Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharide-binding protein (LBP)-dependent extraction and transfer of individual endotoxin molecules to CD14 in Toll-like receptor 4 (TLR4)-dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gram-negative bacteria and shed membrane vesicles ("blebs"). However, the ability of LBP and CD14 to efficiently promote TLR4-dependent cell activation by membrane-associated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membrane-associated endotoxin with LBP, CD14, and endotoxin-responsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI > 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP-, CD14-, and TLR4/MD-2-dependent fashion, but the blebs were 3-10-fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBPdependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin⅐sCD14 reacts with MD-2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane.

Preeclampsia is a prevalent and potentially devastating disorder of pregnancy. Characterized by a sudden spike in blood pressure and urinary protein levels, it is associated with significant obstetric complications. BPH/5 is an inbred mouse model of preeclampsia with borderline hypertension before pregnancy. BPH/5 mice develop hypertension, proteinuria, and endothelial dysfunction during late gestation (after E14.5). We hypothesized that BPH/5 mice might exhibit early feto-placental abnormalities before the onset of maternal disease. All placental cell lineages were present in BPH/5 mice. However, the fetal and placental weights were reduced, with abnormalities in all the placental zones observed starting early in gestation (E9.5-E12.5). The fractional area occupied by the junctional zone was significantly reduced at all gestational timepoints. Markedly fewer CDKN1C-stained trophoblasts were seen invading the proximal decidual zone, and this was accompanied by reductions in Cdkn1c gene expression. Trophoblast giant cell morphology and cytokeratin staining were not altered, although the mRNA levels of several giant cell-specific markers were significantly downregulated. The labyrinth layer displayed decreased branching morphogenesis of endothelial cells, with electron microscopy evidence of attenuated trophoblast layers. The maternal decidual arteries showed increased wall-to-lumen ratios with persistence of actin-positive smooth muscle cells. These changes translated into dramatically increased vascular resistance in the uterine arteries, as measured by pulse-wave Doppler. Collectively, these results support the hypothesis that defects at the maternal-fetal interface are primary causal events in preeclampsia, and further suggest the BPH/5 model is important for investigations of the underlying pathogenic mechanisms in preeclampsia.

Bacterial invasion triggers sensitive, specific molecular alarms that result in the mobilization of host defenses designed to recognize and eliminate invading bacteria and their remnants. The major inducer of host responses to Gram-negative bacteria (GNB) 1 is endotoxin, i.e. lipopolysaccharide, LPS, the unique glycolipid that comprises the bulk of the outer leaflet of the GNB outer membrane (1). These amphipathic molecules are composed of a unique, conserved hydrophobic moiety, the lipid A region, that is a disaccharide of N-acetylglucosamine substituted with saturated fatty acids and attached to a highly charged acidic carbohydrate region of varying size and composition (1, 2). The chemical structure of endotoxin promotes the formation of highly ordered, but potentially malleable aggregated state(s). Interaction with host endotoxin-binding proteins alters the physical presentation of endotoxin and its ability to activate cell responses (2-5). The cumulative work of many laboratories has implicated lipopolysaccharide-binding protein (LBP), CD14, Toll-like receptor 4, and MD-2 as key factors in cell activation by LPS (3, 6 -13). LBP facilitates delivery of LPS to both membrane-bound, GPI-linked (mCD14) and soluble CD14 (sCD14) (14 -19). Through interaction with CD14, LPS activates cells via a transmembrane receptor capable of promoting signal transduction. This recognition/response cascade includes the Toll-like receptor family of proteins, most notably Toll-like receptor 4, that serve as the primary mediator of endotoxin signaling (20, 21).The host also utilizes defense mechanisms that blunt endotoxin-triggered inflammatory responses by eliminating viable GNB and by neutralizing endotoxin. One potent host protein that blocks LPS activity is bactericidal/permeability increasing protein (BPI), a basic protein residing in azurophilic granules of polymorphonuclear (PMN) leukocytes and in the extracellular fluid of PMN-rich inflammatory exudates (22). BPI efficiently neutralizes LPS and is also potently cytotoxic and opsonic, especially toward GNB (22)(23)(24)(25). Although the functional properties of LBP and BPI differ markedly, they share 45% amino acid identity, are encoded within the same region of chromosome 20, and belong to a family of lipid-binding proteins that include phospholipid transfer protein and cholesteryl ester transfer protein (26 -30). The three-dimensional crystal structure for BPI reveals an unusual "hinged" two-domain boomerang-like molecular structure. The extensive sequence homology between LBP and BPI predicts a nearly superimposable threedimensional structure for LBP (30) suggesting that despite their different bioactivities, BPI and LBP have a similar organization of structure and function.In support of this view, studies on the interaction of LBP and BPI with LPS have demonstrated that the NH 2 -terminal domain of each protein is responsible for binding LPS (28,(31)(32)(33)(34). In BPI, both affinity for LPS and antibacterial activity are concentrated in this portion of the protein (31-...

Platelet activation in response to dual stimulation with collagen and thrombin results in the formation of a subpopulation of activated platelets known as coated platelets. Coated platelets are characterized by high surface levels of ␣-granule proteins and phosphatidylserine, which support the assembly of procoagulant protein complexes. Using murine models, we tested the hypothesis that the collagen receptor-associated molecule FcR␥ and the transglutaminase factor XIIIA are required for the formation of coated platelets. Following dual stimulation with the collagen receptor agonist convulxin and thrombin, 68% of platelets from C57BL/6 mice acquired the coated platelet phenotype, defined by high surface levels of fibrinogen and von Willebrand factor and decreased binding of the ␣IIb␤3 activation-dependent antibody PE-JON/A. In FcR␥ ؊/؊ mice, only 10% of platelets became "coated" after dual stimulation with convulxin plus thrombin (P < .05 vs C57BL/6 platelets). Decreased coated platelet formation in FcR␥ ؊/؊ platelets was accompanied by decreased annexin V binding (P < .01) and decreased platelet procoagulant activity (P < .05). Platelets from FXIIIA ؊/؊ mice did not differ from control platelets in coated platelet formation or annexin V binding. We conclude that FcR␥, but not factor XIIIA, is essential for formation of highly procoagulant coated platelets following dual stimulation with collagen and thrombin.