The role of the endothelium in immune reactions is greatly amplified when endothelial cells (EC) express activation molecules including MHC class II antigens and cell adhesion molecules (CAMs). Immune-mediated vascular injury limits both the viability of allografted organs, and the use of xenografts. The comprehensive objective of this work is to study the molecular mechanisms of intercellular contact-dependent endothelial activation by cytotoxic lymphocytes, in particular by natural killer (NK) cells. We have previously demonstrated that, in a beta2 integrin-ICAM adhesion-dependent fashion, human CD56+, CD3- NK cells efficiently induce activation of EC MHC class II genes. Despite involvement of the transcription factor STAT1, neither IFN-gamma, the class II transactivator (CIITA) nor JAK1 play a major role in this induction. This may be a general feature of NK-EC interactions, as human NK cells also induce MHC II on porcine EC, trigger EC nuclear translocation of NF-kappaB and induce both human and porcine endothelial CAM expression. The unifying hypothesis is that NK cell adhesion, through engagement of EC adhesion receptors, results in recruitment of signaling complexes and the generation of novel signal transduction cascades, leading to EC activation. Specific proposals now include to: (1) define molecules which interact with the cytoplasmic domain of both human and pig ICAM-1, utilizing yeast 2-hybrid and GST pull down approaches; (2) perform in vivo footprint analysis of the NK-induced EC HLA-DRalpha promoter and screen an EC cDNA library for novel adhesion-induced transcription factors; (3) dissect features of lymphocyte contact-dependent EC NF-kappaB activation with EMSAs, supershift assays, ICAM-1 engagement experiments and analysis of the kappaB sites in the MHC II-associated invariant chain promoter; and (4) further identify an adhesion-activated, novel CAM-inducing NK soluble factor, by screening an LFA-1 activated, NK cell soluble protein expression cDNA library in a CAM induction assay. All the molecular pathways referred to above will be evaluated with human and pig EC targets, and adenoviral EC transduction will be utilized to manipulate expression/function of defined involved molecules. This work may provide direction for pharmacologic and/or genetic manipulations, thereby interfering with endothelial activation and immune-mediated vasculopathy in clinical transplants.
|Rossetti, Grazisa; Collinge, Mark; Bender, Jeffrey R et al. (2002) Integrin-dependent regulation of gene expression in leukocytes. Immunol Rev 186:189-207|
|Sadeghi, M M; Tiglio, A; Sadigh, K et al. (2001) Inhibition of interferon-gamma-mediated microvascular endothelial cell major histocompatibility complex class II gene activation by HMG-CoA reductase inhibitors. Transplantation 71:1262-8|
|Sadeghi, M M; Collinge, M; Pardi, R et al. (2000) Simvastatin modulates cytokine-mediated endothelial cell adhesion molecule induction: involvement of an inhibitory G protein. J Immunol 165:2712-8|
|Russell, K S; Stern, D F; Polverini, P J et al. (1999) Neuregulin activation of ErbB receptors in vascular endothelium leads to angiogenesis. Am J Physiol 277:H2205-11|
|Collinge, M; Pardi, R; Bender, J R (1998) Class II transactivator-independent endothelial cell MHC class II gene activation induced by lymphocyte adhesion. J Immunol 161:1589-93|
|Blum, M S; Toninelli, E; Anderson, J M et al. (1997) Cytoskeletal rearrangement mediates human microvascular endothelial tight junction modulation by cytokines. Am J Physiol 273:H286-94|