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The mechanisms that regulate the adhesion and migration of NK cells to and across endothelium have been studied under nonflow conditions; however, the involvement of these processes in vivo is poorly understood. The present studies investigated the potential vascular adhesion ligand interactions that determine the in vivo recruitment of NK cells to pulmonary and hepatic parenchyma, and s.c. tumor after treatment of mice with biologic response modifiers. Seventy-two hours after a single injection of the cytokine-inducing agent poly-L-lysine stabilized in carboxylmethyl cellulose (poly-ICLC), pulmonary NK cell lytic activity and N-alpha-carbobenzoxy-L-lysine thiobenzyl ester (BLT)-esterase were augmented 29- and 14-fold, respectively, and the number of lung-associated NK cells was increased from 2.3 x 10(5) to 7.4 x 10(5). Similar fold increases in NK cell number and activity were observed in the liver and s.c. B16 melanoma after poly-ICLC injection or in the lungs and liver of mice treated with IL-2. Concomitant treatment of mice with alpha-VCAM-1 or alpha-VLA-4 mAb, but not alpha-ICAM-1 or alpha-LFA-1, abrogated the poly-ICLC and IL-2-induced increase in organ-associated NK activity and percentage of tumor-associated NK cells, resulted in a 61 to 76% decrease in pulmonary and hepatic NK cell number, and was independent of T and/or B cells. The decrease in NK cell number in organ parenchyma and tumor lesions was correlated to an increase in the number of NK cells in peripheral blood, but not bone marrow. These results demonstrate that VCAM-1/VLA-4 interaction is critically involved in the infiltration of newly recruited NK cells in to lung, liver, and progressively growing tumor after mobilization from the bone marrow.

Fibroblast growth factor 1 (FGF-1)-coated collagen-gelatin sponges were affixed to various tissues to generate vascular beds, in which the vessels originated in the tissue to which the sponges were affixed. Organ-derived endothelium was obtained from vascularized sponges implanted in or on the skin, peritoneal wall, abdominal mesentery, epimysium, spleen, and liver. Collagenase digestion yielded single-cell suspensions that were analyzed by flow cytometry. Approximately 25% of the cells were positive for the endothelial cell (EC) markers MECA-32 and Sca-1 and for uptake of diIAcLDL. Similar results were obtained when sponges were implanted in several different mouse strains, although there was some evidence of heterogeneity in the degree of vascularization and EC recovery. Long-term cultures of high purity were obtained when the ECs were grown on mitomycin C-treated L929 feeder layers, in medium supplemented with cis-hydroxyproline and FGF-1. These cells have been utilized in preliminary studies of T cell-EC binding. Thus we have developed a generalized method for the recovery and culture of organ-derived murine endothelial cells. This technique should greatly improve the feasibility of studies of the interactions between murine endothelial and immune effector cells.