Figure 4
Figure 4. HGF-R/c-met is the receptor providing the costimulatory signal for mast-cell activation. (A) Purified PMCs (5 × 104) isolated from WT mice were incubated for 1 hour with a washed suspension of Listeria and anti-Listeria antibody alone (LA), Listeria, anti-Listeria antibody plus 50% serum from WT mice (LAS), latex beads coated with bovine serum albumin (BSA) plus anti-BSA antibody alone (BA), BSA plus anti-BSA antibody and 50% WT murine serum (BAS), BSA plus anti-BSA antibody and 50% WT murine serum (BAS) with either LPS (100 ng/mL), Pam3Cys (100 μg/mL), Listeria (108), heat-killed Listeria (108 organisms), or LPS (100 ng/mL), Pam3Cys (100 μg/mL), Listeria (108), heat-killed Listeria (108 organisms) alone. Supernatants were collected and analyzed for IL-6 production by ELISA. (B) Representative flow cytometric histograms of PMCs stained with c-met PMCs from WT (panel A) and α2β1 integrin-deficient (KO, panel B) were stained with phycoerythrin–anti–c-kit and APC–anti–c-met and assessed by flow cytometry. Mast cells were identified as c-kithigh–staining cells and represented 1% to 3% of resident peritoneal cells in both WT and KO mice. (C) Purified PMCs isolated from WT mice were pretreated with inhibitory antibodies toward E-cadherin, c-met, or irrelevant control antibody for 1 hour before stimulation with a washed suspension of Listeria, anti-Listeria antibody alone, or Listeria, anti-Listeria antibody, and 50% WT murine serum. Supernatants were collected and analyzed for IL-6 production by ELISA. (D) Purified PMCs (5 × 104) isolated from WT mice were incubated with Listeria and anti-Listeria antibody alone (LA), Listeria, and anti-Listeria antibody plus 50% serum from WT mice (LAS), latex beads coated with BSA, plus anti-BSA antibody alone (BA), or latex beads coated with BSA, anti-BSA antibody, plus 50% serum (BAS) plus or minus the addition of Listeria (108) or HGF (2 mg/mL), or Listeria (108), or HGF (2 mg/mL) alone. Supernatants were collected and analyzed for the concentration of IL-6 by ELISA. (E) Purified PMCs (5 × 104) isolated from WT mice were allowed to adhere to a matrix of Listeria and anti-Listeria antibody (LA), Listeria, anti-Listeria antibody plus serum (LAS), type I collagen (25 mg/mL), C1q (25 mg/mL), or tissue culture plastic (No Matrix) with or without either Listeria (108) or HGF (2 mg/mL). Supernatants were collected and analyzed for IL-6 production by ELISA. All experiments were carried out in the presence of 2 mM MgCl2. Results are presented as means plus or minus SEM from triplicate wells of a single experiment and represent 1 of at least 3 experiments demonstrating similar results. The P values were determined by unpaired Student t test (*P < .05, **P < .01).

HGF-R/c-met is the receptor providing the costimulatory signal for mast-cell activation. (A) Purified PMCs (5 × 104) isolated from WT mice were incubated for 1 hour with a washed suspension of Listeria and anti-Listeria antibody alone (LA), Listeria, anti-Listeria antibody plus 50% serum from WT mice (LAS), latex beads coated with bovine serum albumin (BSA) plus anti-BSA antibody alone (BA), BSA plus anti-BSA antibody and 50% WT murine serum (BAS), BSA plus anti-BSA antibody and 50% WT murine serum (BAS) with either LPS (100 ng/mL), Pam3Cys (100 μg/mL), Listeria (108), heat-killed Listeria (108 organisms), or LPS (100 ng/mL), Pam3Cys (100 μg/mL), Listeria (108), heat-killed Listeria (108 organisms) alone. Supernatants were collected and analyzed for IL-6 production by ELISA. (B) Representative flow cytometric histograms of PMCs stained with c-met PMCs from WT (panel A) and α2β1 integrin-deficient (KO, panel B) were stained with phycoerythrin–anti–c-kit and APC–anti–c-met and assessed by flow cytometry. Mast cells were identified as c-kithigh–staining cells and represented 1% to 3% of resident peritoneal cells in both WT and KO mice. (C) Purified PMCs isolated from WT mice were pretreated with inhibitory antibodies toward E-cadherin, c-met, or irrelevant control antibody for 1 hour before stimulation with a washed suspension of Listeria, anti-Listeria antibody alone, or Listeria, anti-Listeria antibody, and 50% WT murine serum. Supernatants were collected and analyzed for IL-6 production by ELISA. (D) Purified PMCs (5 × 104) isolated from WT mice were incubated with Listeria and anti-Listeria antibody alone (LA), Listeria, and anti-Listeria antibody plus 50% serum from WT mice (LAS), latex beads coated with BSA, plus anti-BSA antibody alone (BA), or latex beads coated with BSA, anti-BSA antibody, plus 50% serum (BAS) plus or minus the addition of Listeria (108) or HGF (2 mg/mL), or Listeria (108), or HGF (2 mg/mL) alone. Supernatants were collected and analyzed for the concentration of IL-6 by ELISA. (E) Purified PMCs (5 × 104) isolated from WT mice were allowed to adhere to a matrix of Listeria and anti-Listeria antibody (LA), Listeria, anti-Listeria antibody plus serum (LAS), type I collagen (25 mg/mL), C1q (25 mg/mL), or tissue culture plastic (No Matrix) with or without either Listeria (108) or HGF (2 mg/mL). Supernatants were collected and analyzed for IL-6 production by ELISA. All experiments were carried out in the presence of 2 mM MgCl2. Results are presented as means plus or minus SEM from triplicate wells of a single experiment and represent 1 of at least 3 experiments demonstrating similar results. The P values were determined by unpaired Student t test (*P < .05, **P < .01).

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