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

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

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

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

Sensitivity to change of MRP8/14 serum levels compared to DAS28 and CRP serum level.

<p>Standardized response mean (SRM) of the change in myeloid related protein 8/14 (MRP8/14), C-reactive protein (CRP) and disease activity score evaluated in 28 joints (DAS28-CRP) 4 weeks after treatment with infliximab (n = 34), adalimumab (n = 85), rituximab (n = 20) and treatment with placebo or ineffective therapy (n = 28). The solid line indicates the 0.2 SRM cut off point (low), the thick dotted line indicates the 0.5 SRM cut off point (moderate) and the thin dotted line indicates the 0.8 SRM cut off point (high).</p

Baseline characteristics of patients enrolled in the effective and placebo/ineffective treatment groups.

<p>Median and interquartile range or percentages are shown. EULAR responder = good or moderate responder according to the European League Against Rheumatism Response, TJC68 =  tender joint count of 68 joints, SJC68 =  swollen joint count of 68 joints, ESR = erythrocyte sedimentation rate, CRP = C-reactive protein, DAS28-CRP = disease activity score based on CRP. P-value <0.05 was considered statistically significant. Significance of the comparison is determined by the chi-square test or the Mann-Whitney test.</p><p>Baseline characteristics of patients enrolled in the effective and placebo/ineffective treatment groups.</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

Western blot of supernatant of HEK293 transfected cells showing CD40:Fc expression.

<p>HEK293 cells were transduced with AAV2-CD40:Fc or were mock transduced. The fusion protein was detected by anti murine-CD40 (left panel) and anti-human-Fc (right) and showed a band ∼90 kD (size of non-reduced dimer) and a ∼45 kD (reduced monomer) band.</p

Characteristics of the study population.

<p>Characteristics of the study population.</p

Cross-validation values for the discrimination between patients with RA, PsA and controls.

<p>Cross-validation values for the discrimination between patients with RA, PsA and controls.</p