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The selectin family of molecules (L-, P-, and E-selectin) mediates adhesive interactions between leukocytes and endothelial cells required for recruitment of leukocytes to inflammatory sites. Soluble E-selectin levels are elevated in inflammatory diseases and act to promote neutrophil beta(2)-integrin-mediated adhesion by prolonging Ca(2+) mobilization. Although soluble E-selectin alone was unable to initiate Ca(2+) signaling, it allowed a novel "permissive" store-operative calcium entry (SOCE) following the initial platelet-activating factor (PAF)-induced release of Ca(2+) from inositol 1,4,5-trisphosphate (IP(3))-sensitive stores. This induction of permissive SOCE in response to soluble E-selectin and PAF was shown to act through a G protein-coupled receptor (GPCR) coupled to pertussis toxin-insensitive G(q/11). Furthermore, we demonstrated that permissive SOCE was mediated by canonical transient receptor potential channel (TRPC) due to its sensitivity to specific inhibition by MRS1845 and Gd(3+) and that TRPC6 was the principal TRPC family member expressed by human neutrophils. In terms of mechanism, we demonstrated that soluble E-selectin activated Src family tyrosine kinases, an effect that was upstream of phosphatidylinositol 3'-kinase in a signaling pathway that regulates permissive SOCE following exposure of neutrophils to PAF. In summary, this report provides the first evidence for communication between an inflammatory mediator and adhesion receptors at a molecular level, through selectin receptor ligation allowing permissive SOCE to occur following PAF stimulation of human neutrophils.

To study rolling of mouse neutrophils on P- and E-selectins in whole blood and without cell isolation, we constructed an autoperfused flow chamber made from rectangular microslides (0.2x2 mm) perfused from a carotid artery catheter. A differential pressure transducer served to measure wall shear stress. Green fluorescent neutrophils rolled on P-selectin but not E-selectin coated at 50 ng/ml, with some rolling on E-selectin at 150 ng/ml. However, when P- and E-selectins were coimmobilized, the resulting number of rolling neutrophils was sixfold and fourfold higher than on P- or E-selectin alone. Velocity and flux analysis shows that P-selectin initiates neutrophil rolling, and a small amount of E-selectin, unable to capture many neutrophils, reduces the rolling velocity of all neutrophils by more than 90%. The unexpected synergism between E- and P-selectins explains why neutrophil recruitment is enhanced when both selectins are expressed.

Objective: To study granulocyte adhesion to E-selectin, VCAM-1 and ICAM-1 in patients with primary Sjögren's syndrome (pSS). In previous studies diminished neutrophil adhesion has been shown as measured by the nylon fiber method., Methods: Neutrophil and eosinophil adhesion to the adhesion molecules E-selectin, VCAM-1 and ICAM-1 were measured using transfected fibroblasts. The cell surface expression of the integrin proteins CD11a, CD11b, CD18 and CD29 on neutrophils was assayed by means of flow cytometry., Results: Neutrophils and eosinophils from patients with pSS had elevated basal adhesion in the presence of Mn2+ as compared with controls (basal adhesion was considered to be the adhesion to untransfected fibroblasts). Granulocyte adhesion to E-selectin was also elevated. No differences were seen between patients and controls in cell surface expression of the integrin proteins CD11a, CD11b, CD18 and CD29 on neutrophils, nor was there any difference in these parameters between patients with and without extra glandular symptoms., Conclusions: These results suggest that blood neutrophils and eosinophils are activated in pSS. Accordingly they do not confirm results from earlier studies of impaired neutrophil adhesion in pSS.

Appropriate recruitment of neutrophils to sites of infection or tissue injury is a key event in the inflammatory response. A number of studies have shown the critical role of selectins in tethering and rolling of neutrophils on vascular endothelium, as well as a more complex regulatory role, since they have the potential to alter leukocyte recruitment by triggering beta2 integrin-mediated adhesion. In this study, we report that in contrast to patients "at risk" of developing acute respiratory disease syndrome (ARDS), elevated plasma levels of soluble E-selectin are found in patients with established disease. Since neutrophil granulocytes are implicated in ARDS pathogenesis, we have investigated the possibility of a link between elevated soluble plasma E-selectin levels and disease progression by examining the effects of soluble recombinant E-selectin (E-zz) upon neutrophil function. In this paper, we describe the novel finding that exposure of neutrophils to E-zz potentiates a number of neutrophil functions which may act to drive inflammatory processes. Although neutrophil deformability, an important parameter determining retention within the lung microvasculature, was not affected by E-zz, neutrophil polarization was observed. In addition, neutrophil beta2 integrin-mediated adhesion was found to be augmented by E-zz without alteration in levels of surface expression of alphaMbeta2 or the "activation" reporter epitope defined by monoclonal antibody 24. Concomitantly with increased beta2 integrin-mediated adhesion, we observed an inhibition of formyl-Met-Leu-Phe-directed chemotaxis. Together with an augmentation of neutrophil reactive oxidant species production and release of superoxide anions, these data raise the possibility that soluble E-selectin exerts pro-inflammatory effects upon neutrophil function at sites of inflammation, thereby exacerbating disease processes.

