Depletion of Alveolar Macrophages Abrogates Prolongation of Cardiac Allograft Survival Induced by Intratracheal Delivery of Alloantigen

BACKGROUND: We previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory cells in mice. In this study, we examined the role of alveolar macrophages (AM) in our ITD model. METHODS: Some CBA mice were given ITD of C57BL/6 splenocytes and underwent transplantation of C57BL/6 hearts 7 days later. In others, AM were depleted with clodronate-loaded liposomes 3 days before ITD. In adoptive transfer studies, whole splenocytes were obtained from ITD-treated CBA mice and administered to naïve CBA secondary recipients, which were given C57BL/6 hearts immediately afterward. Interleukin-10 concentrations in bronchoalveolar lavage fluid were assessed by enzyme-linked immunosorbent assays. Immunohistologic and flow cytometric studies were performed after ITD. RESULTS: C57BL/6 splenocytes given by ITD were ingested by AM in 2 days and undetectable in paratracheal lymph nodes or spleen tissue. CBA mice given ITD of C57BL/6 splenocytes had markedly prolonged allograft survival (median survival time [MST], 86 days), whereas naïve CBA mice rejected allografts acutely (MST, 8 days). AM-depleted, ITD-treated mice also rejected allografts (MST, 5.5 days). Naïve secondary recipients given adoptive transfer of splenocytes from ITD-treated mice had prolonged allograft survival (MST, >100 days), whereas secondary recipients given adoptive transfer of splenocytes from AM-depleted, ITD-treated mice rejected the grafts (MST, 15.5 days). Interleukin-10 expression in bronchoalveolar lavage fluid was down-regulated in AM-depleted mice compared with naïve mice. CONCLUSIONS: AM have an important role in the induction of regulatory cells in our model of ITD of alloantigen.