The bactericidal/permeability-increasing protein (BPI) is thought to play an important role in killing and clearance of Gram-negative bacteria and the neutralization of endotoxin. A possible role for BPI in clearance of cell-free endotoxin has also been suggested based on studies with purified endotoxin aggregates and blood monocytes. Because the interaction of BPI with cell-free endotoxin, during infection, occurs mainly in tissue and most likely in the form of shed bacterial outer membrane vesicles (“blebs”), we examined the effect of BPI on interactions of metabolically labeled ([14C]-acetate) blebs purified from Neisseria meningitidis serogroup B with either human monocyte-derived macrophages or monocyte-derived dendritic cells (MDDC). BPI produced a dose-dependent increase (up to 3-fold) in delivery of 14C-labeled blebs to MDDC, but not to monocyte-derived macrophages in the presence or absence of serum. Both, fluorescently labeled blebs and BPI were internalized by MDDC under these conditions. The closely related LPS-binding protein, in contrast to BPI, did not increase association of the blebs with MDDC. BPI-enhanced delivery of the blebs to MDDC did not increase cell activation but permitted CD14-dependent signaling by the blebs as measured by changes in MDDC morphology, surface expression of CD80, CD83, CD86, and MHC class II and secretion of IL-8, RANTES, and IP-10. These findings suggest a novel role of BPI in the interaction of bacterial outer membrane vesicles with dendritic cells that may help link innate immune recognition of endotoxin to Ag delivery and presentation.

Glycosphingolipids (GSLs) are complex macromolecules on cell membranes that have been shown to play a role in neutrophil differentiation, activation, phagocytosis, and adhesion to both microorganisms and vascular endothelium. Because GSLs are often cryptic antigens on cell membranes, little is known regarding GSL expression in early myelopoiesis. To study the latter, myeloblasts were collected from patients with acute nonlymphocytic leukemia (ANLL) who required therapeutic leukocytopheresis for hyperleukocytosis. The neutral GSLs were isolated and identified by high-performance thin-layer chromatography (HPTLC), HPTLC immunostaining, gas chromatography, nuclear magnetic resonance, and fast atom bombardment-mass spectrometry. Like mature peripheral blood neutrophils, myeloblasts expressed glucosylceramide, lactosylceramide, and the neolacto-family GSLs, lactotriaosylceramide and neolactotetraosylceramide. Unlike neutrophils and chronic myeloid leukemia, most ANLL samples also expressed the globo-series GSLs, globotriaosylceramide and globotetraosylceramide. Globo GSL expression was strongly associated with a myeloblastic (ANLL M0-M2) and monoblastic phenotype (M5). A weak association was also noted with expression of either lymphoid (P < .

Activated platelets are known to express P-selectin, a lectin-like adhesion receptor (CD62), through which they bind to sialyl Lewis X (sLex) ligands displayed on the membranes of leukocytes. To determine whether direct platelet-platelet interactions via P-selectin/sLex interactions are also possible, we have examined the ganglioside extract of human blood platelets for the presence of sLex ligands. Using the sensitive method of high-performance thin-layer chromatography (HPTLC)-immunostaining with the monoclonal antibody (mAb) CSLEX or with sialidase followed by mAbs MC480 or PM81, eight sLex bands were demonstrated at Rf 0.01, 0.03, 0.05, 0.06, 0.08, 0.10, 0.14 and 0.21 in the solvent 45:55:10 chloroform-methanol-aqueous 0.02% CaCl2. The sensitivity of all eight bands to sialidase or endoglycoceramidase confirmed that they were gangliosides. Comparison of the HPTLC mobilities and densities of platelet bands with those from five other human tissues (granulocytes, monoblasts, kidney, aortic endothelium and erythrocytes) in three different solvents revealed three major bands associated with platelets: 3 (Rf0.03), 6 (0.08) and 14 (0.21). Platelet bands were demonstrated not to have resulted from granulocyte contamination. Partial purification of platelet sLex gangliosides by high-performance liquid chromatography and their reaction with 14 oligosaccharide-specific mAbs (FH4, FH5, LM112-161, LM119-181, A5, 1B2, BR55-2, BE2, ES4, MC631, MH04, SH34, P001 and MC813-70) revealed that band 6 is a multifucosylated neolacto ganglioside and band 14 is a branched, disialo neolacto fucoganglioside. Platelet band 3 combined the features of both bands 6 and 14, and reacted differently than granulocyte band 3. These partial structures resemble gangliosides associated with adhesion in other cell systems. It is concluded that platelets express tissue-specific sLex gangliosides (sLex ligands). Thus, it is possible that platelet-platelet binding may be mediated at least partially through P-selectin/sLex interactions, especially after platelet activation.