Christine A. Teitsma

The value of rheumatoid factor and anti-citrullinated protein antibodies as predictors of response to infliximab in rheumatoid arthritis: an exploratory study

OBJECTIVE It remains unclear whether autoantibodies are useful biomarkers to tailor the choice of biological treatment in RA. We investigated the relationship between the presence and levels of different RF and ACPA isotypes and the response to TNF blockade in an exploratory study. METHODS A total of 101 active RA patients were prospectively treated with infliximab (3 mg/kg). Changes in disease activity were monitored by the 28-joint DAS (DAS-28). Serum levels of different isotypes [immunoglobulins M, G and A (IgM, IgG and IgA)] of RF and anti-citrullinated peptide antibodies were measured by ELISA. RESULTS The mean DAS-28 decreased from 5.9 (1.1) at baseline to 4.0 (1.3) at Week 16 of infliximab treatment (P < 0.001). High baseline levels of different isotypes of RF (all P < 0.008), ACPA IgM (P = 0.008) and ACPA IgG (P = 0.07) were associated with an absolute decrease in DAS-28 after TNF blockade. This relationship persisted after adjusting for DAS-28 at baseline. However, the different isotypes of baseline RF and ACPA levels accounted for only a small proportion of variance in treatment response (RF: R² between 7 and 12% and ACPA: R² between 4 and 7%). The simultaneous presence of all three isotypes of RF or ACPA had no additive value. CONCLUSION Presence as well as the titres of RF and IgM ACPA at baseline are significantly correlated with better response to infliximab treatment. However, this correlation is not strong enough to allow a reliable prediction in individual patients. Trial Registration. ISRCTN Register, http://www.controlled-trials.com/isrctn/, ISRCTN36847425.

Presence and Role of Anti–Citrullinated Protein Antibodies in Experimental Arthritis Models

OBJECTIVE Anti-citrullinated protein antibodies (ACPAs) are the serologic hallmark of rheumatoid arthritis. Functional studies on the role of ACPAs in experimental arthritis have yielded conflicting results, and therefore the present study was undertaken to assess systematically whether citrullinated proteins can really induce ACPAs and modulate arthritis in mice. METHODS Balb/c, SJL, and DBA/1 mice were immunized with either native or citrullinated fibrinogen, myelin basic protein (MBP), and type II collagen (CII). ACPAs were detected with a peptide-based enzyme-linked immunosorbent assay (ELISA) and with Western blotting using fibrinogen as substrate. Arthritis was induced in mice by immunization with CII in Freunds complete adjuvant or by injection of anticollagen antibodies. RESULTS Analysis of the sera of mice immunized with citrullinated proteins revealed false-positive results with the citrulline peptide-based ELISA. In contrast, Western blot analysis using either citrullinated or native fibrinogen as substrate reliably detected ACPAs in Balb/c mice immunized with citrullinated fibrinogen, MBP, and CII. However, these ACPAs failed to induce or aggravate disease in Balb/c mice in the anticollagen antibody-induced arthritis model. Immunization with citrullinated fibrinogen induced ACPAs but did not lead to arthritis development in SJL and DBA/1 mice. In contrast, immunization with citrullinated CII failed to induce ACPAs or enhance disease in these strains in the collagen-induced arthritis model. CONCLUSION Mice can develop genuine ACPAs, but detection of ACPAs is highly dependent on strain, immunogen, immunization protocol, and detection assay. Murine ACPAs are not overtly pathogenic, since neither preexisting ACPAs nor the use of citrullinated collagen as immunogen modulates the clinical course of arthritis.

Are polymorphisms of the β3-adrenoceptor gene associated with an altered bladder function?†‡

As the presence of a Trp64Arg polymorphism of the gene encoding the β3‐adrenoceptor (B3AR) has been linked to the presence of overactive bladder, we investigated whether additional polymorphisms are detectable in this gene and explore their relationships parameters related to lower urinary tract function.

Polymorphisms in human muscarinic receptor subtype genes.

