Endothelial cell adhesion molecule expression in gene-targeted mice.

Gene-targeted mice are now routinely employed as tools for defining the contribution of different leukocyte and endothelial cell adhesion molecules to the leukocyte recruitment and tissue injury associated with acute and chronic inflammation. The objective of this study was to determine whether gene-targeted mice that are deficient in CD11/CD18, intracellular adhesion molecule-1 (ICAM-1), or P-selectin exhibit an altered constitutive or induced expression of the endothelial cell adhesion molecules E- and P-selectin. The gene-targeted mice were all developed in the 129Sv mouse strain and backcrossed into C57B1/6J mice. The number of backcrosses ranged between 8 (P-selectin) and 10 (CD18 and ICAM-1) generations. The dual-radiolabeled monoclonal antibody technique was used to quantify E- and P-selectin expression in different vascular beds. In the unstimulated state, E-selectin expression was significantly elevated (relative to wild-type mice) in the stomach, large intestine, and brain of mutants deficient in ICAM-1. In general, constitutive expression of P-selectin did not differ between wild-type, ICAM-1-deficient, and CD11/CD18-deficient mutants. In CD11/CD18-deficient mice, tumor necrosis factor-alpha (TNF-alpha) administration elicited a more profound upregulation of P-selectin in several vascular beds, compared with wild-type and ICAM-1-deficient mice. E-selectin expression in brain of TNF-alpha-stimulated, ICAM-1-deficient, and P-selectin-deficient mice was attenuated compared with wild-type mice. These findings indicate that chronic deficiency of some of the adhesion glycoproteins that mediate leukocyte recruitment alters basal and induced surface expression of other adhesion molecules on endothelial cells.

Generation and characterization of a novel adhesion function blocking monoclonal antibody recognizing both rat and mouse E-selectin.

The prerequisite for the recruitment of circulating leukocytes to sites of inflammation is adhesion to vascular endothelial cells. Selectins play a significant role in the initiation of this multistep process by mediating "rolling" of the leukocytes on the endothelium. Investigation of selectin-dependent cell interactions using function blocking monoclonal antibodies (MAb) provides insights into the mechanisms involved in leukocyte migration into inflammation. Until now most studies in inflammation models in rats have relied on cross-reactive or polyclonal antibodies against rat E-selectin. In an E-selectin knockout mouse, we aimed to generate an adhesion function blocking MAb to rat E-selectin by immunization with rat E-selectin transfected Chinese hamster ovary cells (RESEC). An IgG1 kappa MAb was identified that reacts with RESEC but not with untransfected Chinese hamster ovary cells, as well as with recombinant mouse E-selectin protein as assessed by ELISA. This MAb is designated RME-1. It does not cross-react with rat L-selectin or rat P-selectin or E-selectin expressed on human umbilical vein endothelium. Adhesion of the HL-60 myeloid cells to immobilized mouse E-selectin was completely inhibited by MAb RME-1 under static conditions and adhesion of rat polymorphonuclear leukocytes to recombinant mouse E-selectin was blocked under rotation condition. This novel antibody thus recognizes a function-related epitope on rodent E-selectin.

Characterization of a novel adhesion function blocking monoclonal antibody to rat/mouse P-selectin generated in the P-selectin-deficient mouse.

P-selectin is an important adhesion molecule involved in leukocyte migration. However, to date, no monoclonal antibodies (MAb) generated against rat P-selectin have been identified which block P-selectin mediated leukocyte adhesion. Most studies in the rat have utilized crossreacting antibodies generated against P-selectin in higher species. In a P-selectin deficient mouse we generated an anti-rat/mouse P-selectin MAb, designated RMP-1, by immunization with activated rat platelets. This IgG2a MAb immunoprecipitates a 140 kDa protein under reducing conditions from rat platelet lysate. By ELISA and immunofluorescence flow cytometry, MAb RMP-1 reacts with thrombin-activated but not unactivated rat platelets. In addition, by ELISA MAb RMP-1 binds to activated mouse platelets and recombinant rat and mouse P-selectin. MAb RMP-1 inhibited adhesion of HL-60 myeloid cells to immobilized mouse P-selectin by 97% and to activated rat and mouse platelets by 100% under static conditions, confirming the adhesion function blocking activity of MAb RMP-1. This novel MAb should be useful for studying P-selectin function in vitro and in vivo in both rat and mouse inflammation models.

