrophil Ca 2ϩ signaling (5). Using previously established quantitative microfluorometry and high-speed fluorescence microscopy methods, we have studied the Ca 2ϩ signaling events associated with spontaneous neutrophil motility.…”

“…Therefore, the neutrophil lamellipodium is highly enriched in lipid raft markers. Previous studies have shown that lipid rafts can be disrupted by m␤CD, which abstracts cholesterol from cell membranes (5,6). Cells were exposed to 5 mM m␤CD for 5 min at 37°C followed by immunofluorescence microscopy.…”

“…However, these conclusions are based largely on fractionated cell components isolated by differential detergent ultracentrifugation. To deflect concerns regarding potential artifacts of cell fractionation, methyl-␤-cyclodextrin (m␤CD) is frequently used, because it binds to cholesterol and thereby disrupts lipid rafts in plasma membranes (5,6). Because m␤CD treatment provides no direct structural information regarding lipid rafts, it would be useful to have an independent means of verifying and studying lipid rafts in cell membranes.…”

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

“…Because immune cell function depends heavily on Ca 2ϩ 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 Ca 2ϩ entry and subsequent activation, potentially linking inflammation mechanistically to the pathogenesis of cholesterol-dependent clinical disease states.…”

“…Thus TRPC-based channels can mediate agonist-induced Ca 2ϩ entry but the mechanisms linking phospholipase activation and store depletion to Ca 2ϩ 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).…”