Our laboratory is in a unique position to determine whether the two types of TNF and IL-1 receptors regulate the synthesis of different protein in human T cells, B cells, monocytes and A375 melanoma cells, and to identify some of these proteins from their amino acid sequences. Only now are such studies feasible because of the presence in our own laboratory of state-of-the-art, computerized 2-D gel image analysis integrated with protein microsequencing from preparative scale gels (by either Edman degradation or high performance tandem mass spectrometric techniques) and because of the recent availability from our collaborators of the cDNAs for the individual TNF and IL-1 receptors and antibodies that act as either antagonists or agonists. Specifically, our studies will ascertain whether cells known to have type I receptors for IL-1 (i.e., human T-cells and fibroblasts), and cells known to have type II receptors for IL-1 (i.e., human B-cells, monocytes and A375 melanoma cells) have different patterns of proteins induced or suppressed in response to both forms of IL-1, using computer based analysis of 2-D gels. As our previous studies had shown that TNF induces the same proteins in fibroblasts as does IL-1, a comparison will be made between the pattern of proteins induced or suppressed by IL-1 with that of TNF in T cells, B cells, monocytes and a375 melanoma. Next, our studies will determine if the synthesis of those proteins induced or suppresses by TNF are regulated via interaction with either the 55kD TNF receptor, the 75kD TNF receptor, or both. The amino acid sequences of key IL-1 and TNF regulated proteins isolated from preparative-scale 2-D gels will be obtained using Ednam degradation and/or mass spectrometry, and the mRNA expression of such proteins in their cell type of origin will be assessed. Finally, the effect of other cytokines (i.e. IL-4, IL- 6 and IFN-gamma) on the expression of the 55kD and 75 kD TNF receptors and the type I and type II IL-1 receptors will be assessed in these same cells. The latter studies should demonstrate if alterations in receptor expression affects the amount or type of individual proteins induced or suppressed in the respective cell type. The studies proposed are consistent with our long term objective of understanding the mechanisms of action of TNF and IL-1 and their comparative effects on the function of normal and malignant cells. The identification of cellular proteins whose synthesis is regulated by these cytokines will help define their functional roles in mediating the action of TNF and IL-1. Furthermore, these studies will provide new observations and important insights that will be of help to those who design clinical trials employing these cytokines, in which the ultimate aim is to suppress those pleiotropic actions or side effects that are deleterious and amplify those that are beneficial.
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