Liver endothelial E-selectin mediates carcinoma cell adhesion and promotes liver metastasis.

E-selectin is a cytokine-inducible endothelial cell adhesion receptor which is involved in the process of leukocyte rolling, the first in a cascade of interactions leading to leukocyte transmigration. Several studies have implicated this receptor in carcinoma cell adhesion to the endothelium, an interaction thought to be required for tumor extravasation during metastasis. To study the role of this receptor in the process of metastasis, we utilized a murine carcinoma line H-59 which is highly metastatic to the liver in vivo. When adhesion of H-59 cells to primary cultures of murine hepatic endothelial cells was measured, it was found that the tumor cells had a low basal level of adhesion to the sinusoidal endothelial cells, which could be significantly and specifically augmented by pre-activation of the endothelial cells with rTNF alpha. This incremental increase in adhesion to the activated endothelium could be completely and specifically abolished by a neutralizing monoclonal antibody to murine E-selectin (MAb 9A9). Similar results were obtained with 2 highly metastatic human colorectal carcinoma lines, HM 7 and CX-1, but not with a second murine subline, M-27, which is poorly metastatic to the liver. To assess the role of E-selectin in metastasis to the liver in vivo, the effect of MAb 9A9 on experimental liver metastasis was evaluated using the syngeneic H-59 model. We show here that this antibody caused a marked, specific and Fc-independent inhibition of experimental liver metastasis, reducing the median number of metastases by 97% relative to the control groups. Our results provide evidence that endothelial E-selectin is a mediator of carcinoma metastasis to the liver.

Differential effect of E-selectin antibodies on neutrophil rolling and recruitment to inflammatory sites.

The selectins are inducible adhesion molecules critically important for the inflammatory response. We investigate here the functional effects of three monoclonal antibodies (MoAbs) raised against murine E-selectin (9A9, 10E6, and 10E9.6) on neutrophil recruitment in vivo, leukocyte rolling and circulating leukocyte concentrations in vivo, and adhesion of myeloid cells to E-selectin transfectants and recombinant E-selectin-IgG fusion protein in vitro. MoAbs 9A9 and 10E6 map to the lectin and epidermal growth factor (EGF)-like domains of murine E-selectin, whereas 10E9.6 binds to the consensus repeat region. 10E9.6 blocked neutrophil recruitment in a model of thioglycollate-induced peritonitis in Balb/c mice by more than 90% but had no effect in C57BL/6 mice. 9A9 and 10E6 blocked neutrophil recruitment in this assay only when combined with a P-selectin antibody, 5H1. Neither 9A9 nor 10E9.6 alone blocked leukocyte rolling in tumor necrosis factor-alpha-treated venules of Balb/c mice, but 9A9 almost completely inhibited leukocyte rolling when combined with the function-blocking murine P-selectin MoAb, RB40.34. In contrast, 10E9.6 had no effect on leukocyte rolling in RB40.34-treated Balb/c or C57BL/6 mice. 10E9.6 did not affect adhesion of myeloid cells to E-selectin transfectants or attachment, rolling, and detachment of myeloid cells to murine E-selectin-IgG fusion protein. However, adhesion was completely blocked in the same assays by 9A9. Taken together, these results indicate that E-selectin serves a function, other than rolling, that appears to be critically important for neutrophil recruitment to inflammatory sites in Balb/c mice.