Neutrophil emigration through endothelial cells under shear flow involves several adhesion processes including cell rolling, arrest, and transmigration. Rolling is mediated by selectins, while arrest and transmigration both require activated CD18 integrins. One mode of CD18 activation is via selectins expressed on neutrophils and endothelial cells. We have recently reported that cross-linking of L-selectin (CD62L) resulted in the rapid activation of CD18-dependent adhesion. In the current study, we examine whether binding of E-selectin (CD62E) and L-selectin can activate neutrophil CD18-dependent adhesion under shear flow. Human ICAM-1 (CD54) and E-selectin were co-transfected into L cells. Neutrophil capture, rolling, and arrest on these monolayers were quantitated in a parallel plate flow chamber at a wall shear stress of 2.0 dyne/cm2. Under these conditions, E-selectin supported cell capture and rolling on the monolayer, but did not trigger CD18-mediated cell arrest within 200 microm of rolling. However, when neutrophils were treated with anti-L-selectin mAb and cross-linked with a secondary mAb, approximately 50% of the cells arrested within 54 microm. Cell arrest was also observed in response to IL-8 stimulation. A subthreshold level of IL-8 in combination with L-selectin cross-linking potentiated the level of cell arrest due to either stimulus alone. The transition to cell arrest involved both LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Blocking either subunit alone failed to reduce arrest, while blocking both molecules with mAbs reduced the number to baseline levels. These data support the conclusion that L-selectin, but not E-selectin, can signal the transition from neutrophil rolling to cell arrest under shear flow.

The selectin family of adhesion molecules is composed of the L-, E-, and P-selectins, which promote leukocyte rolling during inflammation. Although E-selectin supports neutrophil and lymphocyte rolling, its ability to mediate eosinophil rolling under conditions of flow in vitro and in vivo has not been determined. Using function-blocking mAbs raised against rabbit E-selectin, we have determined whether E-selectin supports human eosinophil rolling in comparison to human neutrophil rolling in IL-1-stimulated rabbit mesenteric venules utilizing intravital microscopy. Anti-rabbit E-selectin mAbs 8B9 and 8G9 were found to inhibit neutrophil rolling but had no significant effect on eosinophil rolling. Likewise, mAb 8B9 F(ab')2 fragments were found to block neutrophil rolling, but did not significantly alter the flux of rolling eosinophils. Isotype-matched Abs and a nonblocking anti-rabbit E-selectin mAb 2A5 failed to inhibit both neutrophil and eosinophil rolling on venular endothelium. In support of these in vivo observations, significant numbers of human neutrophils but not eosinophils were found to avidly roll on monolayers of E-selectin transfectants under physiologic condition of flow in vitro. Under subphysiologic conditions of shear (0.17-0.5 dyn/cm2), eosinophils rolled on E-selectin, albeit in lower numbers (three- to sevenfold) compared with neutrophils. In addition, the rolling velocity of eosinophils was significantly higher compared with neutrophils on E-selectin transfectants. These studies suggest that at physiologic shear rates, E-selectin is likely to function as a major vascular adhesion receptor in mediating neutrophil but not eosinophil rolling in inflamed postcapillary venules.

Objective: Neutrophil deposition in tissues (leukosequestration) after shock may produce local tissue injury from proteases and high-energy oxygen species released from sequestered neutrophils. The initial step in the binding of neutrophils to capillary endothelium is the interaction of adhesion molecule (selectin) receptors between neutrophils and endothelial cells. We quantified leukosequestration in the tissues of burned rats using two methods of analysis: a) measurement of lung myeloperoxidase; and b) measurement of radiolabeled neutrophils and erythrocytes deposited in multiple tissues. We then determined the ability of a selectin receptor blocking agent to affect neutrophil deposition in tissues after burn injury., Design: Prospective, controlled, laboratory study., Setting: University research laboratory., Subjects: Male Wistar rats (200 to 300 g)., Interventions: After tracheostomy and venous cannulation, rats received 17% total body surface area full-thickness contact burns and were resuscitated with saline (20 mL i.p.). Experimental animals received 2 mg/kg body weight i.v. administration of a P- and E-selectin blocking monoclonal antibody, CY-1747, immediately after burn. Lung tissue neutrophils were estimated by measuring myeloperoxidase in lung tissue. Neutrophil retention in lung, liver, spleen, gut, skin, muscle, kidney, and brain tissues was determined by removing (preburn) and differentially radiolabeling neutrophils (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hrs after burn, and measuring tissue radioactivity 30 mins later. Edema was estimated by measuring extravasated 125 I-labeled albumin in the various tissues. Peripheral blood neutrophils were analyzed for intracellular hydrogen peroxide content, utilizing a fluorescent dye that reacts with hydrogen peroxide, coupled with analysis of cell fluorescence by flow cytometry., Measurements and Main Results: Myeloperoxidase concentration was increased in lungs 5 hrs after burn (p < .05), indicating neutrophil deposition. Radioisotope studies demonstrated significant (p < .05) leukosequestration into the lung, gut, kidney, skin, and brain tissues at 5 hrs after burn. Flow cytometry showed increased intracellular hydrogen peroxide content in peripheral blood neutrophils 5 hrs after burn. Tissue edema, manifested by radiolabeled albumin retention, was not seen in any tissues. Postburn neutrophil deposition in lungs and liver was blocked (p < .05) by administration of CY-1747 after burn, but maximal neutrophil hydrogen peroxide content was unaffected., Conclusion: Burn injury in rats results in accumulation of neutrophils in multiple tissues. Neutrophil deposition in the lungs and liver is blocked by administration of the E/P-selectin blocking antibody, CY-1747. Since sequestration of metabolically active neutrophils may induce tissue injury, therapies that block postburn leukosequestration may improve clinical outcomes by limiting remote tissue injury.