Extravasation of leukocytes into peripheral tissues is a multi-step cascade of events, involving recognition of chemotactic signals released from the affected areas, reversible adherence to capillary endothelium and penetration through extracellular matrix. Treatment of mice with BRMs results in a recruitment of natural killer (NK) cells to the liver and spleen. The mechanism for these events is being studied using via a unique assay that measures the adherence of unstimulated NK cells to a murine endothelioma cell line, eEND2. Treatment of eEND2 cells with recombinant human (rh) interleukin (IL)-1alpha, rhIL-6 and rmurine (rm) tumor necrosis factor alpha (rmTNFalpha) induced adherence of murine splenic NK cells in a dose and time-dependent fashion. In contrast, rhIL-7, rhIL-8, rm interferon/gamma (rmIFNgamma) and rhTNFbeta were inactive in this assay. Binding of NK cells to cytokine stimulated endothelium was found to differ from peripheral lymph node T cells and bone-marrow derived neutrophils. Adherence of T cells to eEND2 was stimulated only after overnight treatments with rmTNFalpha, rhIL-1alpha or rmIFNgamma, while both neutrophil and NK cell binding reached maximal effects after 2 hrs of cytokine treatment, with rmIFNgamma being ineffective in stimulating NK cell adherence at any time point tested. These data suggest that cytokines act on eEND2 cells to induce the expression of specific surface adhesion molecules involved in regulating leukocyte binding. Monoclonal antibodies directed against these ligands were studied for their ability to block NK cell adherence. Antibodies against the intracellular adhesion molecule, ICAM-1, and the glycoprotein sialyl Lewis X (SiLeX), the carbohydrate ligand for E- selection, were ineffective in blocking NK cell adherence. Pretreatment of the NK cells with antibodies directed against the ligands CD11a, CD11b, CD44 and L-selection was also ineffective in reducing NK cell adherence to EEND2 cells stimulated by rmTNFalpha, suggesting that a novel molecule(s) may be responsible for the enhanced binding of NK cells stimulated by this cytokine. Future studies will focus on the nature of these molecules and their counter-ligands present on NK cells.