Blocking both E-selectin and P-selectin inhibits endotoxin-induced leukocyte infiltration into the eye.

The initial contact between leukocytes and the vascular endothelium at sites of inflammation is mediated by selectins. The purpose of this study was to investigate the role of the two selectins expressed on the vascular endothelium, E-selectin and P-selectin, in the pathogenesis of endotoxin-induced uveitis. Endotoxin-induced uveitis was produced in female C3H/HeN mice using Salmonella typhimurium endotoxin injected into one hind footpad. At the time of endotoxin injection mice were treated with an intraperitoneal injection of a monoclonal antibody against E-selectin or P-selectin, a combination of both anti-selectin antibodies, or isotype-matched control antibodies. In a second set of experiments, antibody treatment was administered 6 hr after endotoxin injection, when inflammatory cells are already entering the eye. Ocular inflammation was graded histologically by a masked observer. When administered at the time of endotoxin injection, anti-P-selectin antibody decreased ocular inflammation by 37% compared to control animals (P = 0.05). There was no statistical decrease in ocular inflammation in animals treated with anti-E-selectin antibody. The combination of anti-P-selectin and anti-E-selectin antibodies decreased infiltrating inflammatory cells by 61% (P < 0.01). When treatment was delayed until 6 hr after endotoxin injection, the combination of anti-P-selectin and anti-E-selectin antibodies again decreased ocular inflammation by 60% (P < 0.01). Immunohistochemical staining showed decreased ICAM-1 expression in the eyes of animals treated with the combination of anti-P-and anti-E-selectin antibodies. Blocking both P-selectin and E-selectin resulted in a significant decrease in endotoxin-induced intraocular inflammation.

The association between alpha4-integrin, P-selectin, and E-selectin in an allergic model of inflammation.

In this study, we examined the relationship between the endothelial selectins (P-selectin and E-selectin) and whether they are critical for alpha4-integrin-dependent leukocyte recruitment in inflamed (late phase response), cremasteric postcapillary venules. Animals were systemically sensitized and 2 wk later challenged intrascrotally with chicken ovalbumin. Leukocyte rolling flux, adhesion, and emigration were assessed at baseline and 4 and 8 h postantigen challenge. There was a significant increase in leukocyte rolling flux, adhesion, and emigration in sensitized and challenged mice at both 4 and 8 h. At 8 h, the increase in leukocyte rolling flux was approximately 50% inhibitable by an anti-alpha4-integrin antibody, 98% inhibitable by fucoidin (a selectin-binding carbohydrate), and 100% inhibitable by an anti-P-selectin antibody. P-selectin-deficient animals displayed no leukocyte rolling or adhesion at 8 h after challenge. However, at 8 h there were many emigrated leukocytes in the perivascular space suggesting P-selectin-independent rolling at an earlier time point. Indeed, at 4 h postantigen challenge in P-selectin-deficient mice, there was increased leukocyte rolling, adhesion, and emigration. The rolling in the P-selectin-deficient mice at 4 h was largely alpha4-integrin dependent. However, there was an essential E-selectin-dependent component inasmuch as an anti-E-selectin antibody completely reversed the rolling, and in E-selectin and P-selectin double deficient mice rolling, adhesion and emigration were completely absent. These results illustrate that P-selectin underlies all of the antigen-induced rolling with a brief transient contribution from E-selectin in the P-selectin-deficient animals. Finally, the antigen-induced alpha4-integrin-mediated leukocyte recruitment is entirely dependent upon endothelial selectins.

E-selectin preferentially supports neutrophil but not eosinophil rolling under conditions of flow in vitro and in vivo.

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.

Selectins mediate eosinophil recruitment in vivo: a comparison with their role in neutrophil influx.

