Adenosine inhibits ischemia-reperfusion injury (IRI), i.e. tissue damage that occurs during reperfusion bllowing ischemia in liver, heart and other tissues. Preliminary data indicate that bone marrow-derived CD4+ T cells appear to be the most important targets of adenosine-mediated protection from IRI. The overall goal of this proposal is to better understand how adenosine markedly protects liver and heart from IRI. We hypothesize that endogenous glycolipid mediators and other factors produced during acute ischemia- reperfusion injury are presented to certain T lymphocytes by antigen presenting cells (APCs) resulting in their rapid activation. This activation is inhibited by adenosine produced in inflamed tissues. Possible targets of AaA agonists are CD4+ T effector cells (CD25-) NKT cells, antigen presenting cells (including macrophages and dendritic cells) and T regulatory cells (CD25+).
Aim 1 is to create tissue specific deletions n granulocytes, T cells, cardiomyocytes, endothelial cells and hepatocytes of the A^AR gene through the useiof the Cre/lox system. In addition, we will label various T cell populations in vitro with 111-ln and follow their, trafficking non-invasively with y-camera imaging following their transfer into wild-type mice. Hypothesis 1_is that cre/lox deletion of the A2AAR from T lymphocytes will greatly reduce adenosine-mediated tissue protection and enhance the trafficking of effector T cells into injured tissues. We hypothesize that T cell activation during reperfusion injury my initiate an inflammatory cascade in which macrophages and neutrophils are also activated.
Aim 2 is to investigate the role of macrophages in adenosine-mediated tissue protection. The activation of T cells during IRI appears to require the participation of antigen presenting cells (APCs) such as macrophages or dendritic cells. We will deplete macrophages from various mouse lines, using liposomal clodronate, and then to reconstitute mice with monocytic RAW cells that express various numbers of A^ARs. Hypothesis 2 is that the contribution of macrophages to tissue protection will be largely dependent on their expression of A2AARs and that the sensitivity of mice to protection by AaAagonists will be enhanced by prior inflammatory stimuli.
Aim 3 is to investigate the role of the A2AAR in vitro on T eel activation/proliferation in co-cultures of activated T effector cells or NKT cells, Treg cells and antigen- presenting splenocytes or dendritic cells, each derived from wild-type and/or A2AAR ko mice. Hypothesis 3 is that AaAreceptors on all of these cells contribute to the regulation of T cell activation and proliferation These experiments will results in increased understand about the role of the innate immune system in IRI and will provide new insights into the treatment of ischemic diseases and tissue transplantation.
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