ORIGINAL PAPER Cholesterol is obligatory for polarization and chemotaxis but not for endocytosis and associated signaling from chemoattractant receptors in human neutrophils Jeremy J. Rose Æ John F. Foley Æ Ling Yi Æ Gina Herren Æ Sundararajan Venkatesan Received: 8 February 2008 / Accepted: 13 February 2008 / Published online: 3 March 2008 Ó National Science Council Taipei 2008 Abstract Plasma membrane cholesterol is critical for neutrophil chemotaxis, although how cholesterol affects chemotactic signaling pathway has not been clearly delineated. Here we demonstrate that cholesterol was absolutely required for polarized redistribution of key chemotactic mediators in human neutrophils in response to all chemoattractants tested (fMet-Leu-Phe, and the che- mokines CXCL1, CXCL8 and CXCL12). In particular, PI3K and phosphatidylinositol-3,4,5 triphosphate (PIP 3 ) failed to accumulate at the front and phosphatase and tensin homolog (PTEN) at the back of chemoattractant-stimulated neutrophils after cholesterol depletion. Cholesterol deple- tion did not affect early chemoattractant signaling events such as G-protein activation, intracellular calcium flux or G-protein-independent endocytosis-linked signaling, including the activation of mitogen-activated protein kinase (MAPK), Hck and Fgr transduced by b-arrestin. During cell polarization, F-actin assemblies redistributed the cholesterol-rich microdomains and cytoskeleton- anchored proteins, including CD16 and CD44 from the leading edge. These data suggest that spatial polarization of chemotactic mediators is orchestrated by protein:protein interactions that organize cholesterol-rich domains of the plasma membrane. Keywords Neutrophils Á Chemotaxis Á Cholesterol Á Lipid rafts Á Chemoattractants Á G-proteins Á Endocytosis Á Arrestin Á Degranulation Á Protein kinases Á F-actin Abbreviations APC Allophycocyanin DRMs Detergent-resistant membranes EGFR Epidermal growth factor receptor fMLF N-Formyl-methionine-leucine-phenylalanine FPR N-Formyl peptide receptor GPCR G-protein-coupled receptor GPI Glycosyl phosphatidylinositol GRK G-protein-coupled receptor kinase GROa Growth related oncogene a HMG-CoA Hydroxy-methyl glutaryl CoA IL-8 Interleukin-8 IP 3 Inositol-1,4,5 trisphosphate Lat-B Latrunculin B LTB 4 Leukotriene B 4 MAPK Mitogen-activated protein kinase MbCD Methyl-b-cyclodextrin PIP 2 Phosphatidyl inositol 4,5 bisphosphate PIP 3 Phosphatidylinositol-3,4,5 triphosphate PTEN Phosphatase and tensin homolog PTX Pertussis toxin SDF Stromal cell-derived factor TM Transmembrane Introduction Cholesterol is a ubiquitous component of animal cell membranes, making up 30–50% of plasma membrane lipids, scant amounts in the endoplasmic reticulum (ER) and mitochondria and intermediate amounts in the Golgi [1]. As well as providing structural integrity to the plasma membrane and decreasing passive membrane permeability, cholesterol plays a direct or an indirect role in various signaling pathways and is a precursor to signaling J. J. Rose Á J. F. Foley Á L. Yi Á G. Herren Á S. Venkatesan (&) Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 6A05, Bethesda, MD 20892-1576, USA e-mail: sv1s@nih.gov; aradhana@helix.nih.gov 123 J Biomed Sci (2008) 15:441–461 DOI 10.1007/s11373-008-9239-x