Thy-1 expression in human fibroblast subsets defines myofibroblastic or lipofibroblastic phenotypes

Abstract

Fibroblasts represent a dynamic population of cells, exhibiting functional heterogeneity within and among tissues. Fibroblast heterogeneity also results from phenotypic differences and may arise from activation or differentiation processes taking place in the cells. We previously reported that human fibroblasts were heterogeneous with respect to surface Thy-1 expression and that separation into Thy-1(+) and Thy-1(-) subsets resulted in functionally distinct subpopulations, leading to the concept of fibroblast subset specialization. In this report we investigated whether Thy-1(+) and/or Thy-1(-) fibroblasts were capable of differentiating into myofibroblasts or lipofibroblasts. Fibroblast subsets were used from human myometrium and orbit to test this hypothesis. Only Thy-1(+) human myometrial and orbital fibroblasts were capable of myofibroblast differentiation after treatment with TGFbeta or platelet concentrate supernatant, assessed by alpha smooth muscle actin expression. Interestingly, only Thy-1(-), but not Thy-1(+) subsets differentiated to lipofibroblasts, as determined by the accumulation of cytoplasmic lipid droplets after treatment with 15-deoxy-Delta(12, 14)-PGJ(2) or ciglitazone. We propose that fibroblast Thy-1 display pre-determines lineage to a contractile or lipid-like phenotype in the human myometrium and orbit. This additional distinction between Thy-1(+) and Thy-1(-) human fibroblast subtypes has important consequences in normal tissue homeostasis and in pathogenesis of orbital and myometrial diseases characterized by persistent myofibroblasts or fat accumulation, such as occurs in Graves' ophthalmopathy, tissue fibrosis, abnormal wound healing, and scarring.

Figure 1.

αSMA is only expressed in Thy-1⁺ human myometrial fibroblasts. Primary myometrial fibroblasts were double-stained by immunofluorescence for αSMA (PE; red) and Thy-1 (FITC; green) after a 4-day TGFβ treatment (5 ng/ml), as described in Materials and Methods. Isotype controls for αSMA and Thy-1 were mIgG2a and mIgG1 respectively, and stained negative for PE or FITC. Fibroblasts within the parental culture stained positive for both αSMA and Thy-1. Fibroblasts negative for Thy-1 were also negative for αSMA, and they are indicated in the phase picture by arrows. Original magnification, ×20.

Figure 2.

αSMA is only expressed in Thy-1⁺ human orbital fibroblasts. Primary Graves’ orbital fibroblasts were treated with TGFβ for 4 days and then double-stained for αSMA by PE labeling (red) and for Thy-1 with FITC (green), as described in Materials and Methods. Isotype controls mIgG2a and mIgG1 stained negative for PE or FITC. Fibroblasts within the parental orbital culture that expressed Thy-1 were also positive for αSMA. Fibroblasts negative for Thy-1 did not stain for αSMA, and they are indicated in the phase picture by arrows. Original magnification, ×20.

Figure 3.

TGFβ and platelet concentrated supernatant (PCS)-induced αSMA expression in Thy-1⁺, but not Thy-1⁻ human myometrial fibroblasts; IFNγ antagonizes αSMA induction. Myometrial fibroblasts were left unstimulated or treated with TGFβ (5 ng/ml), PCS (1:50), IFNγ (500 U/ml), TGFβ with IFNγ or PCS with IFNγ for 4 days. Cells were then stained for αSMA as described in Materials and Methods. A: Unstimulated Thy-1⁺ myometrial fibroblasts express constitutive αSMA in a heterogeneous fashion. TGFβ and PCS induce αSMA expression in Thy-1⁺ fibroblasts, and this up-regulation is reduced by IFNγ. Original magnification, ×40. B: Thy-1⁻ myometrial fibroblasts do not express αSMA, even after TGFβ or PCS treatments. Original magnification, ×20.

Figure 4.

αSMA expression is up-regulated in TGFβ-and PCS-treated Thy-1⁺, but not Thy-1⁻ human orbital fibroblasts; IFNγ inhibits αSMA up-regulation. Graves’ orbital fibroblasts were left unstimulated or treated with TGFβ (5 ng/ml), PCS (1:50), IFNγ (500 U/ml), TGFβ with IFNγ, or PCS with IFNγ for 4 days. Cells were then stained for αSMA as described in Materials and Methods. A: Unstimulated Thy-1⁺ orbital fibroblasts do not express αSMA. After TGFβ and PCS treatments, αSMA expression is induced and this up-regulation is completely inhibited by IFNγ. Original magnification, ×40. B: Thy-1⁻ orbital fibroblasts are not positive for αSMA staining when treated with TGFβ or PCS. Original magnification, ×40.

Figure 5.

TGFβ induces αSMA protein expression in Thy-1⁺ myometrial and orbital fibroblast subsets and up-regulation is inhibited by IFNγ. Myometrial and orbital fibroblasts were left unstimulated or treated with TGFβ (5 ng/ml) for 24 hours, 48 hours, 4 days, and 6 days, with or without IFNγ. Cells were harvested in lysis buffer and subjected to Western blot analysis, as described in Materials and Methods. Human lung fibroblasts treated with TGFβ for 72 hours, were used as positive control indicates by (+). Unstimulated cells are designated by “untx”. A: Myometrial Thy-1⁺ fibroblasts express constitutive αSMA, which is up-regulated as early as 24 hours with TGFβ treatment, and is sustained through 6 days. IFNγ reduces the induced αSMA to basal levels. Thy-1⁻ myometrial fibroblasts do not express αSMA under the same treatments. B: αSMA in Thy-1⁺ orbital fibroblasts is up-regulated at 48 hours and peaks to high levels at 4 and 6 days after TGFβ treatment. IFNγ completely blocks αSMA induction. Thy-1⁻ orbital fibroblasts do not display αSMA expression.

Figure 6.

PCS up-regulates αSMA protein in Thy-1⁺ myometrial and orbital fibroblasts and induction is reduced by IFNγ. Myometrial and orbital fibroblasts were left unstimulated or treated with PCS (1:50) for 24 hours, 48 hours, 4 days, and 6 days, with or without IFNγ. Cells lysates were subjected to Western blot analysis, as described in Materials and Methods. Human lung fibroblasts treated with TGFβ for 72 hours, were used as positive control indicated by (+). Unstimulated cells are designated by “untx”. A: PCS induces αSMA in myometrial Thy-1⁺ fibroblasts after 4 and 6 days of treatment. IFNγ reduces the induced αSMA to basal levels. Thy-1⁻ myometrial fibroblasts do not express αSMA under PCS treatments. B: αSMA in Thy-1⁺ orbital fibroblasts peaks at 4 and 6 days after PCS treatment. IFNγ inhibits αSMA induction. Thy-1⁻ orbital fibroblasts do not express αSMA under the same conditions.

Figure 7.

Differentiation to a lipofibroblastic phenotype is restricted to Thy-1⁻ fibroblasts from human myometrium and orbit. Human myometrial and orbital fibroblast subsets were treated with DMSO (vehicle control) or 15-day-PGJ₂ (10 μmol/L) every other day for 6 days. Cells were then stained for cytoplasmic lipid droplets by Oil Red O. A: Only Thy-1⁻ myometrial fibroblasts acquire lipid droplets after treatment with 15-day-PGJ₂. B: Thy-1⁻, but not Thy-1⁺ orbital fibroblasts accumulate lipid droplets in their cytoplasm when treated with 15-day-PGJ₂.