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Proteomic analysis of secreted proteins in early rheumatoid arthritis: anti-citrulline autoreactivity is associated with up regulation of proinflammatory cytokines

Wolfgang Hueber^1,2, Beren H Tomooka^1,2, Xiaoyan Zhao^1,2, Brian A Kidd^1,2, Jan W Drijfhout³, James F Fries¹, Walther J van Venrooij⁴, Allan L Metzger⁵, Mark C Genovese¹, William H Robinson^1,2

¹ Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California, USA
² GRECC, Veterans Affairs Palo Alto Health Care System, Palo Alto, California, USA
³ Leiden University Medical Center, Leiden, The Netherlands
⁴ Radboud University Nijmegen, Nijmegen, The Netherlands
⁵ Rheumatology Diagnostics Laboratory, Los Angeles, California, USA

Correspondence to:
Dr W Hueber
Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Palo Alto VA Health Care System, MC 154R, 3801 Miranda Avenue, Palo Alto, CA 94304, USA;whueber{at}stanford.edu

Objectives: To identify peripheral blood autoantibody and cytokine profiles that characterise clinically relevant subgroups of patients with early rheumatoid arthritis using arthritis antigen microarrays and a multiplex cytokine assay.

Methods: Serum samples from 56 patients with a diagnosis of rheumatoid arthritis of <6 months’ duration were tested. Cytokine profiles were also determined in samples from patients with psoriatic arthritis (PsA) and ankylosing spondylitis (n = 21), and from healthy individuals (n = 19). Data were analysed using Kruskal–Wallis test with Dunn’s adjustment for multiple comparisons, linear correlation tests, significance analysis of microarrays (SAM) and hierarchical clustering software.

Results: Distinct antibody profiles were associated with subgroups of patients who exhibited high serum levels of tumour necrosis factor (TNF), interleukin (IL)1ß, IL6, IL13, IL15 and granulocyte macrophage colony-stimulating factor. Significantly increased autoantibody reactivity against citrullinated epitopes was observed in patients within the cytokine "high" subgroup. Increased levels of TNF, IL1, IL12p40 and IL13, and the chemokines eotaxin/CCL11, monocyte chemoattractant protein-1 and interferon-inducible protein 10, were present in early rheumatoid arthritis as compared with controls (p<0.001). Chemokines showed some of the most impressive differences. Only IL8/CXCL8 concentrations were higher in patients with PsA/ankylosing spondylitis (p = 0.02).

Conclusions: Increased blood levels of proinflammatory cytokines are associated with autoantibody targeting of citrullinated antigens and surrogate markers of disease activity in patients with early rheumatoid arthritis. Proteomic analysis of serum autoantibodies, cytokines and chemokines enables stratification of patients with early rheumatoid arthritis into molecular subgroups.

Abbreviations: ARAMIS, Arthritis, Rheumatism, and Aging Medical Information System; CCP2, cyclic citrullinated peptide 2; FDR, false discovery rate; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN, interferon; IP-10/CXCL10, interferon-inducible protein 10; MCP-1/CCL2, monocyte chemoattractant protein-1; PsA, psoriatic arthritis; SAM, significance analysis of microarrays; TNF, tumour necrosis factor

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