Olga N. Karpus

Shear Stress–Dependent Downregulation of the Adhesion-G Protein–Coupled Receptor CD97 on Circulating Leukocytes upon Contact with Its Ligand CD55

Adhesion G protein–coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane β subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is poorly understood. In this study, we investigated the interaction between the pan-leukocyte aGPCR CD97 and its ligand CD55. We found that leukocytes from CD55-deficient mice express significantly increased levels of cell surface CD97 that normalized after transfer into wild-type mice because of contact with CD55 on both leukocytes and stromal cells. Downregulation of both CD97 subunits occurred within minutes after first contact with CD55 in vivo, which correlated with an increase in plasma levels of soluble CD97. In vitro, downregulation of CD97 on CD55-deficient leukocytes cocultured with wild-type blood cells was strictly dependent on shear stress. In vivo, CD55-mediated downregulation of CD97 required an intact circulation and was not observed on cells that lack contact with the blood stream, such as microglia. Notably, de novo ligation of CD97 did not activate signaling molecules constitutively engaged by CD97 in cancer cells, such as ERK and protein kinase B/Akt. We conclude that CD55 downregulates CD97 surface expression on circulating leukocytes by a process that requires physical forces, but based on current evidence does not induce receptor signaling. This regulation can restrict CD97–CD55-mediated cell adhesion to tissue sites.

Triggering of the dsRNA Sensors TLR3, MDA5, and RIG-I Induces CD55 Expression in Synovial Fibroblasts

Background CD55 (decay-accelerating factor) is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS. Methods FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR) ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C)) and 5′-triphosphate RNA were used to activate the cytoplasmic double-stranded (ds)RNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry. Results CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1β and especially by the TLR3 ligand poly(I:C). Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55. Conclusions Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocytes.

Silencing the Expression of Ras Family GTPase Homologues Decreases Inflammation and Joint Destruction in Experimental Arthritis

Changes in the expression and activation status of Ras proteins are thought to contribute to the pathological phenotype of stromal fibroblast-like synoviocytes (FLS) in rheumatoid arthritis, a prototypical immune-mediated inflammatory disease. Broad inhibition of Ras and related proteins has shown protective effects in animal models of arthritis, but each of the Ras family homologues (ie, H-, K-, and N-Ras) makes distinct contributions to cellular activation. We examined the expression of each Ras protein in synovial tissue and FLS obtained from patients with rheumatoid arthritis and other forms of inflammatory arthritis. Each Ras protein was expressed in synovial tissue and cultured FLS. Each homolog was also activated following FLS stimulation with tumor necrosis factor-α or interleukin (IL)-1β. Constitutively active mutants of each Ras protein enhanced IL-1β-induced FLS matrix metalloproteinase-3 production, while only active H-Ras enhanced IL-8 production. Gene silencing demonstrated that each Ras protein contributed to IL-1β-dependent IL-6 production, while H-Ras and N-Ras supported IL-1β-dependent matrix metalloproteinase-3 and IL-8 production, respectively. The overlap in contributions of Ras homologues to FLS activation suggests that broad targeting of Ras GTPases in vivo suppresses global inflammation and joint destruction in arthritis. Consistent with this, simultaneous silencing of H-Ras, K-Ras, and N-Ras expression significantly reduces inflammation and joint destruction in murine collagen-induced arthritis, while specific targeting of N-Ras alone is less effective in providing clinical benefits.

Mice overexpressing CD97 in intestinal epithelial cells provide a unique model for mammalian postnatal intestinal cylindrical growth

Transgenic mice overexpressing CD97 in intestinal epithelial cells develop an upper megaintestine with normal microscopic morphology after birth and before weaning. Intestinal enlargement by CD97 depends on signaling but does not require binding of its ligand, CD55.

