Anti-Citrullinated Protein Antibodies Promote Synovial Fibroblasts Migration and Adhesion through a Peptidylarginine Deiminases (PAD) Dependent Pathway

Meng Sun¹, Vijay Joshua¹, Akilan Krishnamurthy¹, Yanying Liu², Aase Hensvold¹, Sergiu-Bogdan Catrina³, Caroline Ospelt⁴, Vivianne Malmström⁵, Khaled Amara¹, Johanna Steen¹, Muhammad Sohel Mia¹, Marianne Engström¹, Heidi Wähämaa¹, Jimmy Ytterberg¹, Bence Rethi¹ and Anca I Catrina¹, ¹Rheumatology Unit, Department of Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden, ²Rheumatology Unit, Department of Medicine, Peking University People's Hospital, Beijing, China, ³Molecular Medicine and Surgery, Molecular Medicine and Surgery, Karolinska University Hospital, Karolinska Institute, Stockholm, Sweden, ⁴University Hospital Zurich, Center of Experimental Rheumatology, Switzerland, Zurich, Switzerland, ⁵Department of Medicine, Rheumatology Unit, Department of Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden

Meeting: 2016 ACR/ARHP Annual Meeting

Date of first publication: September 28, 2016

Keywords: ACPA, fibroblasts and synovial cells, PAD, synovial fluid

Session Information

Date: Monday, November 14, 2016

Title: Cytokines, Mediators, Cell-Cell Adhesion, Cell Trafficking and Angiogenesis - Poster I

Session Type: ACR Poster Session B

Session Time: 9:00AM-11:00AM

Background/Purpose:

Synovial fibroblasts (SFs) contribute to rheumatoid arthritis (RA) pathogenesis by growing into the synovial space and by producing pro-angiogenic and tissue remodelling factors, chemokines and inflammatory cytokines that recruit and stimulate various immune cells. We have recently demonstrated that anti-citrullinated proteins antibodies (ACPAs) promote osteoclastogenesis through an interleukin-8 (IL-8) dependent autocrine mechanism. In the present work we investigated alterations of synovial fibroblast morphology and behaviour in response to ACPAs, including signalling transduction, cytokine production and mobility.

Methods: SFs were isolated from synovial tissue of RA patients by enzymatic digestion. Polyclonal ACPA and others non-ACPA IgGs were separated from peripheral blood of RA patients by affinity purification on cyclic citrullinated peptide (CCP)-2 column. SF migration capacity were tested by scratch-assays in presence of various stimuli, including ACPAs, non-ACPA IgGs, TNF and IL-8 using starved cells. The results were evaluated by NIH ImageJ software. SF adhesion was analyzed by xCELLigence System Real-Time Cell Analyzer (ACEA bioscience). Cytokine production was detected in supernatant by cytometric bead array. Signaling cascades were targeted using inhibitors of phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), G-protein coupled receptors (GPCRs), focal adhesion kinase (FAK) and peptidylarginine deiminases (PAD) in scratching assays. Protein phosphorylations were monitored by western blot.

Results: Polyclonal ACPAs but not non-ACPA IgGs induced migration (a fold increase of 2.6±0.5, mean±SD, p<0,05) and adhesion (a fold increase of 1.3±0.1 at 6 hours, p<0.05) after starvation. The cytokines TNF and IL-8 synergistically increased SF migration in presence of ACPAs. By inhibiting PADs, the enzymes responsible for protein citrullination, we showed that PAD activity is needed for the ACPA effects but not for baseline SF mobility. GPCR and PI3K blocking inhibited the effects of ACPAs whereas PTEN blocking enhanced migration, indicating important roles for GPCR and PI3K in the ACPA-mediated SF modulation. Immunoblot analysis revealed an increased AKT phosphorylation in ACPA-treated cells, further suggesting the involvement of PI3K in the ACPA-mediated signals.

Conclusion: ACPAs promote SFs migration and adhesion acting synergistically with IL-8 through a PAD-dependent pathway. Our ﬁndings suggest that SFs might have an active role in the ACPA-dependent disease propagation from the bone marrow to synovial tissue during RA.

Disclosure: M. Sun, None; V. Joshua, None; A. Krishnamurthy, None; Y. Liu, None; A. Hensvold, None; S. B. Catrina, None; C. Ospelt, None; V. Malmström, None; K. Amara, None; J. Steen, None; M. S. Mia, None; M. Engström, None; H. Wähämaa, None; J. Ytterberg, None; B. Rethi, None; A. I. Catrina, None.

To cite this abstract in AMA style:

Sun M, Joshua V, Krishnamurthy A, Liu Y, Hensvold A, Catrina SB, Ospelt C, Malmström V, Amara K, Steen J, Mia MS, Engström M, Wähämaa H, Ytterberg J, Rethi B, Catrina AI. Anti-Citrullinated Protein Antibodies Promote Synovial Fibroblasts Migration and Adhesion through a Peptidylarginine Deiminases (PAD) Dependent Pathway [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/anti-citrullinated-protein-antibodies-promote-synovial-fibroblasts-migration-and-adhesion-through-a-peptidylarginine-deiminases-pad-dependent-pathway/. Accessed .

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