The role of selectins in mediating eosinophil recruitment in vivo was assessed in a model of lipopolysaccharide (LPS)-induced mouse pleurisy. LPS administration resulted in significant eosinophil influx at 24 hours, whereas neutrophil recruitment to the cavity peaked at 4 hours and persisted for 24 hours. The anti-L-selectin monoclonal antibody (MoAb) MEL-14 effectively inhibited (by 97%) eosinophil influx at 24 hours and also inhibited neutrophil recruitment at both times (75% to 95%). Eosinophil recruitment was partially reduced (54%) by the anti-P-selectin MoAb 5H1 but, in contrast, was unaffected by the anti-E-selectin MoAb 10E6. Neutrophil influx at 4 or 24 hours was not affected by the anti-P- or anti-E-selectin MoAbs. However, coadministration of anti-P-selectin and anti-E-selectin was very effective at inhibiting eosinophil influx at 24 hours (86%) and neutrophil influx at 4 (93%) and 24 hours (92%). These results show that all three selectins play a role in LPS-induced eosinophil and neutrophil recruitment in vivo, although P- and E-selectin show a degree of functional redundancy. The demonstration that P-selectin mediates eosinophil but not neutrophil influx suggests that suppressing the function of this adhesion molecule may be beneficial in blocking eosinophil accumulation in pleural inflammation.

Relative contribution of the selectins in the neutrophil recruitment caused by the chemokine cytokine-induced neutrophil chemoattractant (CINC).

Rat CINC induced a dose- and time-dependent accumulation of neurophils into murine air-pouches, a response which was inhibited by two selective H1-antagonists, mepyramine and triprolidine (approximately 60%). As pretreatment with fucoidin abolished CINC effect, the relative time-related contribution of selectins on this process was then investigated by using specific monoclonal antibodies (mAb). Anti-CD62L mAb gave a similar degree of inhibition of CINC-induced cell accumulation both at the 2h and 4h time-point (approximately 75%). Anti-CD62P mAb, but not the anti-CD62E mAb, inhibited PMN accumulation at 2h (65%), but only co-administration of these two mAbs inhibited the cell response to CINC at the 4h time-point (90%). Thus endogenous histamine, CD62L, CD62P, and CD62E, though to a different degree, are required for PMN extravasation observed in response to CINC administration.

Mouse bone marrow-derived mast cells roll on P-selectin under conditions of flow in vivo.

Increased numbers of mast cells are noted at sites of wound healing and inflammation. These mast cells are either recruited from the bone marrow or proliferate locally under cytokine stimulation. However, the molecular mechanisms mediating initial adhesive interactions between mast cell precursors and vascular endothelial cells are not well understood. We have used a syngeneic dorsal skinfold chamber model of microcirculation to study early events of mast cell-endothelial cell interactions by intravital fluorescence microscopy. Because "rolling" represents the earliest step of granulocyte adhesion under conditions of flow, our objective was to determine whether vascular selectins promote rolling of immature mouse bone marrow-derived mast cells (MBMMC) on endothelial cells lining murine blood vessels in vivo. In this study, titanium window chambers were implanted on the dorsal skinfolds of BALB/c mice. The passage of injected fluorescently labeled MBMMC within blood vessels of the striated skin muscle was observed by stroboscopic epi-illumination. As previously determined for other leukocytes, MBMMC were observed to roll in venules but not in arterioles or capillaries. Mice were also treated with neutralizing anti-E-selectin (mAb 9A9) and anti-P-selectin (mAb 5H1) antibodies and tested for their ability to block MBMMC rolling on venular endothelial cells. Intravenous administration of mAb 5H1 resulted in a marked decrease in MBMMC rolling, whereas mAb 9A9 and isotype matched control antibodies had no effect on the rolling flux of MBMMC. These studies represent the first identification of P-selectin as a rolling receptor for MBMMC, and demonstrate the use of a dorsal skinfold technique to study MBMMC-endothelial cell interactions under conditions of physiologic flow. Further studies will determine whether vascular selectins participate in the rolling and tissue recruitment of true circulating immature mast cell precursors in vivo.

