Charles D. Mills - US grants

The goal of this investigation is to increase the success of clinical islet transplantation. Two interrelated hypotheses are put forth to achieve this goal. First, Nitric Oxide (NO) is postulated to be a prime mediator of islet dysfunction. Second, a previously unrecognized ability of insulin to decrease NO production is proposed to explain its islet-protective activity. Both of these hypotheses will be tested directly by focusing the experiments in this investigation on the use of inducible Nitric Oxide Synthase "knockout" mice (iNOS-/-). Specific Aim I will determine the role of macrophage NO in islet graft rejection. Based on preliminary and published results from this laboratory, NO is proposed to inhibit islets in 2 "waves": an early wave that occurs 1-2 days posttransplant of syngeneic or allogeneic islets is responsible for early islet dysfunction. A second wave NO-mediated islet dysfunction is proposed to occur during classical allograft rejection. The early wave will be investigated by comparing syngeneic islet function in diabetic iNOS-/- and iNOS-/+ mice; the second wave will be investigated using allogeneic islets in these hosts. Inhibitors of NO will then be used to determine how to best increase islet function. Preliminary and published evidence from this laboratory suggests that inhibiting NO in a clinically applicable way does increase the success of islet transplantation. Islets will be implanted in the intraperitoneal cavity in this investigation because intragraft NO production and allograft rejection responses can readily be followed at this site. Mice will be the primary species used because, like humans, they are "low" producers of NO; rats will be used in selected experiments because of their larger size. Specific Aim II will determine the mechanism by which insulin promotes islet function. A dominant paradigm has been that insulin allows transplanted beta-cells to "rest". However, preliminary evidence indicates that insulin administration to diabetic mice or rats decreases macrophage iNOS mRNA expression and NO production. Therefore, specific experiments will again compare iNOS- /- and iNOS-/+ mice to directly test the hypothesis that the islet- protective activity of insulin results from decreasing NO production. Finally, hyperglycemic clamps will be employed to maintain hyperglycemia in order to determine if the islet-protective activity of insulin depends on its ability to lower blood glucose. The results of this investigation should provide important new information that will increase the success of clinical islet transplantation as well as providing basic information on what role NO plays in rejection of cell transplants, and how NO production is regulated by insulin.

Description (provided by applicant): The goal of this investigation is to more clearly defme M- 1 and M-2 macrophages and how they influence immune responses. The concept of M-1 and M-2 fomented from observations in this laboratory that with the same stimuli, macrophages from certain mice (C57BL/6, B1OD2) preferentially metabolize argimne to NO while other mice (Balb/c, DBAI2) increase ornithine production. NO inhibits cell replication while omithine (via polyamines) promotes cell replication. Therefore, M-1/M-2 does not simply represent activated or unactivated macrophages, but macrophages that have upregulated qualitatively different metabolic programs. M-1/M-2 propensities are also observed in C57BL/6 and Balb/c SCID mice and are thus independent of T or B lymphocytes. Indeed, other evidence indicates that M-1/M-2 macrophages can shepherd T lymphocytes into Thi or Th2 responses, respectively. Macrophages are a major precursor of dendritic cells, which have been reported to influence the Thl/Th2 balance. Therefore, our results suggest that macrophages play much a more important role in orchestrating immune responses than is currently appreciated. The Main Hypothesis is that M-1 is associated with destructive products and Thl responses, while M-2 is associated with the constructive products involved in healing/regeneration and with Th2 responses. It is also hypothesized that overexpression of the M-l phenotype (e.g. NO) can inhibit specific immune responses. To test these hypotheses Specific Aim 1 will more clearly define products and properties of M-1/M-2 macrophages including a) arginine metabolites (NO/ornithine); b) oxygen radicals (O2-, ONOO-, H2O2); and c) cytokines/growth factors (e.g. IL- 12, IFN-g, IL-8, IFN a/b, macrophage stimulating protein). Specific Aim I will also determine if M-1/M-2 macrophage phenotypes are expressed at the single cell level using double color ELISPOT. Specific Aim 2 will determine how M-l/M-2 macrophage responses influence non-specific or specific immune responses in vivo. Specific Aim 2a will determine how M-1/M-2 macrophage responses influence non-specific inflammation associated with injury/danger. Specific Aim 2b will compare innate immunity in M-l/M-2-dominant mice. Specific Aim 2c will determine how M- 1 /M-2 macrophages influence tumor specific or allo specific immune responses. M1 (C578L/6) and M-2 (Balb/c) SCID mice will be used extensively as test subjects and as sources of macrophages for adoptive transfer to directly determine effects due to M-1/M-2 macrophages. Together, this investigation will result in a major increase in our understanding of M-1/M-2 macrophages and how they influence immune responses.