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Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. While numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here, we describe the endothelial-derived secreted molecule, developmental endothelial locus-1 (Del-1), as an anti-adhesive factor that interferes with the integrin LFA-1-dependent leukocyte-endothelial adhesion. Endothelial Del-1-deficiency increased LFA-1-dependent leukocyte adhesion in vitro and in vivo. Del-1-/-mice displayed significantly higher neutrophil accumulation in LPS-induced lung inflammation in vivo, which was reversed in Del-1/LFA-1-double deficient mice. Thus, Del-1 is an endogenous inhibitor of inflammatory cell recruitment and could provide a basis for targeting leukocyte-endothelial interactions in disease.
Leukocyte extravasation is integral to the response to infection or injury and to inflammation and autoimmunity. Leukocyte recruitment comprises a well coordinated cascade of adhesive events including selectin-mediated rolling, firm adhesion of leukocytes to endothelial cells and their subsequent transendothelial migration. The interaction between LFA-1 (αLβ2, CD11a/CD18) and endothelial ICAM-1 is crucial during firm endothelial adhesion of leukocytes (1-5). Whereas numerous adhesion receptors promoting inflammatory cell recruitment have been identified, very little information exists about endogenous inhibitors of the leukocyte adhesion cascade (1-7). Developmental endothelial locus-1 (Del-1) is a glycoprotein that is secreted by endothelial cells and can associate with the endothelial cell surface and the extracellular matrix (8-10). Del-1 is regulated upon hypoxia or vascular injury and has been implicated in vascular remodelling during angiogenesis (10-12). Here, we sought to determine whether endothelial-derived Del-1 participates in leukocyte-endothelial interactions. RT-PCR analysis revealed Del-1 mRNA predominantly in the brain and lung, with no expression in liver, spleen, or whole blood (Fig. 1A and fig. S1A). Del-1 was expressed in WT but not in Del-1-/- murine lung endothelial cells (Fig. 1B, 9). Immunohistochemistry of lung tissues demonstrated the presence of Del-1 in vessels, as observed by co-staining with the endothelial marker PECAM-1 (fig. S1B).
To determine whether Del-1 participates in leukocyte recruitment interactions, we studied adhesion of primary neutrophils to immobilized Del-1. Mouse neutrophils specifically bound to Del-1 under static conditions. Adhesion was inhibited by a blocking monoclonal antibody (mAb) to CD11a (αL-integrin subunit), but not by antibodies to αv-integrin or β1-integrin (Fig. 1C), suggesting that LFA-1 mediates the interaction of neutrophils with Del-1. Consistently, LFA-1-/- neutrophils displayed reduced adhesion to Del-1 (Fig. 1C). The residual, LFA-1-independent binding of neutrophils to Del-1 was blocked by mAb to Mac-1 (fig. S2A), consistent with the fact that LFA-1 and Mac-1 are closely related and share several ligands (13). In addition, αL-transfected but not vector-transfected J-β2.7 cells specifically bound to immobilized Del-1 (Fig. 1D), whereas a direct interaction between Del-1 and the ligand-binding I-domain of LFA-1, locked in the open high-affinity conformation, was observed (Fig. 1E and fig. S2B). These findings indicate that Del-1 is a ligand of LFA-1 integrin.
To address whether Del-1 participates in leukocyte-endothelial interactions, we studied neutrophil and monocyte adhesion to WT and Del-1-/- endothelial cells (14,15). Contrary to our prediction, Del-1-/- endothelial cells promoted significantly higher neutrophil and monocyte adhesion. LFA-1-deficiency on leukocytes and mAb to LFA-1 abolished the enhanced adhesion to Del-1-/- endothelium (Fig. 2A and fig. S3). Thus, enhanced inflammatory cell adhesion to Del-1-/- endothelium is specifically mediated by LFA-1 on leukocytes.
To understand the unexpected inhibitory role of Del-1 in leukocyte-endothelial adhesion, we addressed whether soluble Del-1 interfered with the interaction of LFA-1 with its major ligand, ICAM-1. Mn2+ -induced binding of ICAM-1-Fc to murine leukocytes in solution was significantly inhibited by soluble Del-1 (Fig. 2B). Moreover, soluble Del-1 inhibited the LFA-1-dependent adhesion of WT neutrophils to immobilized ICAM-1 under physiologic flow conditions, whereas soluble Del-1 did not affect the weaker adhesion of LFA-1-/- neutrophils to ICAM-1 (Fig. 2C).
The finding that endothelial Del-1 antagonizes LFA-1-dependent adhesion (Fig. 2A) appeared to be discordant with the finding that immobilized Del-1 promoted leukocyte adhesion under static conditions (Fig. 1C). We therefore assessed the ability of Del-1 and ICAM-1 to promote adhesion when co-immobilized with P-selectin and the chemokine MIP-2 under physiologic flow conditions at low and high shear rate (0.8 and 2 dynes/cm2). In this system leukocytes first roll on selectin and then arrest on the integrin ligand. Whereas ICAM-1 promoted robust firm adhesion of neutrophils under both shear rates, Del-1 promoted only weak adhesion under the lower shear rate and almost none at the higher shear rate (Fig. 2D). We then analyzed how the presence of plate-bound Del-1 would affect adhesion of neutrophils to ICAM-1 under flow. Increasing concentrations of Del-1 co-immobilized with ICAM-1, P-selectin and MIP-2 significantly inhibited neutrophil adhesion to ICAM-1 (Fig. 2E). Thus, although being a ligand of LFA-1, Del-1 does not promote firm leukocyte adhesion under flow but interferes with leukocyte adhesion to endothelial ICAM-1.