Absence of trauma-induced leukocyte rolling in mice deficient in both P-selectin and intercellular adhesion molecule 1.

Leukocyte recruitment during inflammation is achieved through a multistep paradigm that includes margination, selectin-mediated rolling, beta 2 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-1-deficient mice was not different from wild-type mice, but P-selectin/ICAM-1-deficient mice showed a total absence of rolling for at least 2 h after surgical trauma. Rolling in both wild-type and ICAM-1-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-alpha induced leukocyte rolling in P-selectin/ICAM-1-deficient mice, but the rolling flux fraction was significantly lower than in TNF-alpha-treated ICAM-1-deficient mice. Leukocyte rolling in P-selectin/ICAM-1-deficient mice treated with TNF-alpha for 3 h was completely blocked by an E-selectin mAb (9A9E3), and partially by an L-selectin mAb (MEL-14). This clearly demonstrates E-selectin-dependent rolling in vivo. Leukocyte rolling velocities were significantly reduced after TNF-alpha treatment and were similar in wild-type and gene-targeted strains. We conclude that the residual trauma-induced 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-1-deficient mice at early time points (< or = 4 h).

E-selectin mediates leukocyte rolling in interleukin-1-treated rabbit mesentery venules.

The selectins are lectin-like cell surface glycoproteins that have been implicated in playing a crucial role in the initiation of leukocyte adhesion to endothelial cells (ECs) during inflammation. Binding of selectins under conditions of flow mediates leukocyte rolling, which in vivo is almost exclusively observed in venular microvessels. We have shown in previous experiments that intraperitoneal treatment of rabbits with interleukin-1 beta (IL-1) increases leukocyte rolling in exteriorized mesenteries. In the present study, we used immunohistochemistry of mesenteries and found that IL-1 induced a marked E-selectin immunoreactivity, preferentially in venules. We therefore hypothesized that the increased rolling in response to IL-1 may be related to the induction of E-selectin on venular ECs. Intravital microscopy was used to investigate interactions between leukocytes and ECs after intraperitoneal application of IL-1. The rabbit E-selectin monoclonal antibody (MoAb) 9H9 significantly reduced rolling of leukocytes by approximately 40%. Vehicle alone, class-matched control MoAb or the nonblocking anti-E-selectin MoAb 14G2 had no effect on rolling. These results indicate that leukocytes roll on inflamed venular ECs partly through interactions with E-selectin. Furthermore, we propose that the restricted E-selectin immunoreactivity by venular ECs contributes to the remarkable difference seen between arterioles and venules in exhibiting leukocyte rolling in vivo.

Consensus repeat domains of E-selectin enhance ligand binding.

To study the structural characteristics of E-selectin necessary for mediating cell adhesion, we examined the role of the consensus repeat (CR) domains in E-selectin function. Soluble constructs containing different numbers of CR domains were stably expressed in Chinese hamster ovary cells, purified to homogeneity, and characterized. The minimum functional unit of soluble E-selectin consisted of the lectin (Lec) and epidermal growth factor (EGF) domains alone (Lec-EGF) as indicated by its ability to mediate in vitro HL-60 cell adhesion. However, E-selectin containing all six CR domains (Lec-EGF-CR6) at its COOH terminus was the most potent in blocking neutrophil or HL-60 cell adhesion to either immobilized E-selectin or cytokine-stimulated human umbilical vein endothelial cells. This increased potency of Lec-EGF-CR6 in blocking cell adhesion was not due to CR-mediated oligomerization of the protein. Lec-EGF-CR6 was most likely monomeric in solution, as judged by gel filtration fast protein liquid chromatography, membrane ultrafiltration, and chemical cross-linking analysis. Therefore, although the lectin and EGF domains are necessary and sufficient for mediating cell adhesion, the additional six CR domains, present in native E-selectin, contribute to the enhanced binding of E-selectin to its ligand.

