Supplementary material

Supplementary material

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  • Supplementary material -
    Supplementary materials and methods
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  • Figure S1 - Effects of in vivo exposure to apoptotic cells into unstimulated lungs on production and mRNA expression of HGF. Two hours after intratracheal instillation of saline alone (Sal), or apoptotic Jurkat T cells (ApoJ) into unstimulated lungs, bronchoalveolar lavage was performed. (A, C) HGF production in BAL fluid samples and alveolar macrophage cultures was quantified by ELISA. (B) HGF mRNA levels in lung homogenates were analyzed by relative quantitative RT-PCR and normalized to β-actin mRNA levels. (D) Phagocytic indices (PIs) were measured in BAL alveolar macrophages. Values represent means � SEM from groups of five mice. * Significantly different from saline control, p<0.05.
  • Figure S2 - In vivo production and mRNA expression of HGF in bleomycin-stimulated lungs are increased by apoptotic cell clearance. Two hours after intratracheal instillation of saline alone (BLM+Sal), apoptotic HeLa cells (BLM+ApoH), or viable HeLa cells (BLM+ViaH) into bleomycin-treated lungs (day 2), bronchoalveolar lavage was performed. (A, C) HGF production in BAL fluid samples and alveolar macrophage cultures was quantified by ELISA. (B) HGF mRNA levels in lung homogenates were analyzed by relative quantitative RT-PCR and normalized to β- 15 actin mRNA levels. (D) Phagocytic indexes (PIs) were measured in BAL alveolar macrophages. Values represent means � SEM from groups of five-ten mice. * Significantly different from saline control, p<0.05. +Significant differences between the BLM+ApoH group versus the BLM+Sal or BLM+ViaH group, p<0.05.
  • Figure S3 - Anti-HGF antibody abrogated the anti-inflammatory and anti-apoptotic effects of apoptotic cell instillation. Rabbit anti-HGF neutralizing antibody (anti-HGF Ab) or normal goat IgG (Isotype Ab) was i.p. coadministrated with saline (Sal) alone or apoptotic Jurkat T cells (ApoJ) into bleomycin-stimulated lungs (2 days), and mice were euthanized on days 3 following bleomycin treatment. To inhibit sequential induction of HGF, the anti-HGF antibody or isotype antibody was administrated once more sequentially at 5 days after bleomycin treatment and mice were euthanized on days 7 following bleomycin treatment. (A) TNF-α, (B) MIP-2, and (C) TGF-β1 levels in BAL fluid were quantified by ELISA. (D) Neutrophil and (E) total protein levels in BAL fluid. (F, G) Caspase-3 and 9 activities in lung tissue. Values represent means � SEM from groups of 5 mice. +Significant differences between the groups, p<0.05.
  • Figure S4 - Contribution of HGF to the inhibition of proinflammatory cytokines which occurs after apoptotic cell treatment to LPS or bleomycin-stimulated macrophages in vitro. (A-E) RAW 264.7 macrophages were treated with 1 ng/ml LPS or 5 μg/ml bleomycin at the same time, apoptotic cells, viable Jurkat cells, or apoptotic cells and anti-HGFR were added. Supernatants were collected 18 h later. (A, D) TNF-α, (B, E) MIP-2, and (C) HGF levels in the conditioned medium were quantified by ELISA. Values represent means � SEM of five separate experiments. + p<0.05.
  • Figure S5 - Proliferating cell nuclear antigen (PCNA) protein expression and immunostaining in lung tissue. Mice were i.t. instilled with saline (BLM+Sal) or apoptotic Jurkat T cells (BLM+ApoJ) 2 days after bleomycin treatment and lungs were harvested on days 3-21. (A) Lung tissue homogenates were immunoblotted for PCNA. Relative values of PCNA versus β-actin are indicated below the gel. (B) Representative photomicrographs showing PCNA-positive cells on days 7 and 21 after bleomycin treatment. Dark brown nuclei indicate positive staining. Original magnifications: �200. (C) Relative-quantification of histological assessment of PCNA-positive cells. Values represent means � SEM from groups of five mice. * Significantly different from control, p<0.05. +Significant differences between the BLM+ApoJ group versus the BLM+Sal 17 group, p<0.05.
  • Figure S6 - HGF receptor antibody further enhances TGF-β1 production induced by apoptotic cells. RAW macrophages were pretreated 10 μg/ml anti-HGFR antibody or 10 μg/ml IgG for 1 hour and then stimulated with apoptotic Jurkat cells for 18 hours to detect TGF-β1 production. TGF-β1 levels in the conditioned medium were measured by ELISA. Values represent means � SEM of five separate experiments, *p<0.05.