Anti-CD14 Anticorps [MEM-18] (A85711)

Sepsis is a systemic disease resulting from harmful host response to bacterial infections. During the exacerbation of severe sepsis or septic shock, apoptosis of endothelial cells is induced in susceptible organs such as the lung and liver and triggers microcirculatory disorder and organ dysfunction. LPS, an outer membrane component of Gram-negative bacteria, is one of the major virulence factors for the pathogenesis. We previously reported that LL-37, a human anti-microbial cathelicidin peptide, potently neutralizes the biological activity of LPS and protects mice from lethal endotoxin shock. However, the effect of LL-37 on the LPS-induced endothelial cell apoptosis remains to be clarified. In this study, to further elucidate the action of LL-37 on severe sepsis/endotoxin shock, we investigated the effects of LL-37 on the LPS-induced endothelial cell apoptosis in vitro and in vivo using lung-derived normal human microvascular blood vessel endothelial cells (HMVEC-LBls) and D-galactosamine hydrochloride (D-GalN)-sensitized murine endotoxin shock model. LL-37 suppressed the LPS-induced apoptosis of HMVEC-LBls. In addition, LL-37 inhibited the binding of LPS possibly to the LPS receptors (CD14 and toll-like receptor 4) expressed on the cells. Thus, LL-37 can suppress the LPS-induced apoptosis of HMVEC-LBls via the inhibition of LPS binding to the cells. Furthermore, LL-37 drastically suppressed the apoptosis of hepatic endothelial cells as well as hepatocytes in the liver of murine endotoxin shock model. Together, these observations suggest that LL-37 could suppress the LPS-induced apoptosis of endothelial cells, thereby attenuating lethal sepsis/endotoxin shock.

Here we report that soluble CD14 isolated from the urine of nephrotic patients (uCD14) contains a potent cytokine inducing activity. CD14 derived from urine appeared to consist of two major polypeptides of about 54 and 48 kDa. In uCD14 isolated from three different nephrotic patients the cytokine-inducing activity appeared to co-migrate with the 48-kDa polypeptide which upon sequencing had the same N-terminal sequence as native CD14. Treatment of human monocytes and the human astrocytoma cell line U373 with uCD14 resulted in a strong secretion of tumor necrosis factor (TNF) and interleukin-6, respectively. The cytokine-inducing activity of the uCD14 preparations was unaffected by the absence of serum. This is in contrast to the activation of human monocytes and U373 cells by lipopolysaccharide (LPS) which is highly dependent on the presence of serum. The cytokine-inducing activity was not affected by LPS-binding protein (LBP) or polyclonal rabbit antibodies against LBP. The TNF-inducing activity of uCD14 was also heat labile in contrast to the cytokine-inducing activity of LPS, which was relatively heat resistant. The results suggest that CD14 may exist in at least two forms of which one is involved in cytokine induction.

Two monoclonal antibodies, MEM-15 and MEM-18, were prepared which recognize a monocyte 53-kDa antigen. A soluble form of this antigen was found in most normal sera and in urine of some nephrotic patients. Milligram amounts of this glycoprotein were isolated by immunoaffinity chromatography from urine and used for quantitative amino acid and carbohydrate analysis and N-terminal amino acid sequencing. The reactivity of the isolated antigen with several previously described monoclonal antibodies indicates that it is the antigen previously called MY-4 or MY-23. A significant homology exists between the sequenced N-terminal portion of the antigen and sequences found in the kinase-related transforming protein src and in the precursor of epidermal growth factor.

The carboxy-terminal amino acid sequence of the soluble form of the 53,000 mol. wt monocyte surface antigen, CD14, was determined by carboxypeptidase Y digestion and compared with the complete amino acid sequence of this protein as predicted from the structure of cloned cDNA [Goyert et al. Science 239, 497-500 (1988)]. The soluble antigen isolated from urine appears to lack eight C-terminal amino acid residues predicted for the full-size translation product, but possesses a major part of the C-terminal hydrophobic domain originally suggested as the membrane-spanning segment. The CD14 antigen can be removed from the monocyte surface by phosphatidylinositol-specific phospholipase C treatment, indicating that this glycoprotein is anchored in the membrane by a phospholipid and is not a transmembrane protein. The soluble form occurring in serum and in supernatants of cultured monocytes thus probably arises by phospholipase-mediated cleaving off the cell surface antigen. A sensitive sandwich ELISA was developed using a monoclonal anti-CD14 antibody, MEM-18, and polyclonal rabbit anti-CD14 antiserum for quantitation of the soluble antigen concns in sera and cell culture supernatants. Using this assay, the antigen present in the supernatant of phospholipase treated peripheral blood mononuclear cells could be estimated. The assay was also used for estimation of the concns of the soluble form of the CD14 antigen in human sera.