Modulation of an Adhesion-Related Surface Antigen on Equine Neutrophils by Bacterial Lipopolysaccharide and Antiinflammatory Drugs

Abstract

The essential role of the CD11/CD18 family of leukocyte adhesion molecules (LeuCams) in neutrophil-substrate adhesion is well documented. We have found that a monoclonal antibody designated 60.3 (MoAb 60.3) that recognizes the common beta-subunit (CD18) on human neutrophils (PMN) also recognizes a surface antigen on equine PMN. Antigen expression as assessed by immunofluorescence flow cytometry was enhanced by zymosan-activated serum (ZAS) or phorbol 12-myristate 13-acetate (PMA) stimulation. Pretreatment of equine PMN with MoAb 60.3 inhibited ZAS-stimulated aggregation, indicating that the monoclonal recognized a functional epitope on equine PMN involved in adhesion-related functions. Cells pretreated only with bacterial lipopolysaccharide (LPS; 1 microgram/ml) exhibited moderate increased binding of MoAb 60.3 as determined by fluorescence intensity. Preincubation of PMN with LPS resulted in a slight increase in MoAb 60.3 binding after subsequent ZAS stimulation, greater than that with either LPS or ZAS as sole stimulus. Similarly, enhanced binding of MoAb 60.3 was observed with LPS preincubation when PMA was used as a stimulus, but this effect was dose dependent and was observed at only one of three PMA concentrations tested (1 ng/ml). In other experiments, preincubation of PMN with antiinflammatory drugs inhibited 41.5-45.1% of ZAS-stimulated PMN adhesion to monolayers of equine endothelial cells. To determine whether modulation of expression of the adhesion-related antigen recognized by MoAb 60.3 correlated with these observed adhesive responses of PMN, we used immunofluorescence flow cytometry to assess expression of the antigen on drug-treated PMN. Using 10% ZAS as a stimulus, phenylbutazone (PBZ; 100 micrograms/ml) pretreatment of PMN reduced subsequent MoAb 60.3 binding by only 12.3%, and dexamethasone (DEX; 10(-5) M) reduced binding by only 1.0%; reductions of 16.4% with PBZ and 9.3% with DEX occurred when PMA (10 ng/ml) was used as the PMN stimulant. These data suggest that equine PMN express a functional adhesion molecule similar to those found on human PMN and that LPS may enhance the expression of this surface antigen. Expression of this adhesion-related surface antigen on equine PMN does not correlate well with levels of drug-induced diminished adhesion of PMN to endothelium in vitro.