No abstract

We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Tritondetergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton-and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol-but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL 3 additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL 3 reduced the lipid content in Lubrol-as well as in Tritondetergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL 3 -mediated lipid efflux. Key words: ABCA1, CDC42, microvilli, monocytes, rafts Received 12 November 2001, revised and accepted for publication 21 January 2002More than 10 years ago the raft hypothesis was introduced, suggesting that horizontal interaction of certain membrane lipids forms the basis for plasma membrane microdomains 268(termed rafts) that are characterized by the enrichment of cholesterol, sphingolipids and saturated phospholipids (1-3). The specific lipid composition renders these microdomains resistant to solubilization in Triton X-100 (Triton) at 4 aeC, a property that has been widely used to study lipid rafts designated 'Triton rafts' (4). As recently reviewed by Simons and Toomre, the most important role of rafts may be their function in signal transduction and cell regulation by providing a platform for ligand-triggered clustering of receptors and signaling molecules (5). There are, however, still many open issues, including the question whether more than one kind of raft exists on the cell surface of different cell types (6). In support of the existence of different types of rafts, Röper et al. recently identified a novel type of cholesterol-based microdomain that is discriminated from the classical lipid rafts by its solubility in Triton but its resistance to another non-ionic detergent, Lubrol WX (Lubrol) (7). This microdomain has been designated 'Lubrol-raft', and current data suggest that it serves as a building unit for different types of plasma membrane protrusions. The formation of membrane protrusions, like lamellipodia, microvilli, or filopodia...

Recently, we described an 80-kDa lipopolysaccharide (LPS)-binding membrane protein to be identical to CD55 [decay accelerating factor (DAF)]. Here, we demonstrate that CD55 is able to contribute to lipopolysaccharide (LPS) signaling. Transfection of Chinese hamster ovary (CHO) cells with human CD55 resulted in a translocation of NF-‹ B after stimulation with LPS as well as with free lipid A. In addition, interaction of lipid A and CD55 was shown by co-immuno-precipitation of these molecules from CHO-CD55 cells after incubation with lipid A and anti-lipid A monoclonal antibody, as well as by fluorescence resonance energy transfer (FRET) analysis in human monocytes. The comparison of LPS-induced signaling pathways in CHO-CD55 and CHO-CD14 cells revealed that p38, JNK and ERK MAP kinases are activated upon LPS stimulation in both cell lines, and that the activation by LPS can be blocked at the level of Toll-like receptor 4. Finally, through FRET analysis we could demonstrate LPS-induced clustering of CD55 and CD11/CD18 in human monocytes. Our results imply a new functional role of CD55 as a member of a multimeric LPS receptor complex.

Three different methods for MP detection showed good correlations of results, albeit the basis for MP analysis was different. Even if FCM is considered the "gold standard" of MP detection there are still technical limitations concerning detection of small MP. The procoagulant STA-Procoag-PPL assay and the prothrombinase ELISA assay could be useful additional MP tests. Regarding the interpretation of quantitative results of MPs, preanalytical conditions must be optimized and standardized.