Inducible nitric oxide synthase inhibitors prolonged the survival of skin xenografts through selective down-regulation of pro-inflammatory cytokine and CC-chemokine expressions

Abstract

To elucidate the possible immunoregulatory role of nitric oxide (NO) in cellular xenograft rejection we performed rat-to-mouse skin xenotransplantation. The rat skin engrafted mice were treated with the inducible NO synthase (iNOS) inhibitors, aminoguanidine (AMG, 200 mg/kg) and N^G-nitro-l-arginine methyl ester (l-NAME, 60 mg/kg) every other day until rejection. Skin xenograft survival was monitored and immune cell infiltration and intragraft cytokine and chemokine mRNA expressions were analyzed 7 days after grafting. Compared with the control mice, the AMG- and l-NAME treated mice showed delayed xenograft rejection by approximately 3 days (8.9±0.7 days vs. 11.7±1.2 and 12.0±0.9 days, respectively). Infiltrations of CD11b⁺, MOMA-2⁺ cells and neutrophils were significantly reduced in both AMG- and l-NAME treated graft but CD4⁺ and CD8⁺ cells were not. The expression of cytokines such as IL-1β, IL-2, IL-6, IL-12 and IFN-γ in AMG- and l-NAME treated grafts were significantly decreased (P<0.01), whereas IL-10, TNF-α and TGF-β1 were unchanged or enhanced. Additionally, the expressions of CC-chemokines, such as RANTES and MIP-1α, were significantly reduced (P<0.01) whereas the expressions of CXC-chemokines, such as IP-10 and MIG, were unchanged. These results imply that prolonged rat-to-mouse skin xenograft survival by iNOS inhibitors may be due to the selective inhibition of pro-inflammatory cytokines and chemokines and suggest the possible regulatory role of NO in cytokine and chemokine expressions during xenotransplant rejection.

Introduction

Nitric oxide (NO) produced by inducible NO synthase (iNOS) is involved in diverse physiologic and pathophysiologic processes which include vasodilation, neurotransmission and immune defense [1], [2]. In the immune system NO displays various effects including antimicrobial [3], [4], anti-tumor [5], [6], [7], immunosuppressive [8], [9], [10], tissue-damaging [11], [12], regulatory effects on cytokine and chemokine expressions [13], [14], [15], [16] and T helper cell deviation [17], [18].

Studies regarding transplantation immunology have demonstrated abundant NO production [19], [20], [21] and significant iNOS induction [22], [23], [24], [25], [26], [27], [28], [29] during allograft rejection. Moreover, iNOS inhibition by potent inhibitors prolongs allograft survival [30], [31], [32] and iNOS activity is attenuated by conventional immunosuppressants [33], [29]. These studies suggest that NO has an immunoregulatory property in allotransplantation. However, the possible immunoregulatory role of NO in the xenotransplant rejection process, where innate immunity may play a more potent role than in the allotransplant rejection process has not been elucidated. Therefore, we examined the effects of iNOS inhibitors on the immune cell infiltration and the expressions of cytokines and chemokines in rat-to-mouse xenogeneic skingraft rejection.