Recruitment of neutrophils to sites of inflammation is mediated in part by endothelial leukocyte adhesion molecule-1 (ELAM-1), which is expressed on activated endothelial cells of the blood vessel walls. ELAM-1 is a member of the LEC-CAM or selectin family of adhesion molecules that contain a lectin motif thought to recognize carbohydrate ligands. In this report, cell adhesion by ELAM-1 is shown to be mediated by a carbohydrate ligand, sialyl-Lewis X (SLex; NeuAc alpha 2,3Gal beta 1,4(Fuc alpha 1,3)-GlcNAc-), a terminal structure found on cell-surface glycoprotein and glycolipid carbohydrate groups of neutrophils.

No abstract

The leukocyte receptor CD62, which is expressed on activated platelets and endothelial cells, is shown to mediate cell adhesion by binding a sialylated carbohydrate structure, sialyl-Lewis x, found on neutrophils, monocytes, and tumor cells. This structure has previously been identified as the ligand for another member of the LEC-CAM family of cell adhesion molecules, endothelial cell-eukocyte adhesion molecule 1, which also binds neutrophils and monocytes. The results demonstrate that although the two LEC-CAMs differ in their biological activities by their distribution and mode of expression, they are capable of mediating cell adhesion by recognition of the same carbohydrate ligand.Leukocyte trafficking and recruitment to sites of inflammation are mediated by three adhesion receptor families, the integrin and immunoglobulin superfamilies and the recently described LEC-CAM, or selectin family (1)(2)(3)(4) (100 nm) (13). Neutrophils were isolated from fresh human blood by centrifugation at room temperature through mono-poly resolving medium (Flow Laboratories) followed by three washes in ice-cold Hanks' balanced salt solution (GIBCO) containing 20 mM Hepes (GIBCO) and 0.2% glucose (Fisher). Human umbilical vein endothelial cells were obtained as described (6). HL-60 cells were cultured in RPMI 1640 containing 10%6 fetal calf serum. Chinese hamster ovary (CHO-Ki) cells, glycosylation mutants , and the carcinoma line were cultured in the a modification of Eagle's minimal essential medium (a-MEM) containing ribonucleosides, deoxyribonucleosides, and 10%1o fetal calf serum.Assays for CD62-Mediated Adhesion to Activated Platelets. Two assays were used, a fluid phase assay and a plate assay. The fluid phase assay (see Figs. 2-4) was performed essentially as described (10) for PADGEM (CD62). Platelets at 2 x 108 per ml were activated with thrombin at 0.25 units/ml for 20 min at room temperature without stirring. Twenty microliters of the activated platelet suspension was then mixed with an equal volume ofa suspension ofthe cells (2 x 106 cells per ml) to be assessed for adhesion. Adhesion was evaluated microscopically and was scored as the percent ofthe test cells that had bound two or more platelets. The plate assay used was a modification of the endothelial cell-neutrophil adherence assay previously described (20). A suspension of platelets (300 ,l) at 108 per ml was applied to each well of a 48-well plate that had previously been coated with 0.1% gelatin. The Gal431-4 GlcNAc / FUCCY1 3 Le' Abbreviations: LEC-CAM, a family of cell adhesion molecules, each of which contains an N-terminal lectin domain followed by an epidermal growth factor-like domain and a series of consensus repeats similar to those found in complement regulatory proteins; ELAM-1, endothelial cell-leukocyte adhesion molecule

The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion ofthe mesentery with histamine (10-7-10-M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions. (J. Clin. Invest. 1994.93:1508-1515.) Key words: leuko-

Polymorphonuclear neutrophils are important effectors of injury in host defense and inflammation. Many inflammatory diseases are self-limiting, raising the possibility that compounds are generated in vivo during the course of inflammation that inhibit neutrophil recruitment and tissue destruction. Lipoxins, a more recent addition to the families of bioactive eicosanoids, are potential candidates in this regard. Lipoxins are generated via pathways that initially involve the dual lipoxygenation of arachidonic acid and are potent inhibitors of several neutrophil trafficking events in vitro. Here, we present evidence that lipoxin A4 is generated in rat kidneys during experimental immune complex-mediated glomerulonephritis in vivo. Renal lipoxin A4 levels were markedly reduced by prior depletion of animals of either neutrophils or platelets, suggesting that most lipoxin A4 generated in vivo was derived from transcellular biosynthetic pathways during platelet-neutrophil interactions. Electron microscopic examination of glomerulonephritic kidneys revealed areas of intimate contact between neutrophils and platelets within the lumen of glomerular capillaries. P-selectin on platelets is an important mediator of platelet-neutrophil adhesion in vitro and in vivo. Prior treatment of animals with a blocking monoclonal antibody (mAb) against P-selectin (mAb CY1747), but not an isotype-matched non-blocking control mAb (mAb PNB1.6), caused striking inhibition of lipoxin A4 generation without attenuating neutrophil recruitment. Anti-P-selectin mAb also blunted transcellular lipoxin A4 generation during coincubations of activated neutrophils and platelets in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