Insight into E-selectin/ligand interaction from the crystal structure and mutagenesis of the lec/EGF domains.

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.

Characterization of murine E-selectin expression in vitro using novel anti-mouse E-selectin monoclonal antibodies.

A recombinant phage containing the structural exons for mouse E-selectin has been isolated and characterized. Utilizing PCR techniques the lectin and egf domains were fused to form an artificial cDNA for expression in eukaryotic cells. Transient expression in COS cells demonstrated the lectin and egf domains were sufficient to mediate the binding of mouse and human neutrophils as well as HL60 cells. Recombinant soluble mouse E-selectin was purified and used to immunize rats to generate mAbs specific to mouse E-selectin. A panel of mAbs directed against mouse E-selectin was characterized including five that inhibit the adhesion of HL60 cells or mouse neutrophils to COS cells expressing the mouse lectin/egf domains. These mAbs have been used to characterize the expression and function of E-selectin on cytokine stimulated eEnd.2 murine endothelial cells.

P- and E-selectin use common sites for carbohydrate ligand recognition and cell adhesion.

The selectins are a family of three calcium-dependent lectins that mediate adhesive interactions between leukocytes and the endothelium during normal and abnormal inflammatory episodes. Previous work has implicated the carbohydrate sialyl Lewis(x) (sLe(x); sialic acid alpha 2-3 galactose beta 1-4 [Fucose alpha 1-3] N-acetyl glucosamine) as a component of the ligand recognized by E- and P-selectin. In the case of P-selectin, other components of the cell surface, including 2'6-linked sialic acid and sulfatide (galactose-4-sulfate ceramide), have also been proposed for adhesion mediated by this selectin. We have recently defined a region of the E-selectin lectin domain that appears to be directly involved with carbohydrate recognition and cell adhesion (Erbe, D. V., B. A. Wolitzky, L. G. Presta, C. R. Norton, R. J. Ramos, D. K. Burns, R. M. Rumberger, B. N. N. Rao, C. Foxall, B. K. Brandley, and L. A. Lasky. 1992. J. Cell Biol. 119:215-227). Here we describe a similar analysis of the P-selectin lectin domain which demonstrates that a homologous region of this glycoprotein's lectin motif is involved with carbohydrate recognition and cell binding. In addition, we present evidence that is inconsistent with a biological role for either 2'6-linked sialic acid or sulfatide in P-selectin-mediated adhesion. These results suggest that a common region of the E- and P-selectin lectin domains appears to mediate carbohydrate recognition and cell adhesion.

Hypoxia-mediated induction of endothelial cell interleukin-1 alpha. An autocrine mechanism promoting expression of leukocyte adhesion molecules on the vessel surface.

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 approximately 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-1 alpha. Production of IL-1 activity by hypoxic ECs was associated with an increase in the level of mRNA for IL-1 alpha, 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 five-fold 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-1 alpha, 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 approximately 30-40 Torr) displayed increased plasma levels of IL-1 alpha, induction of IL-1 alpha 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-1 alpha, resulting in an autocrine enhancement in the expression of adhesion molecules.

Identification of an E-selectin region critical for carbohydrate recognition and cell adhesion.

E-selectin elicits cell adhesion by binding to the cell surface carbohydrate, sialyl Lewis X (sLe(x)). We evaluated the effects of mutations in the E-selectin lectin domain on the binding of a panel of anti-E-selectin mAbs and on the recognition of immobilized sLe(x) glycolipid. Functional residues were then superimposed onto a three-dimensional model of the E-selectin lectin domain. This analysis demonstrated that the epitopes recognized by blocking mAbs map to a patch near the antiparallel beta sheet derived from the NH2 and COOH termini of the lectin domain and two adjacent loops. Mutations that affect sLe(x) binding map to this same region. These results thus define a small region of the E-selectin lectin domain that is critical for carbohydrate recognition.