Search

CD55 (decay accelerating factor) is best known for its role in the negative regulation of the complement system. Indeed, lack of this molecule leads to disease aggravation in many inflammatory disease models. However, CD55 is abundantly present on fibroblast-like …

Background Autoimmune diseases often result from inappropriate or unregulated activation of autoreactive T cells. Traditional approaches to treat autoimmune diseases have focused on direct inhibition of autoreactive T cells. A key requirement for tolerance is the presentation of antigens in a correct context. Dendritic cells (DCs) are the central antigen-presenting cells (APCs) for the initiation of T cell responses. In this context, stimulation of the Flt3 via Flt3L is known to drive expansion and differentiation of DCs. Moreover, mice lacking Flt3L are have reduced of DC numbers Objectives In the present study, we examined the targeted inhibition of APCs as a mean to downregulate/prevent autoimmune disease in a mouse model for rheumatoid arthritis. Methods Collagen-induced arthritis (CIA) was induced in mice lacking Flt3L (Flt3L-/-) and WT littermates (C57/BL6 background, 9-10 weeks old). The severity of the arthritis was assessed using an established semiquantitative scoring system (0–4). After 60 days, serum, spleen, lymph nodes (LN) and hind paws were collected. Collagen type II (CII) specific antibodies were measured by ELISA. Histological analysis (H&E, Toluidine blue, Safranin O and Trap) was performed on hind paws and phenotypical and functional analysis of spleen and LN was performed: T and B cell markers, FoxP3 expression, activation and co-stimulatory markers and intracellular cytokine staining (after PMA/Ionomycin stimulation). Results Histological analysis of paws showed increased synovial infiltration and joint destruction in WT mice while Flt3L-/- mice showed mild infiltration without inflammation (H&E staining). Cartilage destruction (Safranin O staining) and the number of osteoclast were higher in WT compared with Flt3L-/- mice. Importantly, in steady-state (no CIA induced), Flt3L-/- mice show reduced celularity in both spleen (p=0.007) and LN (p=0.01) and reduced T and B cell numbers compared with WT. CIA induction in Flt3L-/- led to decreased disease incidence and severity (AUC p=0.001) compared with WT littermates. In addition, Flt3L-/- mice showed reduced spleen and LN cellularity (p

Background and Objectives CD55 (decay-accelerating factor) is a complement-regulating protein, expressed at unusually large amounts by fibroblast-like synoviocytes (FLS) in the intimal lining in rheumatoid arthritis (RA). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor, broadly expressed by immune cells invading the inflamed synovium. We previously reported a protective effect of lack of CD55 in experimental models of RA (Hoek et al , Arthritis Rheum 2010; 62(4): 1036–42). Current data cannot explain the high expression of CD55 by FLS. Therefore, we explored in detail the pattern of CD55 expression in RA synovial tissue and 3-D organ cultures. Materials and Methods Synovial tissue was obtained by miniarthroscopy and analysed by immunohistochemistry staining to visualise CD55 and collagen type III, a constituent of extracellular matrix. Reticular fibres were visualised with Gomori silver staining. Expression of CD55 mRNA in synovial tissue was detected using antisense locked nucleic acid (LNA) oligomers. CD55 expression on 2-D-cultured RA-FLS and on blood cells of healthy individuals was detected by flow cytometry and related to mRNA levels measured by qPCR. 3-D micromasses of RA-FLS were generated in matrigel and analysed by immunohistochemistry for CD55 and reticular fibres. Results CD55 was highly expressed in the synovial lining of RA tissue, both at the mRNA and the protein level. Notably, CD55 showed an extracellular staining pattern, which coincided with Gomori silver staining of reticular fibres on sequential sections. CD55 expression on 2D-cultured FLS was less abundant and comparable to PBMCs. In 3D-micromasses of RA-FLS, CD55 was upregulated after 3–4 weeks of culture and showed an extracellular distribution that resembled reticular fibres. Conclusions CD55 mRNA and protein is abundantly expressed by FLS in the intimal lining of RA synovial tissue. We provide evidence that FLS-derived CD55 is deposited in extracellular matrix structures such as reticular fibres, where it may contribute to the synovial stromal address code that facilitates the recruitment of immune cells.

Background and objectives Rheumatoid arthritis is characterised by hyperplasia of the synovial tissue due to local proliferation of stromal and recruitment of inflammatory immune cells. Accumulation of synovial fibroblasts likely is due to an imbalance between cell proliferation, survival and death. Virus RNA receptors can initiate pro-apoptotic signalling in a variety of cell types. Based on our recent observation that fibroblast-like synoviocytes (FLS) are equipped with functional endosomal (TLR3) and cytosolic (MDA5 and RIGI) sensors for double-stranded (ds)RNA, the authors have studied the consequences of stimulation with dsRNA on the survival of FLS. Materials and methods FLS from arthritis patients were stimulated with extracellular poly(I:C) or poly(I:C)/3pRNA complexed with PEI for intracellular uptake. Activation of TLR3, MDA5 and RIG-I was confirmed by semi-quantitative PCR of response genes. Cell death of FLS after triggering of dsRNA sensors was detected by FACS analysis (annexin V/PI staining) and MTT assay. mRNA level of 46 genes involved in apoptosis was tested with a multiplex ligation-dependent probe amplification (MLPA) assay. Changes in protein expression were confirmed by Western blot analysis. Results At concentrations >0.01 µg/ml, stimulation of MDA5 by intracellular delivery of poly(I:C) caused massive cell death in up to 75% of cultured FLS. In contrast, transfection of 3pRNA triggering RIG-I had a much smaller effect on cell viability, and extracellular administration of the TLR3 ligand poly(I:C) induced death only at the high concentration of 100 µg/ml. Type I IFNs did not kill FLS, indicating that cell death was not secondary to IFN receptor signaling. MLPA and biochemical analysis demonstrated a strong induction of the BH3-only pro-apoptotic proteins Puma and Noxa upon dsRNA triggering but was not restricted to MDA5 signaling, suggesting the existence of additional regulatory mechanisms. Treatment of cells with the pan-caspase inhibitor QVD reduced cell death by about 50%, indicating that MDA5-induced cell death in FLS may involve also caspase-independent mechanisms. Conclusions Triggering of the endosomal dsRNA sensor MDA5 induces expression of pro-apoptotic Puma and Noxa and causes death in cultured FLS. Ligation of MDA5 might be a therapeutic target to overcome apoptosis resistance of FLS in rheumatoid arthritis.