Endotoxin shock is the result of activation of the immune system by endotoxin/LPS, a component of Gram-negative bacteria. CD14, a GPI-anchored glycoprotein expressed strongly by monocyte/macrophages, is one of several receptors for endotoxin/LPS. The role of CD14 in bacterial-induced and LPS-induced shock was tested in CD14-deficient mice produced by gene targeting in embryonic stem cells. CD14-deficient mice were found to be highly resistant to shock induced by either live Gram-negative bacteria or LPS; however, at very high concentrations of LPS or bacteria, responses through non-CD14 receptors could be detected. Surprisingly, CD14-deficient mice also showed dramatically reduced levels of bacteremia, suggesting an unexpected role for CD14 in the dissemination of Gram-negative bacteria.

In vitro studies have previously shown that the myelomonocytic differentiation antigen CD14 is a receptor for a complex consisting of lipopolysaccharide (LPS) and LPS-binding protein. To investigate the role of CD14 in vivo and its relationship to induction of LPS-induced endotoxin shock, transgenic mice expressing human CD14 were produced. These mice express human CD14 strongly on the surface of their monocytes, neutrophils, and Thy-l(+) lymphocytes and are hypersensitive to LPS, as evidenced by their increased susceptibility to endotoxin shock. These results document the importance of CD14 in vivo as a primary mediator of this lethal syndrome. Furthermore, these mice provide an important model for testing the therapeutic effects of agents directed specifically against the human, as opposed to the murine, CD14 protein in preventing LPS-induced endotoxin shock.Endotoxin shock is a life-threatening syndrome usually preceded by a Gram-negative infection, which is frequently followed by bacteremia and the release of endotoxin [lipopolysaccharide (LPS)] into the circulation. The endotoxin or LPS is responsible for initiating a cascade of events, including an initial release of cytokines and activation of the coagulation, fibrinolytic, and complement systems, eventually leading to severe pathological sequelae (for review, see ref. 1). Recent studies are beginning to unravel the molecular events occurring in the initial stages ofthis syndrome. In vitro studies have shown that LPS binds to a serum protein, LPS-binding protein (LBP); this complex then binds to a receptor, CD14, present primarily on the surface of monocytes, triggering these cells to become activated and to release cytokines, including tumor necrosis factor a, a primary mediator of the precipitating events occurring in endotoxin shock (2-5). Such activation occurs with very low levels of LPS (>1 ng/ml), similar to circulating levels described in patients undergoing endotoxin shock. Identification of CD14 as a receptor for this complex by using in vitro techniques strongly suggests that CD14 may be a primary mediator of the initial events occurring in vivo in endotoxin shock.CD14 is a 53-to 55-kDa glycoprotein expressed strongly on the surface of monocytes (6-8). Detailed immunofluorescence and biochemical studies have shown that CD14 is also synthesized and expressed by neutrophils, although at lower levels (refs. 9-12 and A.H., B.Z.T., and S.M.G., unpublished work). We previously cloned both the human and murine CD14-encoding genes (13-15); examination of their deduced amino acid sequences revealed that CD14 is not structurally related to any other known receptor proteins identified to date, although it does contain a series of leucinerich repeats found in a number of unrelated proteins from diverse species. CD14 does, however, share one property with several other membrane proteins-i.e., it is anchored to the membrane by linkage to glycosyl-phophatidylinositol (GPI) (16); treatment with phosphatidylinositolphospholipase C (P1-PLC), which cleav...

Kidney transplantation is the most successful treatment option for patients with end-stage renal disease, and chronic antibody-mediated rejection is the principal cause of allograft loss. Predictive factors for chronic rejection include high levels of HLA alloantibodies (particularly HLA class II) and activation of graft endothelial cells (ECs). The mechanistic basis for this association is unresolved. We used an experimental model of HLA-DR antibody stimulation of microvascular ECs to examine the mechanisms underlying the association between HLA class II antibodies, EC activation and allograft damage. Activation of ECs with the F(Ab 0 ) 2 fragment of HLA-DR antibody led to phosphorylation of Akt, ERK and MEK and increased IL-6 production by ECs cocultured with allogeneic peripheral blood mononuclear cells (PBMCs) in an Akt-dependent manner. We previously showed that HLA-DR-expressing ECs induce polarization of Th17 and FoxP3 bright regulatory T cell (Treg) subsets. Preactivation of ECs with anti-HLA-DR antibody redirected EC allogenicity toward a proinflammatory response by decreasing amplification of functional Treg and by further increasing IL-6-dependent Th17 expansion. Alloimmunized patient serum containing relevant HLA-DR alloantibodies selectively bound and increased EC secretion of IL-6 in cocultures with PBMCs. These data contribute to understanding of potential mechanisms of antibody-mediated endothelial damage independent of complement activation and FcR-expressing effector cells.