Natural killer (NK) cells are large granular lymphocytes that spontaneously lyse a limited range of target and may be important in the protection of the host against the development of cancer and certain infectious diseases. In this proposal we will examine how mevalonate (MVA) and its metabolites regulate human NK cytotoxicity and locomotion. MVA is the product of 3-hydroxy-3-methyglutaryl Coenzyme A (HMG CoA) reductase, the major regulatory enzyme of cholesterol and other biosynthetic pathways. We have shown in preliminary experiments that specific inhibition of HMG CoA reductase with inhibitors such as lovastatin impairs NK cell cytotoxicity and locomotion. This inhibition is rapidly reversed by the addition of MVA which indicates that a MVA metabolite is required for these NK cell functions. In addition, we have shown that interleukin-2 (IL-2) can reverse the impairment of cell function associated with reductase inhibition. The kinetics of reversal with IL-2 are much slower than that seen with MVA. The particular focus of this proposal is to examine how MVA and its metabolites regulate NK function.
The specific aims of the proposal are as follows: First, the precise step in NK cytotoxicity which is impaired by inhibition of HMG CoA reductase will be identified. Our preliminary data indicates that step 1 (conjugation) is inhibited as well as a post conjugation step. These studies on step 1 will involve examinations of membrane fluidity and the NK recognition structure using monoclonal antibody and flow cytometry techniques. The post conjugation events will be studied from the viewpoint of intracellular Ca++ changes, cytoskeletal changes, and protein phosphorylation involved in degranulation. Second, the metabolic fate of radiolabelled MVA during short term rescue of HMG CoA reductase inhibition will be examined. This will involve an analysis of the incorporation of (3H)MVA into cellular components. Third, the mechanism by which IL-2 reverses HMG CoA inhibition will be identified. These studies will examine if IL-2 directly modulates either HMG CoA reductase or the pre-reduction enzymes such as HMG CoA synthase. Fourth, the mechanism by which NK cell locomotion is impaired following HMG CoA reductase inhibition will be studied. This will involve studies on whether HMG CoA inhibition interferes with chemoattractant receptor expression, the initial acquisition of locomotor capacity by NK cells, or cytoskeletal events involved in NK cell movement. The use of specific inhibition of HMG CoA reductase will enable us to address major, unanswered questions regarding several aspects of human NK cell regulation.
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