Effect Of ISS And TGF-β1 On MLg Lung Fibroblast Proliferation In Vitro

Abstract

RATIONALE: Since CpG-rich immunostimulatory DNA sequences (ISS) reduced peribronchial fibrosis in the chronic ovalbumin murine asthma model of airway remodeling, we hypothesized that ISS might act in part through a TLR9-dependent effect on fibroblast proliferation.METHODS: RT-PCR was used to determine whether TLR-9 was expressed by MLg, a murine lung fibroblast cell line. MLg fibroblast proliferation was assayed with a BrdU ELISA at 48h. MLg fibroblasts were incubated after a growth arrest phase with ISS 10 μg/ml (a ligand for TLR9), or M-ODN 10 μg/ml (modified oligodeoxynucleotides, which are not TLR9 ligands) with or without TGF-β1 15 ng/ml.RESULTS: MLg fibroblasts expressed TLR-9 as assessed by RT-PCR. MLg fibroblasts incubated with ISS alone had increased proliferation relative to both M-ODN (p<0.01) and untreated media (p<0.02). In contrast, MLg fibroblasts incubated with the combination of ISS and TGF-β1 had reduced proliferation relative to media (p<0.03). The combination of M-ODN and TGF-β1 also inhibited MLg fibroblast proliferation (p<0.03). TGF-β1 alone did not significantly affect proliferation of MLg fibroblasts.CONCLUSIONS: MLg fibroblasts express TLR-9 as assessed by RT-PCR. In the absence of TGF-β1, ISS induced proliferation of MLg fibroblasts in a TLR-9 dependent manner. In the presence of TGF-β1, exogenous DNA (either ISS or M-ODN) inhibited proliferation of MLg fibroblasts in a DNA sequence-independent fashion. Thus in MLg fibroblasts, ISS may either induce proliferation, or inhibit proliferation dependent upon the presence of extracellular TGF-β1. RATIONALE: Since CpG-rich immunostimulatory DNA sequences (ISS) reduced peribronchial fibrosis in the chronic ovalbumin murine asthma model of airway remodeling, we hypothesized that ISS might act in part through a TLR9-dependent effect on fibroblast proliferation. METHODS: RT-PCR was used to determine whether TLR-9 was expressed by MLg, a murine lung fibroblast cell line. MLg fibroblast proliferation was assayed with a BrdU ELISA at 48h. MLg fibroblasts were incubated after a growth arrest phase with ISS 10 μg/ml (a ligand for TLR9), or M-ODN 10 μg/ml (modified oligodeoxynucleotides, which are not TLR9 ligands) with or without TGF-β1 15 ng/ml. RESULTS: MLg fibroblasts expressed TLR-9 as assessed by RT-PCR. MLg fibroblasts incubated with ISS alone had increased proliferation relative to both M-ODN (p<0.01) and untreated media (p<0.02). In contrast, MLg fibroblasts incubated with the combination of ISS and TGF-β1 had reduced proliferation relative to media (p<0.03). The combination of M-ODN and TGF-β1 also inhibited MLg fibroblast proliferation (p<0.03). TGF-β1 alone did not significantly affect proliferation of MLg fibroblasts. CONCLUSIONS: MLg fibroblasts express TLR-9 as assessed by RT-PCR. In the absence of TGF-β1, ISS induced proliferation of MLg fibroblasts in a TLR-9 dependent manner. In the presence of TGF-β1, exogenous DNA (either ISS or M-ODN) inhibited proliferation of MLg fibroblasts in a DNA sequence-independent fashion. Thus in MLg fibroblasts, ISS may either induce proliferation, or inhibit proliferation dependent upon the presence of extracellular TGF-β1.