Circulating autoantibodies, acute phase reactants, and cytokines in relation to HLA genotype and disease activity in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that has a predilection for peripheral synovial joints. The aetiology of the disease is not known, but current evidence indicates that multiple exogenous factors, a genetic predisposition, autoantibodies, especially rheumatoid factor (RF) and anti-citrullinated peptide antibodies, together with an unidentified trigger, conspire to cause an inflammatory response characterised by bone and cartilage destruction in the joint. The genetic predisposition has been linked to the “Shared Epitope” RAA amino acid motif at positions 72-74 of the third hypervariable region of the various HLA-DRβ1 chains. Further subdivision, according to the amino acids at positions 70 and 71, either contributes to (S2, S3P), or negates (S1, S3D) RA susceptibility. The ACR criteria of 2010 acknowledge the contribution of rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA) in the classification of RA. CRP, an acute phase reactant, reflects ongoing inflammation in RA, and is utilised in the clinical assessment score for calculating disease activity in the form of the DAS 28-CRP score. Cytokines form an integral part of the inflammatory process in RA and have been the focus of several treatment strategies utilising monoclonal antibodies against cytokines, as a component of the therapeutic regimen, with notable success. The objective of the laboratory research described in this thesis was to assess possible interactions between these clinical, genetic and circulating biomarkers in RA, particularly those markers which best identify those patients at risk for severe aggressive disease. This is of special significance in the South African, State-administered health environment, in which there is a clear lack of information and insight regarding the diagnostic significance of these markers of RA. Serum cytokines, chemokines, growth factors (IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-17, IFN-γ, TNF, G-CSF, GM-CSF, VEFG, CCL2, CCL4, and IL-1Ra) were measured using a multiplex suspension bead array system in a cohort (n=143, male to female ratio 1:6) of predominantly African (87%) RA patients. Rheumatoid factor (RF), anti-cyclic citrullinated peptide (aCCP) and anti-modified citrullinated vimentin (aMCV) antibodies were measured by nephelometric, immunofluorometric, and ELISA procedures respectively, and the acute phase reactants, CRP, and serum amyloid A (SAA) by nephelometry. Composite disease activity was calculated using the 28 joint (DAS28) score, while HLA-DRB1 allele typing was performed using high-resolution rSSO PCR procedures. The mean levels for all cytokines, chemokines and growth factors were found to be significantly higher in the RA cohort (n=143) than in a smaller group of healthy controls (n=10). Moderate-to-strong positive inter-correlations were observed between Th1/Th2/macrophage/fibroblast-derived cytokines. RF correlated significantly with IL-1β, IL-2, IL-4, IL-10, IL-12, G-CSF, GM-CSF, IFN-γ, and TNF (p<0.0001), while aCCP and aMCV correlated with IL-1β, IL-2, IL-4 and IL-10 (p<0.0002). IL-6 levels correlated best with the acute phase reactants, CRP and SAA (p<0.0001). In a sub-group of patients with high disease activity (HDA), defined as DAS28 scores >5.1, there was a significant correlation of IFN-γ, IL-1β, IL-1Ra, TNF, VEGF and GM-CSF levels with DAS28 scores (p<0.02-0.001). Of the 143 RA patients, 82 (57%) were homozygous (SS) and 50 (35%) were heterozygous (SX) for the SE alleles, with significant over-expression of S2 and S3P (respective odds ratios (ORs) of 5.3 and 5.8, p<0.0001), while 11 (8%) were classified as XX. Both the SS and SX groups showed a strong association with aCCP (OR and p values of 10.21 and 9.2, and p= 0.001and 0.003 respectively) relative to the XX group. Clinical scores and concentrations of the other biomarkers of disease activity (RF, CRP, Th1/Th2/macrophage/fibroblast cytokines) were also generally higher in the SS group relative to the SX and XX groups. In conclusion, these results show that the addition of certain cytokines, aCCP and high-resolution typing of the HLA DRB1 allele in the laboratory diagnostic profile of patients presenting with early RA disease symptoms has the potential to contribute to improved clinical management.