We have developed a therapeutic approach to wound repair involving immobilization of gene transfer vectors within biocompatible matrices (gene-activated matrix, or GAM). The matrix also serves as a scaffold for cellular in-growth and subsequent gene uptake and expression. An adenoviral vector encoding human platelet-derived growth factor-B delivered in collagen (AdPDGF-B/GAM) has demonstrated efficacy in models of wound repair. The safety, biodistribution, and immunogenicity profiles of AdPDGF-B/GAM were examined using a rabbit dermal wound model. Four weekly doses at 1 x 10(10) and 1 x 10(11) viral particles/cm2 of wound surface stimulated dose-related increases in granulation tissue formation and cell proliferation. In situ hybridization and immunostaining demonstrated concordant expression of human PDGF-B mRNA and protein. No treatment-related changes in hematology, serum chemistry, or histopathology were observed. Although AdPDGF-B DNA and PDGF-B mRNA were detected in wounds and axillary lymph nodes of treated animals, no AdPDGF-B was detected in blood or other organs. No immunologic responses against collagen were observed; however, as expected, IgG responses to AdPDGF-B and human PDGF-BB protein were detected. In adenovirus-preimmunized rats, attenuation of the wound healing response was modest (approximately 16%). Collectively, these observations indicate that repeat doses of AdPDGF-B/GAM are well tolerated and lead to robust, localized tissue repair.

Summary:The selectin family of glycoproteins facilitates the early phase of polymorphonuclear leukocyte adhesion to the endothelial cell and, thus, may promote ischemic cell damage, To evaluate E-selectin in the pathogenesis of focal cerebral ischemia and reperfusion injury, we cloned rat E-selectin cDNA and measured the temporal profiles E-selectin mRNA (Northern blot) and protein (immunohistochemistry) during (1 h of ischemia) and after (up to 1 week) transient (2 h) middle cerebral artery (MCA) occlusion in the male Wistar rat We also tested the effect on these rats of administration of CY-1503, an analog of sialyl Lewisx (SLeX), on ischemia cell dam age, mRNA for E-seJectin was first detected in the ischemic hemisphere at 2 h of reperfusion and persisted to 46 h of reperLeukocytes contribute to cerebral ischemic cell dam age (Hallenbeck et aI., 1986;del Zoppo et al., 1991; Garcia et a1., 1994; Zhang RL et aI., 1994a), Accumu lation of leukocytes in an ischemic lesion is a dynamic process that is sequentially regulated by selectin, integrin and immunoglobulin families of related adhesion mol ecules (Patarroyo et aI., 1990;Butcher, 1991; Bevilac qua, 1993). The initial leukocyte endothelial low-affinity interaction promoting leukocyte rolling is mediated by the se1ectin family, including L-selectin, P-selectin, and E-selectin, Subsequently, a high-affinity interaction be-

Several growth factor proteins have been evaluated as therapeutic agents for the treatment of chronic dermal wounds. Unfortunately, most have failed to produce significant improvements in wound healing, in part due to ineffective delivery and poor retention in the wound defect. It has been proposed that gene therapy might overcome the limitations of protein therapy via ongoing transcription and translation, thus prolonging the availability of the therapeutic protein. Reasoning that it would be of further benefit to ensure retention of the DNA vector as well as the therapeutic protein within the wound defect, we have evaluated matrix-enabled gene transfer for cutaneous wound repair (Gene Activated Matrix). Formulations consisting of bovine type I collagen mixed with adenoviral or plasmid gene vectors have been evaluated in 3 in vivo models. The therapeutic transgenes employed encode human platelet-derived growth factor-A or -B, proteins key to each phase of normal wound repair. Increased granulation tissue formation, vascularization, and reepithelialization have been shown compared to controls treated with collagen alone or collagen containing a reporter gene vector. Further enhancements of the tissue repair response have been achieved by combining matrix-enabled gene transfer with molecular targeting, in which the DNA vector is conjugated to a growth factor ligand (basic fibroblast growth factor). These promising results support the clinical evaluation of gene activated matrices for the treatment of chronic dermal wounds.