Implications of lipid raft disintegration: enhanced anti-inflammatory macrophage phenotype.
BACKGROUND
Lipid rafts are membrane microdomains characterized by an enriched cholesterol environment and appear to serve as a platform for signaling. Their role within the macrophage during endotoxin exposure is unknown.


METHODS
THP-1 cells were subjected to lipopolysaccharide stimulation with or without methyl-beta-cyclodextrin (MbetaCD) pretreatment, a cholesterol depleting agent. Cell surface expression of toll-like receptor-4 (TLR4) and platelet-activating factor receptor (PAFr) was determined by flow cytometry. Membrane receptor components and activation of the mitogen-activated protein kinases (MAPK) was determined from lipid raft and cellular protein by immunoblot. Inflammatory mediator production was determined from harvested supernatants by enzyme-linked immunosorbent assay.


RESULTS
Surface expression of TLR4 and PAFr was not affected by MbetaCD. Lipopolysaccharide stimulation led to TLR4 mobilization to lipid rafts, MAPK activation, and inflammatory mediator production. Pretreatment with MbetaCD did not affect TLR4 mobilization to lipid rafts, but did result in lost lipid raft expression of the PAFr coupled G-protein, Galpha1. MbetaCD treatment led to selective attenuation of MAPK activation through ERK 1/2. This dysregulated signaling was associated with attenuated production of tumor necrosis factor-alpha, but increased production of interleukin-10.


CONCLUSIONS
Lipid raft disintegration results in lost expression of Galpha1, dysregulated MAPK signaling, and selective anti-inflammatory mediator production. Therefore, modulation of lipid raft cholesterol content may represent a potential mechanism for regulation of macrophage phenotypic differentiation.
Cuschieri
Joseph
J
Department of Surgery, University of Cincinnati, Ohio USA.
D002460:Cell Line; D004789:Enzyme Activation; D044385:GTP-Binding Protein alpha Subunits / Q000032:analysis; D006801:Humans; D007249:Inflammation / Q000517:prevention & control*; D020935:MAP Kinase Signaling System; D008264:Macrophages / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D010641:Phenotype; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000032:analysis; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
D044385:GTP-Binding Protein alpha Subunits; D008562:Membrane Glycoproteins; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; C039351:platelet activating factor receptor