The present invention relates to compositions and methods for identifying compounds useful in the inhibition of cellular adhesion involved in a number of pathological responses. In particular, the invention relates to assays for inhibitors of a class of receptors which mediate leukocyte extravasation and other responses.
Recent work has established that specialized cell surface receptors (termed here selectins) on endothelial cells and various circulating cells are involved in a number of intercellular interactions. For instance, an adhesion molecule on the surface of leukocytes, lymphocyte homing receptor (LHR), is known to be involved in the adhesive interactions of leukocytes with the endothelial lining of blood vessels. This adhesive interaction is a prerequisite for the movement of leukocytes from the blood to tissue sites where immune reactions and inflammatory reactions occur.
LHR (also known as gp90.sup.MEL, gp100.sup.MEL, gp110.sup.MEL, Mel-14 antigen, Leu8 antigen, TQ1 antigen, DREG antigen, LAM-1, selectin1, LECAM-1 and LEC-CAM-1, depending on animal species, leukocyte, and laboratory preference) is expressed on the surface of leukocytes, such as, lymphocytes, neutrophils, monocytes, and eosinophils (Gallatin, et al., Nature 303:30 (1983) and Lewinsohn, et al., J. Immunol. 138:4313 (1987), which are incorporated herein by reference). LHR is known to mediate the adhesion of lymphocytes to specialized endothelial cells in lymph nodes, leading to the migration of blood-borne lymphocytes into the lymph node. On neutrophils and monocytes, it mediates the early interaction of these cells with endothelium of blood vessels at sites of inflammation.
LHR is a lectin-like protein which performs its adhesive function by recognizing carbohydrate-containing ligands on endothelial cells. Lectin-like receptors have also been found on endothelial cells and platelets. Endothelial leukocyte adhesion molecule-1 (ELAM-1) is present on endothelial cells and is involved in the recognition of various circulating cells by the endothelium. Granule membrane protein-140 (GMP-140) is present on the surface of platelets and endothelial cells, where it mediates platelet-leukocyte and endothelium-leukocyte interactions.
Recent work has established that these receptors share certain structural features. Each of the receptors in this class is a glycoprotein with a lectin-like domain, a region with homology to epidermal growth factor, and a region with homology to complement regulatory proteins (see, Springer, Nature, 346:425, 1989, which is incorporated herein by reference). The term "selectin" is used herein to refer to this class of lectin-like receptors.
There is currently an interest in developing highly specific competitive inhibitors of selectin-mediated cellular adhesion. Such inhibitors are useful in therapeutic regimens to treat various selectin-mediated disease responses. The inhibitors could also be used to target other pharmaceutical compounds, such as anti-inflammatory agents or anti-oxidants, to the sites of injury.
To date, however, insufficient understanding of the interaction of selectin receptors and their ligands has hindered these efforts. In addition, the prior art lacks rapid, economical methods for identifying inhibitors of selectin-mediated interactions. For example, in vitro intercellular adhesion assays have been used to test inhibition (see, e.g., Stamper and Woodruff, J. Exp. Med. 144:828-833 (1976), which is incorporated herein by reference). These assays, however, are difficult to carry out and do not lend themselves to screening large numbers of test compounds. Also, comparisons of active compounds by quantitative dose-response studies is difficult using these assays. The present invention addresses these and related needs.
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