CARBON MONOXIDE INHALATION PROTECTS HEART ALLOGRAFTS FROM CHRONIC REJECTION

Abstract

O69 Aims: Chronic rejection (CR) remains the major obstacle in successful organ transplantation. Heme oxygenase system is shown to provide protection against several inflammatory reactions including CR. Since carbon monoxide (CO), a by-product of heme catalysis, has also been shown to exert similar cytoprotective effects against a variety of stimuli, this study evaluated the efficacy of CO in preventing CR in rat heterotopic heart transplantation (HHT) model. Methods: Fully allogeneic HHT was performed in LEW to BN rat strain combination under short course FK506 (0.5 mg/kg, d0-6). Recipients were either kept in air or exposed to CO (20 ppm) for 28 days continuously after HHT. Cytokine mRNA levels, flowcytometry, mixed lymphocyte reaction (MLR), graft histopathology, and graft survival were analyzed. Results (Table): In CO-treated recipients, blood carboxyhemoglobin (COHb) levels were maintained at 10 % during CO exposure, compared to 1.4 % in air controls. CO treatment was remarkably effective in prolonging heart graft survival to a median of >100 days from 45 days in air control group. Severe histopathological changes associating with CR seen in control allografts were markedly reduced in CO-treated allografts with less numbers of infiltrating ED1+ macrophages. Th1 type cytokine (IL-2, IFNγ) upregulation in the spleen of air group at d28 was significantly downregulated with CO, associating with a considerable reduction of splenic CD25 (IL-2R) expression. Levels of anti-donor IgG antibodies at d50 were also notably reduced in CO-treated recipients. In addition, early short CO exposure (d0-13) was more effective in prolonging graft survival than later exposure (d14-28) (74 vs 57 days), suggesting that CO exposure during the early acute phase was more effective. As CO was shown to exert potent protection against oxidative stress, CO inhibited an early upregulation (3 hours post reperfusion) of inflammatory mediators (TNFα, endothelin-1, IL-1β, IL-6) in this model with significantly lower serum CPK levels. These results suggested that the efficacy of CO in preventing CR in this study was in part mediated via the anti-inflammatory effect of CO on I/R injury, which was known to have strong impacts on subsequent alloimmune responses and CR development. Additionally, direct inhibitory effect of CO on T cell activation was confirmed in vitro assays. When C3H/He mouse T cells were stimulated with anti-CD3 mAb for 48 hours under 20 ppm CO, T cell proliferation was markedly reduced to 78% without affecting T cell viability. Inhibition of T cell proliferation with CO associated with decreased production of IL-2 and IFNγ in the culture supernatant.Table: . (*p<0.05 vs air, mRNA levels; fold-increase vs normal samples).Conclusion: The study for the first time shows that low dose CO inhalation to recipients effectively improves whole organ allograft function and survival by inhibiting the development of CR. CO appears to downregulate alloimmune responses via the direct inhibition of Th1 type cytokine responses and via the indirect regulation of anti-inflammatory responses.