Although inflammation is an integral part of host defense mechanisms, it is also linked to numerous diseases. This is a particularly evident in the lung where inflammation acts as a trigger for the onset of diseases, and is itself the cause of significant morbidity. Inflammation has been intimately linked to idiopathic pulmonary fibrosis, sarcoidosis, and allograft rejection, in addition to pulmonary infections. Such inflammatory processes require the recruitment of immune cells to the endothelium, which involves the interaction of proteins on the surface of endothelial cells with proteins on immune cells. One set of proteins involved in these interactions is the receptor/counter receptor pair consisting of vascular cell adhesion molecule-1 (VCAM-1) on endothelium and the integrin, alpha4Beta1, on immune cells. In order to understand the molecular mechanisms that control the pattern of VCAM-1 expression, we have cloned the promoter region of the gene for VCAM-1 and demonstrated that VCAM-1 expression is under transcription control. Our results show that two cytokine systems control VCAM-1 expression, tumor necrosis factor-alpha (TNF) and interleukin-4 (IL-4). TNF increases VCAM-1 gene transcription through two adjacent NFkappaB-like sites in the VCAM-1 gene promoter. These sites interact with different nuclear proteins and it is this combination of sites that is responsible for the tissue-specific response of VCAM-1 to TNF. Using gel retardation and transfection assays, we propose to characterize the mechanisms important for this response. Interestingly, VCAM-1 is the only member of the set of cell adhesion molecules on endothelial cells that is responsive to IL- 4. Since alpha4Beta1 is only present on a subset of immune cells-- lymphocytes, monocytes, basophils, and eosinophils--IL-4 through its up regulation of VCAM-1 can mediate a selective inflammatory response. Such a response is important in allergic conditions and other eosinophilic conditions, such as asthma. IL-4 has been shown to increase transcription of several genes in B-cells; however, nothing is known about the mechanism of action of IL-4 in cells other than B-cells. We propose a series of studies to examine the mechanism through which IL-4 increases VCAM-1 expression in endothelial cells. Finally, not only is the activation of VCAM-1 important in inflammation, it is also critical to prevent VCAM-1 expression on unstimulated endothelium. To this end, we have found multiple negative elements in the promoter that prevent expression of VCAM-1 on unstimulated cells. TNF overcomes the negative effect of these octamer elements and activates VCAM-1 through the NFkappaB sites discussed above. We propose to study the mechanisms of this repression using gel retardation and transfection assays.
