Detection of nitric oxide by electron paramagnetic resonance spectroscopy during rejection and graft-versus-host disease after small-bowel transplantation in the rat

Abstract

Background: Our previous observation that nitric oxide (NO) is synthesized during antigen-specific immune reactions in vitro led us to investigate whether NO is produced during the in vivo immune response to a vascularized organ allograft.

Methods: Orthotopic small-bowel transplantation in the rat was performed by standard microsurgical techniques in the LBNF1 to Lewis (rejection alone), Lewis to LBNF1 (graft-versus-host disease [GVHD] alone), and a syngeneic strain combination with and without immunosuppressive therapy with FK 506. The recipient serum NO2-/NO3- levels (stable end products of NO metabolism) were measured and erythrocytes were evaluated for the presence of nitrosylferrohemoglobin (specific for NO bound to hemoglobin).

Results: Animals that acutely rejected small-bowel allografts or suffered from acute GVHD showed significantly elevated serum NO2-/NO3- levels on days 6 and 9, and nitrosylferrohemoglobin electron paramagnetic resonance signals of different intensity were detected on days 3, 6, and 9. FK 506-treated allograft recipients and recipients of syngeneic grafts showed normal serum NO2-/NO3- levels and lacked nitrosylferrohemoglobin signals at all time points.

Conclusions: This study indicates that NO is produced early during the course of small-bowel allograft rejection and GVHD and might therefore serve as a simple marker to detect such immune reactions.