Figure 2

Activation of IL-13/TGF-β ₁ pathway in allogeneically transplanted grafts. (A) ELISA of supernants of cultured allograft-infiltrating cells detected significantly elevated IL-13 levels in allografts (day 60, P = 0.0031 and day 100, P = 0.0003)compared to syngrafts or FVB control hearts (P = 0.0055 and P = 0.0042). (B) Immunohistochemistry showed significantly higher and over time increasing numbers of IL-13⁺ cells in FVB hearts transplanted into DBA/1 recipients (day 60, P = 0.0228; day 100, P = 0.0037) relative to syngeneic animals (day 60 versus day 100, P = 0.0083). (C) Representative immunohistochemical stainings showed more IL-13⁺ cells in allografts ( FVB into DBA/1) compared to controls (DBA/1 into DBA/1; day 100). (D) Western blot analysis revealed expression of IL-13Rα₂ only in allograft-infiltrating cells isolated from allogeneically transplanted hearts (FVB into DBA/1) in contrast to cells isolated from FVB controls or syngrafts (DBA/1 into DBA/1) without IL-13Rα₂ expression. (E) Measurement of TGF-β₁ by ELISA detected significantly elevated TGF-β₁ levels in cells isolated from DBA/1 mice grafted with FVB hearts at day 60 (88 ± 5 versus 46 ± 5 pg/mL; P = 0.0010) and at day 100 (133 ± 6 versus 42 ± 7 pg/mL; P <0.0001) in comparison to syngrafts,and FVB control hearts (40 ± 8 pg/mL; P = 0.0048 and P = 0.0009, respectively). (F) Flow cytometry of graft-infiltrating cells extracted from allografts showed a higher percentage of CD11b^highGr1^intermediateTGF-β₁ ⁺ cells (7.3%) than in the syngeneic controls (1.2%; day 100). (G) In the flow cytometric analysis (pre-gated for CD45) these CD11b^high cells were the only source of TGF-β₁ production. The histological score is the mean of 3 HPF (20x magnification); at least five mice per group were analyzed. *P <0.05. ELISA, enzyme-linked immunosorbent assay; HPF, high power field; IL-13, interleukin 13; TGF-β1, transforming growth factor beta 1.