Development of macrophage cytosensors capable of detecting programming signals within models of glomerular inflammation
We have developed a novel TGF-β detection system, using genetically modified macrophages as cytokine biosensors, capable of detecting bioactive TGF-β in vivo and in vitro. Stimulation by TGF-β1 increases secretion of plasminogen activator inhibitor-1 (PAI-1) by activation of the promoter. An adenovirus containing the first 800bp of the PAI-1 promoter fused to the reporter gene β-galactosidase (Ad-PAI1800 βgal) was used to transfect primary cultures of rat bone marrow derived macrophages (BMDM). After transfection the BMDM were stimulated with TGF-β, 2 or 3 or left unstimulated. X-gal histochemical assay was performed 72-hours post-transfection to detect β-galactosidase expression. Macrophages transfected with Ad-PAI1800βgal and stimulated with TGF-β1, 2 or 3 showed a dose-dependent production of β-galacosidase. AdPAI1800βgal BMDM also expressed β-galactosidase when co-cultured with NR8383 rat alveolar macrophages transfected with recombinant adenovirus expressing active TGF-β1. Genetically modified macrophages could detect expression of TGF-β1 in vivo. AdPAI1800βgal transfected BMDM were co-injected with AdTGF-βl transfected NR8383 macrophages into the renal artery of rats with nephrotoxic nephritis (NTN). Isolated glomeruli showed X-gal positive macrophages on assay. AdPAI1800βgal BMDM were also injected into the renal artery of rats 7 days after the induction of Thy 1.1 nephritis, 100% of isolated glomeruli contained X-gal positive macrophages on assay. Signalling macrophages were capable of detecting TGF-β1 expression in sections of kidney tissue containing Ad-TGF-β1 transfected NR8383 macrophages. BMDM showing β-galactosidase were readily detected over the transfected glomeruli. Pre-treatment of Ad-PAI1800βgal transfected BMDM with IFN-γ prevented production of the reporter gene. However when stimulation with TGF-β occurred 20 hours after IFN-γ macrophage programmed unresponsiveness was abrogated. Macrophage responsiveness to TGF-β stimulation in Thy 1.1 nephritic kidneys was also prevented by pre-treatment with IFN-γ. This system demonstrates the use of genetically modified macrophages as both in vivo and in vitro sensors of the bioactive TGF-β providing a novel approach to assess cytokine activity.