Antisense RNA complementary to c-fos mRNA was produced in mouse 3T3 cells by gene transfer techniques. Transcriptional units were constructed consisting of a steroid-inducible mouse mammary tumor virus (MMTV) promoter, mouse or human 5' c-fos gene fragments in either the sense (5' to 3') or antisense (3' to 5') orientation, and splice and poly(A) signals from the human J3-globin gene. A gene that confers neomycin resistance was included in the vectors to allow isolation of stable transformants. Dexamethasone caused a marked induction of hybrid MMTV-fos-globin RNA. Induction of the hybrid transcript containing antisense c-fos RNA decreased colony formation following DNA transfer and inhibited the proliferation of cells into which the antisense transcriptional unit had been integrated. In contrast, colony formation and cell proliferation were not inhibited by induction of hybrid RNA containing c-fos RNA sequences in the sense orientation. These results indicate that the strategy of generating antisense RNA to inhibit gene expression may be useful in delineating the function of protooncogenes. The c-fos gene product appears to have a required role in normal cell division.

Abstract. T cell adhesion to endothelium is critical to lymphocyte recirculation and influx into sites of inflammation. We have systematically analyzed the role of four receptor/ligand interactions that mediate adhesion of peripheral human CD4+ T cells to cultured human umbilical vein endothelial ceils (HUVEC): T cell LFA-1 binding to ICAM-1 and an alternative ligand ("ICAM-X'), T cell VLA-4 binding to VCAM-1, and T cell binding to ELAM-1. Contributions of these four pathways depend on the activation state of both the T cell and HUVEC, and the differentiation state of the T cell. ELAM-1 plays a significant role in mediating adhesion of resting CD4+ T cells to activated HUVEC. LFA-1 adhesion dominates with PMA-activated T cells but the strength and the predominant LFA-1 ligand is determined by the activation state of the HUVEC; while ICAM-1 is the dominant ligand on IL-l-induced HUVEC, "ICAM-X" dominates binding to uninduced HUVEC. Adhesion via VLA-4 depends on induction of its ligand VCAM-1 on activated HUVEC; PMA activation of T cells augments VLA-4-mediated adhesion, both in the model of T/HUVEC binding and in a simplified model of T cell adhesion to VCAM-l-transfected L cells. Unlike LFA-1 and VLA-4, ELAM-l-mediated adhesion is not increased by T cell activation. Differential expression of adhesion molecules on CD4+ T cell subsets understood to be naive and memory cells also regulates T/HUVEC adhesion. Naive T cell adhesion to HUVEC is mediated predominantly by LFA-1 with little or no involvement of the VLA-4 and ELAM-1 pathways. In contrast, memory T cells bind better to HUVEC and utilize all four pathways. These studies demonstrate that there are at least four molecular pathways mediating T/HUVEC adhesion and that the dominance/hierarchy of these pathways varies dramatically with the activation state of the interacting cells and the differentiation state of the T cell.

The induction of an ensemble of adhesion molecules on endothelial cells by inflammatory cytokines is likely to be crucial to the differential migration of T-lymphocyte subsets into inflammatory sites. Two molecular pathways involving the VLA-4 and LFA-1 integrins are known to mediate T-cell adhesion to activated endothelium. Here we show that a third pathway involving the rapidly inducible endothelial cell-surface adhesion molecule ELAM-1 contributes to the binding of resting CD4+ T cells to IL-1-induced human endothelial cells. All three pathways contribute to the greater adhesion to endothelium of memory T cells than naive T cells. There are two unique features of T-cell adhesion to purified ELAM-1: first, ELAM-1 exclusively mediates adhesion of memory T cells; second, memory T-cell binding to ELAM-1 is independent of acute activation events that regulate integrin-mediated adhesion. Thus, ELAM-1 may be of primary importance in the initial attachment of memory T cells to inflamed endothelium in vivo and to the preferential migration of memory T cells into tissue and inflammatory sites.