Transendothelial migration of neonatal and adult bovine neutrophils in vitro

Abstract

We have compared and quantitated transendothelial migration of neonatal neutrophils (N-PMNs) and adult bovine peripheral-blood PMNs (A-PMNs) in vitro using monolayers of endothelium and a two-chamber apparatus. Bovine aortic endothelial cells were cultured to confluence on polycarbonate filters perforated with 3.0-micron-diameter pores. 51Cr-labeled PMNs were added to the upper chamber, with or without an anti-CD18 antibody (monoclonal antibody 60.3). Chemotactic stimuli in the lower chambers included recombinant human interleukin-8 (rhIL-8; 75 ng/ml), rhC5a (10(-7) M), and zymosan-activated bovine serum (ZAS; 10%). At 60 min incubation with rhIL-8, greater numbers (P < .01) of N-PMNs (24.70 +/- 5.95%) than of A-PMNs (15.77 +/- 3.66%) had migrated across the endothelial barrier, and a similar difference was present at 90 min. Migration rates of N-PMNs and A-PMNs were similar (P > .05) at all time points when using rhC5a and ZAS as stimuli. Anti-CD18 monoclonal antibody significantly decreased migration (P < .01) of both N-PMNs and A-PMNs to low levels when IL-8 and ZAS were used as stimuli. Because leukocyte integrin expression on PMNs affects transendothelial migration, we also compared surface expression of CD18, CD11a, and CD11c on PMNs from the two age groups. We found no significant quantitative differences in integrin expression between PMNs from the two age groups, regardless of whether the PMNs were incubated with buffer alone or with chemotaxins (rhIL-8, rhC5a, ZAS).