We then assessed the ability of soluble Del-1 expressed as an Fc fusion protein to inhibit neutrophil recruitment in vivo in acute thioglycollate-induced peritonitis (14). Intravenous administration of Del-1-Fc 30 min prior to thioglycollate injection significantly reduced neutrophil accumulation, as compared to Fc control protein (Fig. 2F). Similarly, ICAM-1-Fc reduced neutrophil recruitment into the peritoneum (Fig. 2F).
To provide further evidence for the role of Del-1 in inflammatory cell recruitment in vivo, we performed intravital microscopy using the dorsal skinfold chamber model (16). Del-1-/- mice displayed increased numbers of leukocytes adherent to postcapillary venules both at baseline and upon TNF-α stimulation (Fig. 3A and B). Besides firm arrest, the interaction between LFA-1 and ICAM-1 contributes to slow rolling processes (17). A significant decrease in rolling velocity accompanied by an increase in the fraction of slow rolling leukocytes was observed in Del-1-/- mice (Fig. 3C and 3D).
We further studied whether Del-1 could regulate inflammatory cell recruitment in vivo, by performing LPS-induced lung inflammation. Del-1-/- mice displayed significantly higher accumulation of neutrophils in the bronchoalveolar lavage (BAL) fluid, as compared to WT mice (Fig. 4A). LFA-1-/- mice displayed reduced neutrophil accumulation in the BAL upon LPS-induced lung inflammation (Fig. 4A), consistent with a previous report (18). The increased neutrophil recruitment in vivo due to Del-1 deficiency required the presence of LFA-1, as neutrophil accumulation in the BAL in Del-1-/-LFA-1-/- mice equaled accumulation of these cells in LFA-1-/- mice (Fig. 4A). The increased leukocyte recruitment due to Del-1 deficiency could not be attributed to an alteration in peripheral blood counts, since constitutive leukocyte numbers were comparable in WT and Del-1-/- mice (fig. S4). In addition, i.v. administration of soluble Del-1 efficiently reversed the increased neutrophil recruitment in Del-1-/- mice (Fig. 4B). Furthermore, we found that Del-1-deficiency resulted in an upregulation of baseline ICAM-1 protein expression by lung endothelial cells, which was overridden upon TNF-α stimulation, whereas VCAM-1 expression was unaffected (fig. S5). No significant increase in ICAM-1 expression, under baseline or inflammatory conditions, was found in Del-1-/- lungs (fig. S6), suggesting that altered ICAM-1 expression is not involved in the increased leukocyte recruitment to Del-1-/- lungs. Moreover, whereas the increased neutrophil recruitment to the lung upon Del-1-deficiency was completely reversed by leukocyte LFA-1-deficiency (Fig. 4A), inhibition of ICAM-1 by a blocking mAb (18,20) decreased neutrophil recruitment by the same extent in both WT and Del-1-/- mice (Fig. 4C), implying an involvement of other LFA-1 ligands. These findings suggest that Del-1 deficiency enhances LFA-1-dependent leukocyte recruitment in vivo.
We found Del-1 to act in an anti-inflammatory fashion, however the expression of Del-1 in inflammation has not been previously elucidated. We therefore analyzed Del-1 mRNA expression in the lung and in endothelial cells upon inflammatory stimulation. Upon LPS administration, lung Del-1 mRNA was significantly reduced (Fig. 4D). Likewise, TNF-α stimulation of endothelial cells induced a significant decrease in Del-1 expression (fig. S7).
Endogenous inhibitors exist in many aspects of inflammation and immunity (21,22), attenuating exuberant inflammatory and immune activation. To date, no endogenous inhibitor was known in the leukocyte adhesion cascade, a central paradigm of inflammation and immunity. Here, endothelial-derived Del-1 was identified to intercept LFA-1-dependent leukocyte-endothelial interactions. Given the importance of LFA-1-dependent leukocyte recruitment in several inflammatory and autoimmune disorders (13, 23-25), Del-1 may provide a platform for designing novel attractive therapeutic modalities to target leukocyte-endothelial interactions in disease.
We thank X. Feng and M. Sardy for generating the Del-1-Fc protein, N. Hogg for the antibody mAb24, Valentis Inc. for recombinant Del-1 and the antibody to mouse Del-1, T. Veenstra for help with mass spectrometry, D. Winkler for help with genotyping, I. Okwumabua for technical assistance and D. Singer for critically reading the manuscript. This research was supported by the Intramural Research Program of the NIH, NCI (T.C. and M.U.), by NIH grants AI067254 (S.C.), RO1 HL082927 (W.C.A), and by the Deutsche Forschungsgemeinschaft (FOR809, TP6 to C.W.; and TR-SFB23; Exc 147/1 to S.D. and E.C.). A patent application on the anti-inflammatory actions of Del-1 has been filed.
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