J. Pugin

Protection from septic shock by neutralization of macrophage migration inhibitory factor.

Identification of new therapeutic targets for the management of septic shock remains imperative as all investigational therapies, including anti-tumor necrosis factor (TNF) and anti-interleukin (IL)-1 agents, have uniformly failed to lower the mortality of critically ill patients with severe sepsis. We report here that macrophage migration inhibitory factor (MIF) is a critical mediator of septic shock. High concentrations of MIF were detected in the peritoneal exudate fluid and in the systemic circulation of mice with bacterial peritonitis. Experiments performed in TNFα knockout mice allowed a direct evaluation of the part played by MIF in sepsis in the absence of this pivotal cytokine of inflammation. Anti-MIF antibody protected TNFα knockout from lethal peritonitis induced by cecal ligation and puncture (CLP), providing evidence of an intrinsic contribution of MIF to the pathogenesis of sepsis. Anti-MIF antibody also protected normal mice from lethal peritonitis induced by both CLP and Escherichia coli, even when treatment was started up to 8 hours after CLP. Conversely, co-injection of recombinant MIF and E. coli markedly increased the lethality of peritonitis. Finally, high concentrations of MIF were detected in the plasma of patients with severe sepsis or septic shock. These studies define a critical part for MIF in the pathogenesis of septic shock and identify a new target for therapeutic intervention.

CD14 Is a pattern recognition receptor

Septic shock caused by a diverse group of bacterial pathogens is a serious human disease. Recognition of bacterial envelope constituents is one mechanism used by mammalian cells to initiate responses leading to bacterial killing or, unfortunately, responses that also cause fatal septic shock. Here we show that CD14 plays a key role in initiating cell activation by a group of bacterial envelope components from Gram-negative and Gram-positive microorganisms, as well as mycobacteria. We propose that CD14 is a receptor used by mammalian cells to recognize and signal responses to a diverse array of bacterial constituents. This finding defines the molecular basis for innate microbial immunity; implicit in these findings are new possibilities for therapeutics.

PHAGOCYTOSIS OF GRAM-NEGATIVE BACTERIA BY A UNIQUE CD14-DEPENDENT MECHANISM

THP‐l‐derived cell lines were stably transfected with constructs encoding glycophosphatidylinositol (GPI) ‐anchored or transmembrane forms of human CD14. CD14 expression was associated with enhanced phagocytosis of serum (heat‐inactivated) ‐opsonized Escherichia coli(opEc). Both the CPI‐anchored and transmembrane forms of CD14 supported phagocytosis of opEc equally well. Lipopolysaccharide‐binding protein (LBP) played a role in CD14‐dependent phagocytosis as evidenced by inhibition of CD14‐dependent phagocytosis of opEc with anti‐LBP monoclonal antibody (mAb) and by enhanced phagocytosis of E. coliopsonized with purified LBP. CD14‐dependent phagocytosis was inhibited by a phosphatidylinositol (PI) 3‐kinase inhibitor (wortmannin) and a protein tyrosine kinase inhibitor (tyrphostin 23) but not a protein kinase C inhibitor (bisindolylmaleimide) or a divalent cation chelator (ethylenediaminetetraacetate). Anti‐LBP mAb 18G4 and anti‐CD 14 mAb 18E12 were used to differentiate between the pathways involved in CD14‐dependent phagocytosis and CD14‐dependent cell activation. F(ab′)2 fragments of 18G4, a mAb to LBP that does not block cell activation, inhibited ingestion of opEc by THP1‐wtCD14 cells. 18E12 (an anti‐CD14 mAb that does not block LPS binding to CD14 but does inhibit CD14‐dependent cell activation) did not inhibit phagocytosis of LBP‐opEc by THP1‐wtCD14 cells. Furthermore, CD14‐dependent phagocytosis was not inhibited by anti‐CD 18 (CR3 and CR4 β‐chain) or anti‐Fcγ receptor mAb. J. Leukoc. Biol. 62: 786–794; 1997.

Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro.

Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 0111:B4 LPS on movement of 14C-BSA across bovine pulmonary artery EC monolayers. In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations > or = 0.5 ng/ml, and LPS (10 ng/ml) exposures of > or = 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 micrograms/ml failed to increase 14C-BSA flux at 6 h. The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold. LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of > or = 2.5%. LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function. LPS-LBP had a more rapid onset than did LPS-sCD14. The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone. These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface. However, in the presence of serum both molecules are required.

Human endothelial cell responses to endotoxin are dramatically enhanced by human blood

Vascular endothelia activated by endotoxin (lipopolysaccharide, LPS) or cytokines actively participate in local and systemic inflammation such as those observed during Gram-negative sepsis, by secreting cytokines and chemokines, and upregulating adhesions molecules for blood leukocytes. LPS can directly activate endothelial cells via its interaction, with soluble CD14, but may also activate endothelial cell indirectly through LPS-induced, monocyte-derived cytokines. Here we show that the addition of blood cells to plasma rendered the endothelial cells sensitive to LPS concentrations 1000 times lower than when only plasma was present. We demonstrated that this effect was carried by monocytes and more precisely by soluble factors produced by monocytes, and they were further identified as tumor necrosis factor (TNF) and interleukin-1 (IL-1).