Although the changes and heterogeneity in functional identity of human B cells during aging are well-described, the biochemical mechanisms underlying these changes are poorly understood. Our investigations demonstrate that stimulated B cells from substantial proportions of elderly subjects display impairments of early activation events not seen in B cells from young subjects. These include i) decreased G-O/G-1 progression, ii) reduced expression of early B cell activation molecules and iii) impaired cytosolic free calcium [Ca2+]i responses. Recent data directly demonstrate that stimulated B cells from approximately 50% of elderly subjects display impaired expression of tyrosine kinase (PTK) and protein kinase C (PKC) enzymatic activity not observed in B cells from young subjects. The overall objective of this revised proposal is to determine the biochemical mechanisms responsible for the differential expression of kinase activity observed with aging. This objective will help understand whether age-related imbalances can take place in selected biochemical events which affect the normal plasticity of early signaling and may modify the functional identity of human B cells.
The specific aims are 1) to quantitate the expression of PTK and PKC enzymatic activity in resting and stimulated B cells from elderly and young subjects, 2) to determine whether age-related changes take place in the levels and isozyme composition of PKC, 3) to investigate whether age-related alterations occur in the normal proteolytic conversion and degradation of PKC, and 4) to determine whether PKC inhibitors or PP1/PP2A phosphatases alter the expression of PKC enzymatic activity during aging. The methodologies for quantitating the alterations in PTK and PKC are enzymatic assays and immunoblots utilizing radiolabeled indicator systems operational in our laboratories. The design of the project now defines the age-related heterogeneity and impairments in the expression of kinase enzymatic activity among elderly humans using quantitative analyses and standard biostatistical procedures to assess the precision of measurements and to reach valid conclusions. The results from this project will provide new information about alterations in early biochemical events and signaling mechanisms which can occur in B cells from a substantial proportion of elderly humans. The work is intended to define the molecular mechanisms and biochemical events which can affect the plasticity and balance of early signaling events during aging. Defining age-related changes in the biochemical events and in the plasticity of early signals important for the functional identity of B cells will provide insights into the molecular basis of the immunobiology of aging. Furthermore, the definition of heterogeneity and of alterations in important biochemical events of human B cells during aging may serve to eventually understand the molecular basis for the heterogeneous effects of the aging process on the immune system of different elderly individuals.
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