Lloyd, Katie L., and Paul Kubes. GPI-linked endothelial CD14 contributes to the detection of LPS. The inflammatory endothelial response to LPS is critical to the host's surviving a gram-negative bacterial infection. In this study we investigated whether human endothelial cells express the functional coreceptor for LPS, CD14, and most importantly whether it is glycosylphosphatidylinositol (GPI) linked. We also examined whether plasma proteins could reconstitute an LPS response in CD14-inhibited

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... ndothelium. RT-PCR-and CD14-specific MAbs demonstrated CD14 expression on primary human umbilical vein endothelial cells (HUVEC) but not passaged HUVEC. The amino acid sequence of endothelial CD14 was 99% homologous to CD14 on monocytes. Endothelium responded to relatively low levels of LPS in the absence of plasma, and this was entirely dependent on CD14. Removal of GPI-linked proteins with phosphatidylinositol-phospholipase C prevented LPS detection and subsequent protein synthesis (E-selectin expression). Endothelial CD14 was sufficient to initiate functional leukocyte recruitment, an event inhibited by blocking its LPS binding epitope and also by removing CD14 from the endothelial surface. Plasma proteins restored only ϳ30% of the LPS response in CD14-inhibited endothelium. In conclusion, our results strongly support an important role for endothelial membrane CD14 in the activation of endothelium for leukocyte recruitment. human; human umbilical vein endothelial cells; inflammation; leukocyte recruitment; glycosylphosphatidylinositol LPS is a major outer membrane constituent of gram-negative bacteria, highly immunogenic and shed during infection. This shed LPS is used as a detection system for local infection by our immune system, leading to important inflammatory responses, including leukocyte recruitment. However, under some conditions, LPS can enter the circulation, causing a systemic inflammatory response that is detrimental to the host. The human immune system is able to detect LPS by its trimeric LPS receptor: Toll like receptor 4 (TLR-4), myeloid differentiation protein 2 (MD-2), and membrane bound CD14 (mCD14). CD14 is described as a glycosylphosphatidylinositol (GPI)-linked 55-kDa glycoprotein on monocytes (9, 26), which was first demonstrated to be an LPS receptor on human monocytes (32). Pugin et al. (21) showed that responses to gram-negative LPS on murine macrophage cell lines were inhibited with CD14 blocking antibodies. The role of CD14 as an LPS receptor was confirmed by Goyert and colleagues (10) in a seminal study demonstrating high resistance to LPSinduced shock in gene-targeted CD14 knockout mice. Isoforms of CD14 occur only via posttranslational modifications, including addition of GPI anchor. GPI-linked mCD14 has been demonstrated to be expressed on numerous blood cells: at a high level on monocytes and macrophage subsets (32) and at a lower level on granulocytes and activated or transformed B cells (10, 14, 16, 29, 35) . Other studies have suggested that mCD14 is also expressed on human corneal cells (27) , gingival fibroblasts (28), and epithelial cells (6). Whether endothelium can synthesize and express CD14 remains unclear. Early studies from several groups suggest that CD14 is absent from endothelium and that proinflammatory endothelial responses to LPS require soluble CD14 (sCD14) in place of mCD14 (2, 4, 22, 23) . A more recent study suggested that CD14 may be present on endothelium and that its previously reported absence was due to the use of passaged endothelial cells that have reduced CD14 expression (11). Endothelium is the critical cell for recruitment of leukocytes into tissues. In fact, recent in vivo data underscored the essential role for endothelial (but not leukocyte) TLR-4 in the early sequestration of neutrophils into lungs (1). Since TLR-4 and CD14 are closely linked partners in the LPS response, CD14 must clearly also contribute to LPS responses in endothelium. Moreover, endothelial detection of LPS may not only be relevant to acute inflammatory responses but also to more prolonged conditions such as those associated with atherosclerosis. Indeed, the absence of TLR-4 diminished development of atherosclerosis in mice (3). However, whether CD14 is necessary for endothelial responses to LPS and whether CD14 was endothelial in origin is unclear. In this study we hypothesized that functional GPI-anchored CD14 is present on and originates from within endothelium. Using primary human umbilical vein endothelial cells (HUVEC) as our model system, we demonstrate that CD14-specific mRNA can be detected in amounts half of that in monocytes and that the protein sequence is 99% identical to monocyte CD14. Moreover, we demonstrate that CD14 is GPI anchored to the cell surface but is lost on passaging. Last, we demonstrate that primary HUVEC can initiate a maximal proinflammatory response via membrane CD14 in response to concentrations as low as 1 ng/ml of smooth Escherichia coli LPS entirely in the absence of plasma proteins and that this results in leukocyte recruitment. However, plasma proteins reconstituted ϳ30% of the LPS response in CD14inhibited endothelium. We therefore suggest that GPI-linked CD14 is present on endothelium and that its presence is important to the endothelial response to LPS. MATERIALS AND METHODS Reagents. CD14 antibody used for ELISA and blocking studies was unconjugated clone MEM-18 monoclonal antibody supplied by Hycult Biotechnology. The isotype (IgG 1) control MOPC-21 was from