FACS analysis
FACS analysis showed that at T0 the percentage of fibrocytes, identified as CD45+COL I+CXCR4+ cells, was 1.0 ± 1.2% in SSc patients and 0.5 ± 0.2% in HSs (50% less) (Fig. 1a). Moreover, in this fibrocyte population, the percentage of HLA-DR+ cells was very low (22.1 ± 21.1% and 13.1 ± 4.7%, respectively), whereas the percentage of CD86+ cells was higher in both SSc patients and HSs at T0 (34.4 ± 21.4% and 68.9 ± 27.6%) (Fig. 1a).
At T8, fibrocytes showed an adherent spindle-shaped morphology, and FACS analysis demonstrated that the percentage of CD45+COL I+CXCR4+ fibrocytes was significantly higher in both SSc patients and in HSs compared with T0 (up to 52.8 ± 27.1% vs. 1.0 ± 1.2% and up to 61.9 ± 24.4% vs. 0.5 ± 0.2%, respectively) (p < 0.01) (Fig. 1b).
At the same time, in this fibrocyte population, the HLA-DR+ cells were significantly increased in SSc patients and HSs compared with T0 (90.1 ± 22.7% vs. 22.1 ± 21.1% and 97.9 ± 1.9 vs 13.1 ± 4.7%, respectively) (p < 0.01) (Fig. 1b).
Similarly, the percentage of CD86+ fibrocytes was higher in SSc patients and HSs compared with T0 (60.4 ± 25.6% vs. 34.4 ± 21.4%, and 90.7 ± 10.9% vs. 68.9 ± 27.6%, respectively) with a greater increment in SSc fibrocytes (Fig. 1b).
Quantitative real-time PCR
SSc fibrocytes
At T8, in the absence of CTLA4-Ig, SSc fibrocytes showed higher gene expression levels of CD86, αSMA, S100A4, TGFβ, and COL I compared with HS fibrocytes (Fig. 1c).
The SSc fibrocytes treated for 3 h with various concentrations of CTLA4-Ig (10, 50, 100, and 500 μg/ml) did not show any significant variations in the gene expression levels of TGFβ, IL-1β, and CXCR2 compared with CNT (Fig. 2a). In these cells, CD86 gene expression decreased (not significantly) after treatment with CTLA4-Ig 500 μg/ml (Fig. 2a).
Interestingly, the gene expression of COL I was significantly lower in SSc fibrocytes treated with CTLA4-Ig even at 10 μg/ml compared with CNT (p < 0.05) (Fig. 2a). Of note, αSMA gene expression also decreased after CTLA4-Ig treatment (significantly after CTLA4-Ig 10 μg/ml treatment, p < 0.05, and CTLA4-Ig 500 μg/ml treatment, p < 0.01), whereas S100A4 gene expression was significantly higher compared with CNT (p < 0.01) excluding at the concentration of 500 μg/ml (Fig. 2a).
Moreover, while treatment with CTLA4-Ig 500 μg/ml did not significantly reduce the gene expression of CXCR4, it did significantly reduce that of CD11a as compared with CNT (p < 0.05) (Fig. 2a).
HS fibrocytes
Unlike SSc fibrocytes, HS fibrocytes treated for 3 h with various concentrations of CTLA4-Ig (10, 50, 100, and 500 μg/ml) did not show any significant modulation in the gene expression levels of CD86 (Fig. 2b).
In addition, gene expression levels of TGFβ, CXCR2, COL I, CXCR4, and CD11a remained unchanged after CTLA4-Ig treatment compared with CNT, as did gene expression of αSMA and S100A4 (Fig. 2b).
SSc fibroblasts
Cultured SSc fibroblasts showed very low gene expression levels of CD86 compared with cultured macrophages obtained from the PBMCs of SSc patients, which were taken as positive controls for CD86 expression (Additional file 1).
Nevertheless, cultured SSc fibroblasts treated for 24 h with CTLA4-Ig (10, 50, 100, and 500 μg/ml) did not show any significant differences in gene expression levels of CD86 compared with CNT (a nonsignificant increase after treatment with CTLA4-Ig 10 μg/ml was observed) (Fig. 3a). At the same time, gene expression of COL I and FN was higher only in the cultured SSc fibroblasts which had been treated with the lowest concentration of CTLA4-Ig (10 μg/ml) (Fig. 3a).
In addition, in these cultured CTLA4-Ig-treated SSc cells, the gene expression levels of αSMA, TGFβ. and S100A4 were higher compared with CNT (significantly for αSMA and S100A4; p < 0.01) (Fig. 3a).
On the other hand, the cultured SSc fibroblasts treated for 48 h with CTLA4-Ig (10, 50, 100, and 500 μg/ml) did not show any significant modulation in the gene expression levels of CD86, FN, αSMA, TGFβ, and S100A4 compared with CNT, whereas the gene expression of COL I was significantly downregulated by the highest dose of CTLA4-Ig (p < 0.05) (Fig. 3b).
HS fibroblasts
Cultured HS fibroblasts treated with CTLA4-Ig for 24 h showed a significant decrease in the gene expression of CD86, although this was limited to the highest dose (500 μg/ml) compared with CNT (p < 0.05) (Fig. 3a).
Of note, the gene expression of TGFβ and S100A4 was significantly reduced by the higher doses of CTLA4-Ig (p < 0.05 after 100 and 500 μg/ml for TGFβ; p < 0.05 after 500 μg/ml for S100A4) (Fig. 3a).
Similar to the results obtained after 24 h of CTLA4-Ig treatment, cultured HS fibroblasts showed a significant decrease in the gene expression levels of CD86 compared with CNT after 48 h of treatment with CTLA4-Ig, though this was limited to the highest dose (500 μg/ml) (p < 0.05) (Fig. 3b).
After 48 h of treatment with the highest dose of CTLA4-Ig (500 μg/ml), gene expression of TGFβ and S100A4 was significantly reduced compared with CNT (both p < 0.05) (Fig. 3b).
Gene expression of COL I, FN, and αSMA was unchanged after both 24 and 48 h of CTLA4-Ig treatment in cultured HS fibroblasts (Fig. 3a, b).
Western blotting
The protein expression of COL I and FN in cultured SSc and HS fibroblasts was unchanged even after treatment with CTLA4-Ig at both 24 and 48 h compared with CNT (Fig. 4).