Characterization of a Computerized Assay for Rapid and Easy Determination of Leukocyte Adhesion to Endothelial Cells

Abstract

Adhesion of polymorphonuclear neutrophils (PMNs) to activated endothelial cells is a prerequisite for the recruitment of PMNs that may cause acute or chronic pathological reactions as observed in reperfusion injuries, stroke, psoriasis, rheumatoid arthritis, respiratory diseases and angiogenesis.1) Expression of a number of different cell adhesion molecules (CAMs) on PMNs and endothelial cells is required for the multistep adhesion process. 2)Because of the sequential characteristics of leukocyte accumulation in inflammation, blockade of any of the binding steps may have strong effects on inflammatory disease. Therefore, it is of great pharmaceutical interest to block interactions between leukocytes and endothelial cells by antagonizing the process of recognition. The function of inflammatory CAMs can be modulated via a number of different mechanisms including competitive blockade, altered expression on the cell surface or, for integrins, interference with integrin activation. 3,4) In order to study the effect of CAM-inhibition on leukocyte adhesion to endothelial cells or to isolated CAMs, various assays under static or dynamic conditions have been developed. These assays often suffer from a lack of reproducibility because of heterogeneities in the components of the assays. Moreover the evaluation and data acquisition are often time consuming (reviewed by Simanek et al.).5) Due to their simplicity and rapidity, static assays are the most widely used ones. In these assays the quantification of adherent leukocytes is often made using leukocytes pre-labeled with radioactive material, usually 51 Cr,6) or leukocytes stained with fluorescent dyes 7) like calcein-AM, 8) BCECF-AM 6) or TDA-AM.9) Other techniques including those using ELISA, 10) horseradish peroxidase 11) or biotinylated cells 12) require additional steps in the assay procedure (e.g. leukocyte manipulating) that may cause undesired activation or leakage of the labeling compounds. Assays based on non-labeled leukocytes and conventional microscopy to quantify cell adhesion have also been described (reviewed by Simanek et al.). 5)However, these techniques require time-consuming manual counting procedures, which lack automated, observer-independent evaluation. The benefits of a computer assisted image analysis combined with an automated data evaluation system have been described using fluorescence-labeled leukocytes and confocal laser scan microscopy in a transmigration assay.13) Here, we present a rapid method for quantification of leukocyte adhesion using unlabelled leukocytes, conventional microscopy and semi-automated computational image analysis that allows high-throughput to study interactions between leukocytes and endothelium and subsequent rapid testing of pharmaceutical adhesion inhibitors. MATERIALS AND METHODS Chemicals and ReagentsThe following cell culture media and reagents were purchased from Sigma (München, Germany): Medium 199, fetal bovine serum, penicillin-streptomycin solution, amphothericin B solution, L-glutamine solution, trypsin-EDT...

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