Avijit Roy

Date of Graduation

1998

Document Type

Dissertation/Thesis

Abstract

Isobutyl nitrite (IBN) is a volatile compound that causes vasodilation and smooth muscle relaxation. A history of abuse of this compound by HIV positive homosexual men has been associated with an increased incidence of Kaposi's Sarcoma, a tumor whose growth is responsive to cytokines. Previous work with the murine pulmonary model has demonstrated that exposure to IBN can suppress cytotoxic T cell ability, reduce the production of nitric oxide by macrophages and impair their ability to kill P815 targets. The overall aim of this project was to further characterize the nature of the immune alteration in the murine macrophages. In particular the effect of IBN exposure on macrophage inflammatory cytokine production, were studied. Macrophages from exposed animals produced significantly less TNF-{dollar}\\alpha{dollar}, IL-1{dollar}\\beta{dollar} and IL-6 protein than macrophages from control animals. Analysis of mRNA levels of all three cytokines found no changes in magnitude comparable to the differences observed at the protein level. In addition, the kinetics of both the message and protein levels was unchanged. There were also no significant changes in message stability of TNF-{dollar}\\alpha{dollar} and IL-1{dollar}\\beta.{dollar} The subsequent analysis of cell associated cytokines found no change in the level of TNF-{dollar}\\alpha{dollar} in contrast to the reduced levels of IL-1{dollar}\\beta.{dollar} These findings indicated that IBN likely affected post-transcriptional processes involved in cytokine production. Immunoblot analysis indicated that there were no detectable changes in both the secreted and cell associated TNF-{dollar}\\alpha{dollar} protein further supporting this contention. Flow cytometric analysis of peritoneal and spleen cells revealed no discernable changes in cell populations ruling out the possibility that the reduced production was due to altered cell numbers. In addition, the ability of macrophages to up regulate MHC Class II molecules was not altered suggesting that the damage caused by IBN was restricted and unlikely mediated by nitric oxide. This contention was further supported because additional chemical analysis found that only a small fraction of IBN was converted to nitric oxide. A direct exposure to nitric oxide did not elicit the same immune suppression seen in IBN exposed animals confirming that IBN did not mediate its effects via nitric oxide. The cumulative findings in this project indicated that IBN alters TNF-{dollar}\\alpha{dollar} production post-translationally.

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