Adult rat MSCs inhibit allogeneic responses in vitro and in vivo through HO-1 and iNOS activity. (A) LEW.1A T lymphocytes (105) were cocultured with LEW.1W APCs (0.25 × 105) in the presence of graded numbers of adult LEW.1A rat MSCs. Proliferation was measured by thymidine incorporation. The results of 1 experiment representative of 3 are shown Error bars represent SD. (B) Adult rat MSCs (LEW.1W) were cultured alone or in the presence of IFN-γ (0.5μg/mL) or T lymphocytes (LEW.1A) stimulated by allogeneic APC (LEW.1W; ratio 1:4). Cells were used for immunostaining with HO-1– and iNOS-specific antibodies (green staining, thin arrow) and anti-CD45 antibodies (red staining, large arrow). Thus, HO-1– and iNOS-positive MSCs are CD45−. Negative controls (preimmune rabbit serum) are shown in the inserts. MSCs and the same antibodies were also used for Western blots. The results are representative of 2 experiments. See “Image acquisition” for image acquisition infomation. (C) T lymphocytes (105) were activated by allogeneic APCs (0.25 × 105) in the absence or presence of adult rat MSCs at an MSC/T cell ratio of 1:1. SnPP (50 μM; Porphirin Products, Logan, UT) or L-NMMA (10 mM; Sigma-Aldrich, Saint Quentin Fallavier, France), the specific inhibitors of HO-1 and iNOS, respectively, were added alone or together to the culture. Proliferation was measured by thymidine incorporation. The results are representative of 7 experiments. Error bars represent SD. (D) Heart allografts from LEW.1W donors were transplanted heterotopically into LEW.1A recipients. Lewis rat recipients were left untreated (control group; dotted line; n = 5) or received intravenous administration of LEW.1W MSCs (12 × 106) 7 days before (●; n = 5), at the time of LEW.1W heat transplantation (▴; n = 5) or at both times (5 × 106 and 7 × 106 LEW.1W MSCs, respectively; ▵; n = 5). Rats receiving 2 donor MSC injections were treated from the day of MSC administration by intraperitoneal injections of SnPP (15 μg/rat) once every 4 days and aminoguanidine (Sigma-Aldrich; 0.1 g/rat) twice a day until rejection (◇;n = 9), or with SnPP (15 μg/rat) alone (□; n = 5) or aminoguanidine (0.1 g/rat) alone (○). Graft survival was monitored daily by abdominal palpation.
