The three-dimensional structure of the ligand-binding region of human E-selectin has been determined at 2.0 A resolution. The structure reveals limited contact between the two domains and a coordination of Ca2+ not predicted from other C-type lectins. Structure/function analysis indicates a defined region and specific amino-acid side chains that may be involved in ligand binding. These features of the E-selectin/ligand interaction have important implications for understanding the recruitment of leukocytes to sites of inflammation.

A novel technique involving radiolabeled monoclonal antibodies was used to characterize and compare the expression of E- and P-selectin on unstimulated, histamine-challenged, and endotoxin-challenged endothelial cells in various tissues of the mouse. Under unstimulated conditions, E-selectin was absent in all organs, but significant expression of P-selectin was observed in several organs. Histamine induced a rapid time-dependent upregulation of P-selectin, with the largest responses observed in mesentery and lung. Significant fold elevations in P-selectin expression occurred as early as 5 minutes after the histamine injection and remained elevated up to 1 hour. Histamine-induced P-selectin upregulation was inhibited by the H 1 receptor antagonist diphenhydramine, whereas the H 2 receptor antagonist cimetidine had no effect. Endotoxin (lipopolysaccharide [LPS]) also induced a time-dependent expression of P-selectin that reached a maximum between 4 and 8 hours after endotoxin administration. LPS-induced upregulation of P-selectin was greatest in heart and stomach, which exhibited insignificant constitutive expression of P-selectin. LPS also induced a time-dependent upregulation of E-selectin, with maximal expression occurring 3 to 5 hours after intraperitoneal administration. The lung and small intestine exhibited the largest responses to LPS challenge. Histamine administration did not affect E-selectin expression in any tissue. E- and P-selectin–deficient mice were used to test the specificity of monoclonal antibody binding in unstimulated, histamine-challenged, and LPS-stimulated tissues. Vascular binding of the radiolabeled E-selectin and P-selectin monoclonal antibodies was not observed in the respective deficient mice. These findings suggest that P-selectin is constitutively expressed on vascular endothelium in some tissues of the mouse and that there are significant regional differences in the magnitude and time course of histamine- and endotoxin-induced P-selectin expression. In contrast, E-selectin appears to be absent on unstimulated vascular endothelium but is upregulated within 3 hours after the administration of endotoxin in most tissues.

No abstract

SummaryLeukocyte recruitment during inflammation is achieved through a multistep paradigm that includes margination, selectin-mediated rolling, 82 integrin-mediated firm adhesion, emigration, and migration into the site of inflammation. We have used the mouse cremaster muscle as a model of trauma-and cytokine-induced inflammation to study the possible role of intercellular adhesion molecule (ICAM) 1 in leukocyte rolling using gene-targeted mice deficient in ICAM-1, P-selectin, and a combination of P-selectin and ICAM-1. Rolling flux and average leukocyte rolling velocity in ICAM-l-deficient mice was not different from wild-type mice, but P-selectin/ICAM-l-deficient mice showed a total absence of rolling for at least 2 h after surgical trauma. Rolling in both wild-type and ICAM-l-deficient mice 60-120 min after trauma was significantly inhibited by a P-selectin monoclonal antibody (mAb) (RB40.34). In contrast, an mAb (KAT-1) blocking ICAM-1 binding to leukocyte function-associated antigen 1 did not block residual rolling in P-selectin-deficient mice. TNF-0t induced leukocyte rolling in P-selectin/ICAM-l-deficient mice, but the rolling flux fraction was significantly lower than in TNF-0~-treated ICAM-l-deficient mice. Leukocyte rolling in P-selectin/ICAM-l-deficient mice treated with TNF-ot for 3 h was completely blocked by an E-selectin mAb (9A9E3), and partially by an L-selectin mAb . This clearly demonstrates E-selectin-dependent rolling in vivo. Leukocyte rolling velocities were significantly reduced after TNF-o~ treament and were similar in wild-type and gene-targeted strains. We conclude that the residual traumainduced leukocyte rolling seen in P-selectin-deficient mice is completely abolished by concomitant ICAM-1 deficiency. This severe defect in leukocyte rolling may explain the absence of leukocyte recruitment into the inflamed peritoneal cavity of P-selectin/ICAM-l-deficient mice at early time points (~<4 h).T he recruitment of leukocytes into a site of inflammation is pivotal to the eventual successful defense and subsequent healing of the host organism. This recruitment is achieved through a multistep paradigm that includes hemodynamic and rheologic margination in the vasculature, transient adhesive interactions with the vascular endothelium, resulting in leukocyte rolling, firm adhesion to the vascular endothelium, emigration through the vascular wall into the extracellular matrix, and the ensuing chemotactic migration to the inflammatory locale (1-3). With the exception of rheological margination, which is thought to be essentially nonlimiting, the aforementioned steps are mediated by specific interactions between constitutive and inducible adhesion molecules found on the leukocyte and the vascular endothelium.Leukocytes from patients with the clinical syndrome leukocyte adhesion deficiency type II, which is characterized by a defect in fucose metabolism and the inadequate recruitment ofgranulocytes into sites of inflammation, have a reduced ability to roll along vascular endothelium treated with th...

Tissue injury that accompanies hypoxemia/reoxygenation shares features with the host response in inflammation, suggesting that cytokines, such as IL-1, may act as mediators in this setting. Human endothelial cells (ECs) subjected to hypoxia (Po2 12-14 Torr) elaborated IL-1 activity into conditioned media in a time-dependent manner; this activity was completely neutralized by an antibody to IL-la. Production of IL-1 activity by hypoxic ECs was associated with an increase in the level of mRNA for IL-ia, and was followed by induction of endothelial-leukocyte adhesion molecule-1 and enhanced expression of intercellular adhesion molecule-1 (ICAM-1 ) during reoxygenation. During reoxygenation there was a three-to fivefold increased adherence of leukocytes, partly blocked by antibodies to endothelial-leukocyte adhesion molecule-1 and ICAM-1. Suppressing endothelial-derived IL-1, using either antibodies to IL-la, specific antisense oligonucleotides or the IL-1 receptor antagonist, decreased leukocyte adherence to reoxygenated ECs, emphasizing the integral role of IL-1 in the adherence phenomenon. Mice subjected to hypoxia (Po2 30-40 Torr) displayed increased plasma levels of IL-la, induction of IL-ia mRNA in the lung, and enhanced expression of ICAM-1 in pulmonary tissue compared with normoxic controls. These data suggest that hypoxia is a stimulus which induces EC synthesis and release of IL-la, resulting in an autocrine enhancement in the expression of adhesion molecules. (J.