A wide range of polymorphisms have been reported in muscarinic receptor subtype genes, mostly in M₁ and M₂ and, to a lesser extent, M₃ receptors. Most studies linking such genetic variability to phenotype have been performed for brain functions, but a more limited amount of information is also available for cardiac and airway function. Unfortunately, for none of the phenotypes under investigation a robust association with genotype has emerged. Moreover, it remains mostly unclear whether a reported association indicates a causative role of the polymorphism under investigation or merely a role as indicator of other polymorphisms affecting expression and/or function of the receptor. Also, most data on genotype-phenotype associations of muscarinic receptor subtypes are based on cross-sectional samples. Mechanistic studies linking polymorphisms to molecular, cellular, and tissue functions are largely missing. Finally, studies on a possible impact of muscarinic receptor polymorphisms on drug responsiveness are also largely missing. Thus, the field of genomics of muscarinic receptor subtypes is still in an early stage and a considerably greater number of studies will be required to judge the role of muscarinic receptor gene variability in physiology, pathophysiology, and drug treatment.

Insulin-Like Growth Factor I Does Not Drive New Bone Formation in Experimental Arthritis

Introduction Insulin like growth factor (IGF)-I can act on a variety of cells involved in cartilage and bone repair, yet IGF-I has not been studied extensively in the context of inflammatory arthritis. The objective of this study was to investigate whether IGF-I overexpression in the osteoblast lineage could lead to increased reparative or pathological bone formation in rheumatoid arthritis and/or spondyloarthritis respectively. Methods Mice overexpressing IGF-I in the osteoblast lineage (Ob-IGF-I+/-) line 324–7 were studied during collagen induced arthritis and in the DBA/1 aging model for ankylosing enthesitis. Mice were scored clinically and peripheral joints were analysed histologically for the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts. Results 90–100% of the mice developed CIA with no differences between the Ob-IGF-I+/- and non-transgenic littermates. Histological analysis revealed similar levels of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the ankle joints. In the DBA/1 aging model for ankylosing enthesitis 60% of the mice in both groups had a clinical score 1<. Severity was similar between both groups. Histological analysis revealed the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the toes in equal levels. Conclusion Overexpression of IGF-I in the osteoblast lineage does not contribute to an increase in repair of erosions or syndesmophyte formation in mouse models for destructive and remodeling arthritis.

Altered BANK1 expression is not associated with humoral autoimmunity in chronic joint inflammation

OBJECTIVE The presence of disease-specific autoantibodies in RA but not spondyloarthritis (SpA) suggests that B-cell tolerance is preserved in the latter condition despite chronic joint inflammation. Which factors control B-cell tolerance vs autoimmunity in chronic arthritis remains incompletely understood. As single nucleotide polymorphisms in the B-cell scaffold protein with ankyrin repeats (BANK1) gene have recently been associated with various autoantibody-positive autoimmune diseases including RA, we explored whether altered expression of BANK1 was associated with humoral autoimmunity in arthritis. METHODS Peripheral B-cell subsets and inflamed synovial tissue were obtained from active SpA and RA. Quantitative PCR was used to assess the expression of full-length BANK1 and delta2 BANK1, a splice variant lacking exon 2 that counteracts BANK1 function. B-cell subsets, autoantibody titres and clinical disease were monitored upon CIA induction in Bank1 knockout (KO) mice. RESULTS Whereas full-length BANK1 was not differentially expressed, the BANK1 delta2 splicing variant was decreased in naïve peripheral B cells as well as in synovial tissue of SpA compared with RA. However, no differences were observed in seropositive vs seronegative RA. Performing functional analysis in mice, we found no differences in B-cell subsets and anti-collagen antibodies upon CIA induction between Bank1 KO mice and littermate controls. Accordingly, the incidence and severity of clinical disease were not altered in Bank1 KO mice. CONCLUSION This study did not reveal a major role for BANK1 in humoral autoimmunity in chronic arthritis. The decreased levels of BANK1 delta2 in SpA, however, warrant more detailed analysis of the functional consequences of an altered BANK1/BANK1 delta2 balance.

