Monocyte cholesterol homeostasis correlates with the presence of detergent resistant membrane microdomains

Abstract

Background: Lipid membrane microdomains are involved in the regulation of biological functions of monocyte membrane proteins. These microdomains show a relative resistance to non‐ionic detergents providing an easy analytical tool to study them. Methods: Here, we applied a rapid detergent‐based flow cytometric assay to investigate microdomain association of proteins on monocytes from whole blood samples. The association of known surface antigens with detergent resistant fraction of membranes (DRMs) was compared using monocytes from healthy blood donors, patients with genetic disorders affecting cellular cholesterol traffic and patients with systemic inflammatory response. Results: All investigated surface antigens of Niemann‐Pick type C (NPC)‐mutant monocytes with impaired cholesterol influx and defective late endosome cholesterol trafficking, presented a strongly increased DRM‐association. Though, membrane antigens of ATP binding cassette transporter A1 (ABCA1)‐mutant monocytes with impaired cholesterol efflux did not show alterations in DRM‐association. Differential CD14‐dependent receptor clustering within microdomains was also investigated in response to in vivo lipopolysaccharide (LPS) and/or atherogenic lipoprotein activation. Increased DRM‐association of the GPI‐anchored proteins CD14, CD55, the Fcγ receptor CD64, the scavenger receptors CD36, CD91 and CD163, the integrin CD11a, and complement receptor 3 complex CD11b/CD18 were observed from patients with systemic inflammatory response syndrome (SIRS)/sepsis or coronary artery disease (CAD)/myocardial infarction. Interestingly, the tetraspanin CD81 presented increased DRM‐association in SIRS/sepsis patients, but not in CAD patients. Moreover, the pentaspanin CD47 and the Fcγ RIII CD16 showed an increased DRM partition in CAD patients but disassembled from DRMs in SIRS/sepsis patients. Conclusions: Our results demonstrate that flow cytometric analysis of short time in situ detergent extraction provides a powerful tool for rapid screening of blood monocyte DRMs to preselect patients with potential raft/microdomain abnormalities for more detailed analysis. © 2007 International Society for Analytical Cytology

Tetraspanin CD9 Negatively Regulates Lipopolysaccharide-Induced Macrophage Activation and Lung Inflammation

“…They further provided data suggesting a coassembly of CD81 and CD14 in monocytes from patients with sepsis (i.e., in vivo LPS stimulation) (15). CD9 was also examined in their recent study (28) but, unlike the present study, CD9 appeared not to be associated with the lipid microdomain in a rapid detergent-based flow cytometric assay. This discrepancy may be due to methodological differences or might be because they used human monocytes from healthy donors (i.e., without in vivo LPS stimulation) in this CD9 experiment (28).…”

“…Lipid rafts are typically insoluble in nonionic detergents, whereas TEMs are mostly soluble in nonionic detergents (8). However, recent data suggest that there may be an overlapping or an interaction between the raft and TEM proteins under certain conditions (9,28,10). Such interactions may result in the close proximity of these distinct sets of membranous molecules and facilitate signal transduction (10).…”

Shedding of Large Functionally Active CD11/CD18 Integrin Complexes from Leukocyte Membranes during Synovial Inflammation Distinguishes Three Types of Arthritis through Differential Epitope Exposure

“…In the same fraction, we could also detect complexes containing the KIM127 epitope together with a CD11a epitope. We considered the possibility that the CD18 integrin oligomerization derived from embedding of CD11/CD18 in ectosomes or other lipid vesicles as described by others (65). However, ultracentrifugation prior to assay analysis could not reduce the sCD18 signal in the plasma samples as would be expected if the integrins were contained in lipid vesicles (50).…”

The effects of detergents on the polymerization properties of actin

“…This raises the question, how the detergents influencing actin polymerization in vitro act on DRMs in intact cells. This approach is appealing, since recently, a flow cytometric method was developed for DRM isolation, called FCDR assay (29)(30)(31)(32)(33)(34). In these works, raft-associated proteins labeled by fluorescently tagged antibodies remained cell associated, after detergent solubilization of the whole cells.…”

“…Lipid rafts involved in cell signaling or membrane and protein trafficking are plasma membrane lipid heterogeneities containing elevated amounts of sphingolipids, cholesterol, GPI-anchored and other particular ''raft'' proteins (23)(24)(25)(26)(27)(28). The methodologically defined detergent-resistant membranes (DRMs), which considered to show comparable chemical properties to rafts, are identified applying the same TX100, Brij and CHAPS detergents and used in recently developed cytometric DRM measurements [flow cytometric detergent resistance (FCDR) assay] (29)(30)(31)(32)(33)(34).…”