The purpose of these studies is to determine which cells are the targets of the noradrenergic innervation found within lymphoid compartments in spleen and lymph node. We will use immunocytochemistry at the light and EM level and computerized image analysis to simultaneously label tyrosine hydroxylase (TH)- positive nerve terminals and specific cells of the immune system in order to determine the relationship of noradrenergic nerve terminals to these cells. The use of the Zeiss 902 electron energy loss spectroscopy (EELS) system using fluorine labeled antibodies will allow eventual automation of the analysis of cellular relationships. Complementary studies will use in vivo dialysis to determine the norepinephrine (NE) concentration present in the extracellular fluid after splenic nerve stimulation and after treatment with pharmacologic agents that enhance or deplete NE. Combining the data from these studies will allow us to propose a simple model to represent relationships of noradrenergic nerves to immune cells. We will then deplete spleen and lymph node of T- lymphocytes, B-lymphocytes, or both, and use the same techniques to determine what happens to NE and noradrenergic terminals in these organs when their putative targets are removed. Recent studies from the P.I.'s laboratory and from others have demonstrated that NE is present in nerve terminals in lymphoid areas of the spleen and lymph node of the rodent, and that these nerve terminals are located very near cells of the immune system. Preliminary evidence at the EM level has shown very close, regular contacts between cells stained immunocytochemically for TH and lymphocytes. Preliminary in vivo dialysis studies have shown that NE is present in the extracellular fluid of the spleen where it could regulate lymphoid cells that have been shown to possess adrenergic receptors. It is particularly important to identify the target cells, and delineate their relationship to NE terminals, because functional studies of the immune system have shown that removing NE terminals through denervation has a profound effect on immune responses. Both interpretation of current studies and design of future studies would be greatly facilitated if we knew which cells were most likely to be regulated by NE, either directly released from the nerve terminal, or at a distance in a paracrine mode. Implications for the effects of neural immune interactions on disease are many. Two states which have been shown to have effect on both the sympathetic system and on the immune system are stress and aging. We have recently shown that aged Fischer 344 rats have decreased noradrenergic innervation and NE content in the spleen compared to young control rats. Certain measures of immune function also decline with aging. The proposed studies will help interpret the relationship between these two events and many similar correlations for which no mechanisms has yet been demonstrated.
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