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Varying Importance of Soluble and Membrane CD14 in Endothelial Detection of Lipopolysaccharide¹

Katie L. Lloyd-Jones^*, Margaret M. Kelly and Paul Kubes^2,*

^* Department of Biophysics and Physiology, Immunology Research Group, and Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, Alberta, Canada

The endothelial response to LPS is critical in the recruitment of leukocytes, thereby allowing the host to survive Gram-negative infection. Herein, we investigated the roles of soluble CD14 (sCD14) and membrane CD14 (mCD14) in the endothelial response to low level LPS (0.1 ng/ml), intermediate level LPS (10 ng/ml), and high level LPS (1000 ng/ml). Removal of sCD14 from serum and sCD14-negative serum prevented low level LPS detection and subsequent response. Addition of recombinant sCD14 back into the endothelial system rescued the endothelial response. GPI-linked mCD14 removal from endothelium or endothelial treatment with a CD14 mAb prevented responses to low-level LPS even in the presence of sCD14. This demonstrates essential nonoverlapping roles for both mCD14 and sCD14 in the detection of low-level LPS. At intermediate levels of LPS, sCD14 was not required, but blocking mCD14 still prevented endothelial LPS detection and E-selectin expression, even in the presence of sCD14, suggesting that sCD14 cannot substitute for mCD14. At very high levels of LPS, the absence of mCD14 and sCD14 did not abrogate TLR4-dependent, E-selectin synthesis in response to LPS. The MyD88 independent pathway was detected in endothelium (presence of TRIF-related adaptor molecule TRAM). The MyD88-independent response (IFN-β) in endothelium required mCD14 even at the highest LPS dose tested. Our results demonstrate an essential role for endothelial mCD14 that cannot be replaced by sCD14. Furthermore, we have provided evidence for a TRAM pathway in endothelium that is dependent on mCD14 even when other responses are no longer mCD14 dependent.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a Canadian Institutes for Health Research operating grant and group grant. P.K. is a Canadian Research Chair and an Alberta Heritage Foundation for Medical Research Scientist and the Snyder Chair in Critical Care Medicine.

² Address correspondence and reprint requests to Dr. Paul Kubes, Institute of Infection, Immunity and Inflammation, University of Calgary, 3330 Hospital Drive Northwest, Calgary AB T2N 4N1, Canada. E-mail address: pkubes{at}ucalgary.ca

³ Abbreviations used in this paper: LBP, LPS-binding protein; mCD14, membrane CD14; sCD14, soluble CD14; PI-PLC, phosphatidylinositol-phospholipase C; TIR, Toll/IL-1 receptor; TRIF, TIR domain-containing adaptor-inducing IFN-β; TRAM, TRIF-related adaptor molecule.