Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


TLR2 Mediates Acute Serum Amyloid A Induced Pro-Inflammatory Effects in Rheumatoid Arthritis.

Connolly3,  Mary, Maratha1,  Ashwini, Ultaigh3,  Sinead Nic an, Miggin2,  Sinead, Fearon3,  Ursula, Veale3,  Douglas J.

Institute of Immunology, National University of Ireland Maynooth, Maynooth, Ireland
Institute of Immunology, National University of Ireland Maynooth
Translational Research Group, Dublin Academic Medical Centre, St. Vincents University Hospital., Ireland

Introduction:

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by progressive joint destruction. Serum amyloid A (A-SAA) is an acute phase protein with cytokine-like properties. Previously we have shown that A-SAA correlates with RA disease activity, is produced at high levels locally in the joint and promotes synovial tissue angiogenesis, migration and MMP production. Recently, A-SAA was identified as a ligand for Toll Like Receptor 2 (TLR2), however no study has examined a role for TLR2 in mediating A-SAA-induced inflammation in RA.

Objective:

To examine A-SAA-induced inflammation via TLR2 in rheumatoid arthritis

Methods:

A-SAA and TLR2 expression in synovial tissue (ST) was examined by immunohistochemistry. Human embryonic kidney (HEK) 293 cells overexpressing TLR2/TLR4 were transfected with the NF-kB luciferase reporter gene plasmid. After 24 hr, cells were stimulated with A-SAA (10 and 50mg/ml), the TLR2 ligand Pam2CSK4 or the TLR4 ligand LPS. After 8 hr, ligand-induced reporter gene activity was assessed. Primary fibroblasts (RASFCs) isolated from synovial biopsies obtained at arthroscopy and human microvascular endothelial cells (HMECs) were stimulated with A-SAA (10mg/ml) ± anti-TLR2 (OPN 301, 1mg/ml). IL-8 was measured by ELISA. Proliferation, invasion and ICAM expression were quantified by crystal violet assay, transwell invasion assays and flow cytometry. Migration and angiogenesis were examined using wound repair and tubule formation assays respectively.

Results:

A-SAA and TLR2 expression were observed in the lining layer and perivascular regions of RA synovium. A-SAA induced a 12-fold increase in NF-kB reporter gene activity in HEK293-TLR2 cells (p<0.05), an effect greater than that observed with Pam2CSK/Pam3CSK. In contrast, A-SAA failed to induce NF-kB reporter gene activity in HEK-TLR4, confirming specificity for TLR2. A-SAA induced IL-8 expression in RASFCs from 2.58±1.51pg/ml to 3453.19±1715.33pg/ml, an effect inhibited in the presence of anti-TLR2, where IL-8 levels fell to 1461.15±819.04pg/ml. A-SAA induced HMEC proliferation by 60.9% (p<0.001) which was significantly reduced by 38.2% in the presence of anti-TLR2 (p<0.05), similar to RASFCs. A-SAA increased ICAM expression on HDECs and RASFCs by 65.4% and 64.3% respectively, which was dramatically inhibited by 59.8% and 38.8% in the presence of anti-TLR2 (p<0.05). Furthermore, A-SAA-induced invasion and wound repair were inhibited by anti-TLR2, with no effect on TNFa-induced events. Finally, A-SAA induced angiogenic tube formation by 31.7%, an effect that was inhibited in the presence of anti-TLR2 by 24.5%.

Conclusion:

A-SAA mediates pro-inflammatory events in RA including chemokine induction, adhesion, migration and angiogenesis, via TLR2. TLR2 blockade may represent a therapeutic strategy for the treatment of arthritis.

To cite this abstract, please use the following information:
Connolly, Mary, Maratha, Ashwini, Ultaigh, Sinead Nic an, Miggin, Sinead, Fearon, Ursula, Veale, Douglas J.; TLR2 Mediates Acute Serum Amyloid A Induced Pro-Inflammatory Effects in Rheumatoid Arthritis. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1533
DOI: 10.1002/art.29299

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