Detection of genuine anti-citrullinated protein antibodies in mice reveals their presence in BALB/c but not DBA/1 and SJL mice hyperimmunised with citrullinated collagen

Antibodies against citrullinated proteins (ACPA) occur in 60–70% of all rheumatoid arthritis (RA) patients with a specificity of 95%. The role of the immune response against citrullinated proteins in the pathophysiology of RA remains unknown, because studies in experimental models yielded contrasting results. It is unclear whether mice develop antibodies crucially dependent on citrullination of the epitope. The authors aimed to develop a reliable method to detect ACPA in mice and to assess the development of ACPA in different mouse …

SAT0251 Endoplasmic reticulum stress is not driving the altered cytokine production by macrophages in HLA-B27+ spondyloarthritis

Background Spondyloarthritis (SpA) is strongly associated with HLA-B27, but the role of this MHC class I molecule in the disease pathogenesis remains largely unknown. Misfolding of HLA-B27 in the endoplasmic reticulum (ER) is described to induce an unfolded protein response (UPR), leading to increased production of pro-inflammatory cytokines upon TLR ligation. However, these data on HLA-B27-induced macrophage ER stress were generated in cell lines and animal models with strongly increased HLA-B27 expression and the relevance of this pathway in HLA-B27+ human SpA remains unknown. Objectives The aim of the study was to investigate the effect of ER stress and HLA-B27 positivity on polarized human macrophages in HLA-B27+ SpA. Methods To analyze the effects of ER stress on macrophage polarization, peripheral blood monocytes from healthy donors (HDs) were stimulated with thapsigargin (TG), polarized for 4 days by IFN-γ, IL-4, or IL-10, and subsequently phenotyped by flow cytometry. To assess the effect of ER stress on cytokine production, polarized peripheral blood derived macrophages (PBDMs) were submitted to ER stress by TG and subsequently stimulated with LPS. Finally, polarized PBDMs from HLA-B27- and HLA-B27+ SpA, rheumatoid arthritis (RA) and HDs were stimulated with LPS. The expression of ER stress markers (BiP, CHOP, ERdj4) and cytokines (IL-23, IL-12, IL-10) was measured by RT-PCR. Results Stimulation of human monocytes with TG prior to in vitro polarization prevented the up-regulation of the macrophage (MΦ)IFN-γ marker CD64 (p<0.05), the MΦIL-4 marker CD200R (p<0.05), and the MΦIL-10 markers CD163 (p<0.001) and CD32 (p<0.05), indicating that ER stress impairs macrophage polarization. When MΦIFN-γ, MΦIL-4 and MΦIL-10 were incubated with TG, they showed up-regulation of BiP, CHOP and ERdj4. The relative expression of IL-23 in comparison with both IL-12 and IL-10 was significantly increased by TG in all three macrophage subsets (IL-23/IL-12 p<0.05 in MΦIFN-γ and p<0.01 in MΦIL-4; IL-23/IL-10 p<0.05 in MΦIL-4and MΦIL-10). LPS stimulation resulted in an important induction of IL-23, IL-12 and to a lesser extent IL-10 expression in MΦIFN-γ and MΦIL-4 (p<0.01), which could not further be up-regulated by TG. Analysis of in vitro polarized macrophages from SpA, RA and HD did not reveal differences in the expression of ER stress marker BiP, both prior and after activation with LPS. Stratification of SpA according to HLA-B27 status also did not reveal differences in BiP expression. However, LPS-stimulated MΦIFN-γ showed an increased relative expression of IL-23 and IL-12 versus IL-10 in HLA-B27+ SpA in comparison with HLA-B27- SpA (p<0.05), indicating a LPS-induced shift towards pro-inflammatory cytokine expression. Conclusions Macrophages from HLA-B27+ SpA patients, and especially MΦIFN-γ, showed increased IL-23 and IL-12 versus IL-10 expression in comparison with macrophages from HLA-B27- SpA patients. However, these differences were not related to increased ER stress in HLA-B27+ SpA macrophages. Disclosure of Interest None Declared