Stromal cell markers are differentially expressed in the synovial tissue of patients with early arthritis

Introduction Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. Methods ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers. Results We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables. Conclusions Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

Absence of Fms-like tyrosine kinase 3 ligand (Flt3L) signalling protects against collagen-induced arthritis

Objective Comprehending the mechanisms that regulate activation of autoreactive T cells and B cell antibody production is fundamental for understanding the breakdown in self-tolerance and development of autoimmunity. Here we studied the role of Fms-like tyrosine kinase 3 ligand (Flt3L) signalling in the pathogenesis of collagen-induced arthritis (CIA). Methods CIA was induced in mice lacking Flt3L (Flt3L−/−) and wild-type (WT) littermates (C57/BL6, 8–10 weeks old). Mice were killed in the initial phase (acute phase: experiment 1) and late phase (chronic phase: experiment 2) of the disease. Arthritis severity was assessed using a semiquantitative scoring system (0–4), and histological analysis of cellular infiltration, cartilage destruction and peptidoglycan loss was performed. Phenotypic and functional analysis of T and B cells, FoxP3 expression, activation and lymphocyte costimulatory markers, and cytokine production were performed ex vivo by flow cytometry in lymph nodes. Serum collagen type II (CII)-specific antibodies were measured by ELISA. Results Flt3L−/− mice showed a marked decrease in clinical arthritis scores and incidence of arthritis in both acute and chronic phases of CIA compared with WT mice. Moreover, decreased synovial inflammation and joint destruction was observed. Both the magnitude and quality of T cell responses were altered in Flt3L−/−. In the acute phase, the amount of CII-specific IgG2a antibodies was lower in Flt3L−/− than WT mice. Conclusions These results strongly suggest a role for Flt3L signalling in the development of arthritis.

Deletion of either CD55 or CD97 ameliorates arthritis

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 …

Intracellular delivery of poly(I:C) induces apoptosis of fibroblast-like synoviocytes via an unknown dsRNA sensor

Fibroblast-like synoviocytes (FLS) express functional membranous and cytoplasmic sensors for double-stranded (ds)RNA. Notably, FLS undergo apoptosis upon transfection with the synthetic dsRNA analog poly(I:C). We here studied the mechanism of intracellular poly(I:C) recognition and subsequent cell death in FLS. FLS responded similarly to poly(I:C) or 3pRNA transfection; however, only intracellular delivery of poly(I:C) induced significant cell death, accompanied by upregulation of pro-apoptotic proteins Puma and Noxa, caspase 3 cleavage, and nuclear segregation. Knockdown of the DExD/H-box helicase MDA5 did not affect the response to intracellular poly(I:C); in contrast, knockdown of RIG-I abrogated the response to 3pRNA. Knockdown of the downstream adaptor proteins IPS, STING, and TRIF or inhibition of TBK1 did not affect the response to intracellular poly(I:C), while knockdown of IFNAR blocked intracellular poly(I:C)-mediated signaling and cell death. We conclude that a so far unknown intracellular sensor recognizes linear dsRNA and induces apoptosis in FLS.

A2.4 Association of CD55 with Reticular Fibres in the Synovial Lining in Rheumatoid Arthritis

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.

SAT0067 Mice lacking FLT3L are protected from collagen-induced arthritis: Regulating the regulators

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<0.0001) but also reduced percentage of CD4+CD25+T cells compared with WT (p=0.03). Flt3L-/- CD4+ T cells produced significantly less IL-17 (p=0.016) and TNF-a (p=0.010) while CD8+ T cells produced less IFN-g (p=0.029) compared with WT. Conclusions Mice lacking Flt3L are protected from CIA. CIA induction in mice that have reduced numbers of DC influenced not only the magnitude (cell numbers) but also the quality (CD25 expression) of T cell responses. Stimulation of lymphocytes by different types of DC, DC at different stages of maturity and producing or responding to different growth factors might contribute for this change in T cell numbers and/or effector functions in Flt3L-/- mice. Targeting this signaling pathway might be considered as a good therapeutic strategy in RA. Disclosure of Interest None Declared