CD55 is upregulated by poly(I:C) and IL-1β on synovial fibroblasts.

<p>RA-derived synovial fibroblasts (<b>A, C-F</b>) and dermal fibroblasts (<b>B</b>) were starved overnight and subsequently stimulated for 2 days with 100 ng/ml TNFα, 100 ng/ml IFNγ, 100 ng/ml IL-1β, 1 ng/ml IL-6, 100 U/ml IFNα, 100 µg/ml LTA (TLR2 ligand), 100 µg/ml poly(I:C) (TLR3 ligand), 10 µg/ml LPS (TLR4 ligand), 100 µg/ml imiquimod (TLR7 ligand), or 10 µg/ml CpG oligonucleotides (TLR9 ligand). Expression of CD55 (<b>A</b> and <b>B</b>), CD46 (<b>C</b>) and CD59 (<b>D</b>) was studied by flow cytometry. <b>E,</b> Upregulation of CD55 in response to increasing concentrations of poly(I:C). <b>F,</b> Inhibition of CD55 upregulation by chloroquine (HCQ), an inhibitor of endosomal acidification, added prior to poly(I:C) stimulation. Indicated is the relative protein expression as percentage of the medium control (mean ± SD, n = 6 (<b>A</b>) and 3–5 (<b>B-F</b>)). *, p<0.05; **, p<0.005.</p

Expression of complement regulatory proteins on cultured FLS of patients with different forms of arthritis.

<p>CD55, CD46, and CD59 expression was measured by flow cytometry on cultured FLS from patients with RA, OA, PsA, and SpA. Indicated is the fold difference in mean fluorescence intensity (MFI) over respective isotype control Ig (cMFI) (mean, n = 4−5).</p

Poly(I:C)-induced upregulation of CD55 on synovial fibroblasts increases the binding capacity for CD97.

<p>Synovial fibroblasts were stimulated for 2 days with 100 µg/ml poly(I:C). Affinity for CD97 was measured with multivalent fluorescent probes loaded with recombinant CD97-3EGF or EMR2-2EGF (control). To confirm specificity, cells were preincubated with mAb CLB-CD97L/1, directed against the first SCR of CD55. On top, representative histogram plots are shown. The bars represent the fold difference in mean fluorescence intensity (MFI) for CD97-3EGF over EMR2-2EGF (mean ± SD, n = 3). *, p<0.05.</p

FLS express functional cytoplasmic dsRNA sensors.

<p>RA-derived synovial fibroblasts were stimulated for 16 h with the indicated concentrations (µg/ml) of poly(I:C), poly(I:C) with fugene, or 3pRNA with fugene to trigger, respectively, TLR3, MDA5, and RIG-I. Transcription levels of (<b>A</b>) TLR3, MDA5, and RIG-I, and (<b>B</b>) the anti-viral/pro-inflammatory response genes IFN β, IP-10, and TNFα was measured by quantitative and semiquantitative PCR, respectively. Depicted is the fold change gene expression compared to medium control (mean ± SD, n = 4) (<b>A</b>) or representative photographs (<b>B</b>). *, p<0.05, **, p<0.005.</p

Stimulation of cytoplasmic dsRNA receptors in FLS upregulates CD55 expression and, through MDA5, induces cell death.

<p>RA-derived synovial fibroblasts were stimulated for 2 days with the indicated concentrations (µg/ml) of poly(I:C), poly(I:C) with fugene, or 3pRNA with fugene to trigger, respectively, TLR3, MDA5, and RIG-I. <b>A,</b> Expression of CD55 analyzed by flow cytometry (mean ± SD, n = 6). <b>B,</b> Representative flow cytometry plots of annexin V and propidium iodide staining. <b>C,</b> Percentages of annexin V and/or propidium iodide-positive cells analyzed by flow cytometry (mean ± SD, n = 6). <b>D, E,</b> Effect of the pan-caspase inhibitor QVD on cell death and CD55 expression induced by intracellular delivery of poly(I:C) (mean ± SD, n = 3). *, p<0.05; **, p<0.005; ***, p<0.001.</p

Relationship between stromal markers and diagnostic group in treatment-naive early arthritis tissue.

<p>Expression of (A) FAP, (B) podoplanin, (C) CD248, (D) CD55 were measured by pixel counting in synovial regions of interest in uninflamed tissue of patients with mechanical joint symptoms undergoing exploratory arthroscopy (Control), and in baseline samples of early arthritis patients, split into patients fulfilling ACR/EULAR 2010 criteria for RA during follow-up (very early rheumatoid arthritis; VERA) and combined non-RA groups made up of patients with spontaneously resolving arthritis and patients with non-RA persistent arthritis (NON-RA). (A) FAP expression was higher in VERA patients (n = 32) compared to other outcome groups (n = 24) (Kruskal-Wallis p = 0.0036, asterisks denote the results of Dunn’s post-test, *p<0.05). Red data points indicate the subgroup of patients developing seronegative, persistent RA. (B) Podoplanin expression also differed between outcome groups (Kruskal-Wallis p = 0.0062), but there was no significant difference in post-testing between VERA (n = 29) and NON-RA groups (n = 23). (C,D) No significant difference was observed in CD55 (lining; 21 VERA vs 13 non-RA) or CD248 (sublining; 16 VERA vs 8 non-RA) expression. Each dot represents a patient; median bars with interquartile ranges are shown.</p

FAP is expressed at high levels throughout the synovium in biopsies of patients developing RA.

<p>(A) Multicolour confocal microscopy images are shown for tissue staining at baseline with FAP (F11-24), podoplanin (D2-40), CD68 (Y1-82A), CD90 (Thy-1A1) antibodies followed by secondary agents, and nuclear (Hoechst) stain in a representative patient presenting with RA whose disease persisted. (B) Higher magnification, merged image. The region representing the lining layer is highlighted by a dotted line.</p

Baseline characteristics of early arthritis patients.

<p>Baseline characteristics of early arthritis patients.</p

Relationship between stromal markers and prognostic outcome in treatment-naive early arthritis tissue.

<p>Expression of (A) FAP (24 self-limiting vs 32 persistent disease), (B) podoplanin (20 self-limiting vs 32 persistent disease), (C) CD248 (10 self-limiting vs 14 persistent disease), (D) CD55 (16 self-limiting vs 18 persistent disease) were measured by pixel counting in synovial regions of interest in baseline samples of early arthritis patients who developed resolving disease (self-limiting) or persistent disease. No significant differences were found. Each dot represents a patient; median bars with interquartile ranges are shown. Significance of the comparisons was determined by the Kruskal-Wallis test.</p

High expression of FAP in tissue from patient developing RA compared to UA control.

<p>Immunohistochemistry was used to stain for FAP positive cells using a sheep anti-FAP antibody in representative tissues from patients developing RA and non-RA disease. (A low power, E high power) FAP staining in patient developing RA vs (C) isotype control; (B low power, F high power) FAP staining in patient with undifferentiated arthritis vs (D) isotype control.</p