Preliminary Study On ICAM-1/LFA-1 Binding Kinetics And Measurement Of Adhesive Strength

Cell adhesion plays a fundamental role in the development of multicellular organisms, immune response and inflammation, which is mediated by the interaction of adhesive molecules. ICAM- 1 is the receptor of LFA- 1. Their kinetics reaction is specific and reversible. During inflammation, leukocytes become adherent to endothelial cell in order to migrate through endothelium. The slow binding kinetics reaction between ICAM-1 and LFA-l leads to the firm adhesion of leukocyte to endothelium. In this study, we use a micropipette technique to determine the percentage of adhesion (adhesion probability) between a pair of cells. The advantage of the method lies in the fact that we can control contact area and contact time. In order to investigate the binding of ICAM-1 to LFA-1 perfectly, we used RBC as microforce transducer to detect adhesion marked by the deformation of RBC. Previously, RBC is coated with anti-human ICAM-1 mAb CA-7, then incubated in human plasma to combine soluble ICAM-1 for the fact that CA-7 was conjugated with the human sICAM- 1 on its domain 5 while the binding site of ICAM- 1 to LFA- 1 is on the domain 1. Another cell is SKW-3 (a kind of human lymphocyte tumor cell line )or normal human lymphocyte cell (HL), which expresses LFA-1. In the experiment, we find adhesion probability is dependent on contact time. In the beginning, adhesion probability increases with contact time, but it doesn抰 increase until it reaches saturation. The results showed that the adhesion probability of PHA stimulating group is markedly higher than that of resting group and the result of flow cytometry also showed that the fluorescent density of the former LFA-1 is higher than that of the latter. TMP(Tetramethylpyrazine) is a herb and had been used clinically for curing cerebral and cardiovascular diseases. We found it has an inhibiting effect for the conjugation of LFA-1 and ICAM-1, which may be one of its curing mechanism. In general, the probability of adhesion of HL to RBC is lower approximately 50% than that of adhesion of SKW-3 to RBC. It can be estimated that PHA and TMP work through changing molecular expression and conformation. A fundamental significance of this novel micromanipulation method is that adhesion image can be recorded combined with image analysis to yield information on the micromechanics of cell-cell adhesion and receptor-ligand bond.