Characterization of the binding of soluble CD14 to human endothelial cells and mechanism for CD14-dependent cell activation by LPS

Abstract

The binding of soluble CD14 (sCD14) to human umbilical vein endothelial cells (HUVEC) was examined in order to understand the role of CD14 in potentiating LPS activity. Both purified sCD14 and [125I]-sCD14 potentiated LPS-stimulated ICAM-1 expression in HUVEC. This potentiation was blocked by anti-CD14 monoclonal antibodies (mAbs) 63D3, UCHM-1 and RM052. Saturation binding assay revealed that [125I]-sCD14 bound to HUVEC with a ligand-acceptor dissociation constant of 290 nM. The binding of [125 I]-sCD14 was inhibited by sCD14 with a sCD14-acceptor dissociation constant of 24 nM. The density of CD14 acceptors was estimated to be 4-8 x 105 sites per cell. The [125I]-sCD14 binding was inhibited by anti-human CD14 mAbs UCHM-1 and RM052 but not 63D3. The bound [125I]-sCD14 could be washed off by acid buffer, pH 3.0, and its localization on the cell membrane was confirmed by light microscopic autoradiography. Based on the previously published description of LPS-sCD14 interactions and our observation that anti-CD14 mAbs inhibiting sCD14-acceptor binding also blocked the potentiation of LPS activity by sCD14, we propose that bridging or crosslinking between the putative LPS-receptor complex with the sCD14-acceptor complex via LPS-sCD14 interactions is the mechanism of CD14-dependent activation of endothelial cells (EC) by LPS. Re-examination of the published data suggests that this mechanism is a universal one for EC, leukocytes and lymphocytes.