Figure 1.

Elevated TLR4 expression in scleroderma skin. A: Skin biopsy specimens from the lesional forearm of patients with scleroderma (n = 19) and forearms of healthy controls (n = 8) were examined by IHC. Left panels: Representative images. Brown staining indicates TLR4-positive cells. Scale bars: 200 μm (top panels); 50 μm (bottom panels). Blue, hematoxylin counterstain. Right panel: Quantitation of dermal cell TLR4 staining. Each dot, number (mean) of immune-positive spindle-shaped interstitial cells (fibroblast-like) from four separate microscopic fields per biopsy. B: Double-immunofluorescence labeling using antibodies to TLR4 and CD31 or α-SMA, or stained with DAPI. Yellow, colocalization of two antibodies. Arrows, colocalization. Scale bar = 50 μm. C: Cell lysates from explanted skin fibroblasts from healthy controls (n = 3) and patients with scleroderma (n = 4) were subjected to Western blot analysis using antibodies against TLR4 and type I collagen (Cgn I). Left panel: Representative immunoblots. Right panel: Quantitation of TLR4 levels. Values represent the mean ± SD, corrected for tubulin in each lane. D: Explanted healthy control (n = 3) and scleroderma (n = 3) fibroblasts were incubated with CLI-095 (3 μmol/L) for 24 hours. RNA and protein were isolated and subjected to qPCR. Left panel: Mean ± SD of triplicate determinations relative to levels in untreated fibroblasts. Right panel: Western blot analysis. Values shown indicate relative levels corrected for tubulin in each lane. E: Immunofluorescence. Antibodies to Fn-EDA or tenascin-C, or HA-binding protein, were used on skin biopsy specimens from healthy controls (n = 3) and patients with scleroderma (n = 5). Scale bar = 50 μm. *P < 0.05.