Phenotype characterization of negatively isolated RA-SFB
To verify whether isolated RA-SFB displayed the features observed in situ in the RASM [1,2,12,13,14,15, 21,22,23,24,25,28], the expression of several surface or intracellular/nuclear molecules was investigated by FACS analysis (Fig. 6 and Table 4). The phenotype was compared with that of primary-culture normal skin-FB and isolated primary-culture OA-SFB (Fig. 6 and Table 4).

Surface antigens
Approximately 74% of RA-SFB expressed the Thy-1 antigen (Fig. 6G and Table 4), compared with 91% of normal skin-FB (Fig. 6A and Table 4). Approximately 77% of the cells expressed MHC-I molecules, compared with 90% of normal skin-FB (Table 4). Strikingly, approximately 66% of the cells expressed MHC-II molecules (Fig. 6H and Table 4), significantly more than OA-SFB (17%; Fig. 6E and Table 4) and normal skin-FB (2%; Fig. 6B and Table 4). At the same time, however, the MFI for MHC-II did not significantly differ between RA-SFB and normal skin-FB (Table 4).
The expression of CD13 (aminopeptidase N; EC 3.4.11.2) on 85% of RA-SFB was comparable with that of normal skin-FB (84%; Table 4).
A low or moderate and variable percentage of normal skin-FB, as well as OA-SFB and RA-SFB, expressed VCAM-1 (using two different anti-VCAM-1 mAbs; see Table 3) without statistically significant differences among these three different FB preparations (Table 4).

Cytoplasmic antigens
Approximately 85% of the RA-SFB expressed the FB marker prolyl-4-hydroxylase (Fig. 6M, and Tables 1 and 4), comparable with the expression in normal skin-FB (71%; Tables 1 and 4).
RA-SFB showed a similar percentage but a higher MFI for the cytoskeletal protein vimentin (Fig. 6I) than normal skin-FB (Fig. 6C and Table 4), although the MFI difference did not attain statistical significance.
A mean of approximately 45% and 50% of the cells, respectively (Table 4), expressed procollagen I (Fig. 6J) and procollagen III (Fig. 6K), as confirmed by double-staining (Fig. 6L). The percentage of cells expressing procollagen I and III was similar to that of normal skin-FB (68% and 55%, respectively; Table 4); however, the MFI for procollagen III was significantly higher in RA-SFB (26 versus 15; Table 4).

Cytoplasmic/intranuclear antigens
Approximately 57% of RA-SFB expressed c-Fos (Fig. 6N and Table 4). Neither the percentage nor the MFI were significantly different from those of normal skin-FB.
The proto-oncogenes c-Jun and Jun-D (both percentage of positive cells and MFI) were also comparably expressed in RA-SFB and normal skin-FB (Fig. 6O and Table 4).
The differences between RA-SFB and normal skin-FB were, in general, not specific to RA since they were also observed in the comparison between the disease control OA-SFB and normal skin-FB (Table 4). In fact, some of the differences in OA were even more pronounced than in RA, attaining statistical significance in the direct comparison between OA and RA (Table 4). Briefly, significantly higher MFI levels in OA-SFB were noted for MHC-I, MHC-II and procollagen I, as well as c-Fos. Opposite differences among the different FB preparations, when considering the percentage of positive cells or the MFI (eg for the comparison of MHC-II and procollagen I and III expression in RA-SFB versus OA-SFB), appeared due to large variations in one or both parameters.

Comparison of FACS analysis and immunohistochemistry
To verify whether the short-term trypsin digestion (2 min at 37°C) employed for removal of the cells from the culture dishes and subsequent FACS analysis altered the expression of the antigens, selected experiments were carried out to compare the expression of surface and intracellular antigens in FACS analysis (Table 1b,e, and Table 4) and immunohistochemistry on chamber slides. Negatively isolated SFB from n = 3 OA and n = 3 RA patients showed comparable percentages of positive cells for surface antigens (RA-Thy-1, 95.3% FACS versus 85% immunohistochemistry; CD14, 1.1% versus 0%; VCAM-1, 0.7% versus 0%) and intracellular antigens (RA-prolyl-4-hydroxylase, 94.7% versus 85%; CD68, 1.7% versus 0%) with both methods, confirming previously published results [47] and excluding the possibility that short-term trypsinization significantly influences antigen expression.