Fig 2: CD248 is abundantly expressed by synovial MSCs in RA inflammatory tissues. (a–o): Frozen synovial tissue sections from an RA patient were stained with affinity-purified mouse (P1A6) or affinity-purified rabbit anti-CD248 (white arrows). Sections were double-stained using antibodies against CD90 (Thy-1), CD31, CD93, and VEGF followed by TRITC-conjugated and FITC-conjugated. Images were viewed using a fluorescent DMLB microscope. Insets in panels a and b depict background stainings using irrelevant antibodies. Magnification ×100 (a–c); ×200 (d–f), and ×400 for panels (g–o). (p–r): RA fibroblasts cultured on glass coverslips were double-stained for CD248 (affinity-purified rabbit antibody) and monoclonal antibodies against either CD141, CD63, or EEA1. Magnification (×400 for (p,q) and ×1000 for panel (r)). Irrelevant antibodies failed to stain fibroblast cells (inset, panel (p)).

Fig 3: CD248 expression by HFFFs in response to cytokines and growth factors: release of a cleaved soluble form. Quiescent HFFFs were treated for 24 h with 20 ng/mL rhVEGF165, 10 ng/mL bFGF, 1 ng/mL TGF-β1, 200 U/mL IL1-β, 50 ng/mL PMA, or were left untreated. (A) Flow cytometric staining of cells with affinity-purified rabbit anti-CD248, and expression of the results as fold changes in the mean of fluorescence over non-treated cells. (B) Western blot of whole cell lysates with affinity-purified rabbit anti-CD248 and comparison of the intensity of the CD248-specific band (160–170 kDa) following treatment with that of the non-treated cells showed no significant change in total cell CD248 expression following any treatment (p > 0.05). Unequal loading was accounted for by blotting for α-tubulin (50 kDa band). The blots are representative of three separate experiments. The graph represents the mean change in intensity compared with non-treated cells ± SEM, n = 3. (C) TCS was collected from treated cells, freeze-dried and subjected to Western blotting with affinity-purified rabbit anti-CD248. Bands of 110 kDa were observed and the intensities of the bands following treatment were compared to the intensity of the band in non-treated cells. Histograms show the analysis of the band intensity of the WB (n = 1). The blot and graph are representative of three separate experiments. The bars represent the fold increase in intensity over non-treated cells.

Fig 4: Perivascular synovial MSCs (pMSCs) express CD248 (endosialin) and may remain at the perivascular level through the multimerin–CD93 anchoring complex. In inflammatory conditions (e.g., RA), interleukin-1 beta produced by macrophages and stimulating the release of MMP will contribute to the shedding of CD248. Membrane CD248 is known to control the PDGF-induced proliferation of MSCs (switching to an aggressive phenotype involved in pannus formation) but the role of sCD248 in this process or as a decoy receptor remains to be ascertained. Microscope image is shown at ×400.

Fig 5: Paraffin wax RA tissue sections double-stained for CD248 and either CD45 (leukocytes) or endothelial (ERG) cell markers. Serial formalin-fixed paraffin-embedded (FFPE) sections were processed using sequential alkaline-phosphatase-based immunostaining with anti-CD248 (clone E9Z7O, red staining) and diaminobenzidine-based immunostaining with (a) anti-CD45 (clone X16/99, Leica) or (b) anti-ERG (clone EPR3864, Ventana).