Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Molecular mimicry wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Adaptive immune system wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Innate immune system wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
X-linked severe combined immunodeficiency wikipedia , lookup
Sjögren syndrome wikipedia , lookup
RHEUMATOLOGY Rheumatology 2015;54:545553 doi:10.1093/rheumatology/keu351 Advance Access publication 5 September 2014 Original article Bone resorption correlates with the frequency of CD5+ B cells in the blood of patients with rheumatoid arthritis Robby Engelmann1, Ni Wang1,2, Christian Kneitz3 and Brigitte Müller-Hilke1 Abstract Objective. The prevention of bone resorption and subsequent joint destruction is one of the main challenges in the treatment of patients suffering from RA. Various mechanisms have previously been described that contribute to bone resorption in tightly defined cohorts. Here we analysed a cross-sectional cohort of RA patients and searched for humoral and cellular markers in the peripheral blood associated with bone resorption. Methods. We enrolled 61 consecutive RA patients positive for ACPA. Blood was analysed by flow cytometry to determine the percentages of regulatory T cells and B cell subpopulations. Cytokine (TNF-a, IL-6, IL-10) and ACPA levels as well as the bone resorption marker CTX-1 were determined from the patients’ sera. Standard clinical disease parameters were included. Results. Multivariate analyses showed that the percentages of CD5+ B cells were positively correlated with CTX-1 serum levels. However, neither low-avidity ACPA nor serum IL-6 levels, both known to be produced by CD5+ cells, were associated with CTX-1 in patients’ sera. There was no correlation between CTX-1 levels and clinical parameters or ACPA levels. Key words: bone resorption, CTX-1, CD5, rheumatoid arthritis, biomarker. Introduction The hallmark of RA is chronic joint inflammation. Cells of the innate and adaptive immune systems infiltrate the synovial membrane and lead to the formation of the highly aggressive pannus tissue that is held to be responsible for joint destruction and local bone erosion [1, 2]. Bone erosion results from the uncoupling of bone formation and bone resorption and several attempts have been made to link chronic inflammation with elevated osteoclast activity [1, 3] and reduced bone formation [4] leading to the focal 1 Institute of Immunology, Rostock University Medical Center, Rostock, Germany, 2Institute of Blood Research, Dalian Blood Center, Liaoning Province, China and 3Klinik für Innere Medizin II, Klinikum Südstadt Rostock, Rostock, Germany. Submitted 31 December 2013; revised version accepted 3 July 2014. Correspondence to: Robby Engelmann, Institute of Immunology, Rostock University Medical Center, Schillingallee 68, 18057 Rostock, Germany. E-mail: [email protected] bone loss characteristic of RA. Indeed, the close phylogenetic relatedness between bone and the immune system allows for an intimate communication that makes use of numerous cellular and humoral factors [5, 6]. Among the cellular factors, the receptor activator of nuclear factor kB ligand (RANKL) is expressed on osteoblasts and Th17 cells alike and initiates osteoclast maturation [79]. Humoral factors include cytokines as well as chemokines and growth factors. Thus pro-inflammatory cytokines such as TNF-a, IL-6 and IL-17, which are predominantly produced by activated macrophages, B cells and T cells, respectively, have been shown to stimulate osteoclast differentiation and thereby enhance bone resorption [7, 10]. TGF-b, which is produced by regulatory T cells (Tregs), Th cells and B cells alike, plays an important and dual role. On the one hand, it impacts directly on the osteoblast to produce osteoprotegerin, which prevents osteoclast maturation [11]. On the other hand, TGF-b balances Th17 and Tregs [6]: while low levels of ! The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected] BASIC SCIENCE Conclusion. In summary, we found that the CD5+ B cell population is associated with bone resorption as measured via serum CTX-1 levels in a cross-sectional cohort of RA patients. However, a possible functional link between CD5+ B cells and bone resorption still needs to be defined. Robby Engelmann et al. TGF-b in combination with IL-6 promote differentiation of inflammatory Th17 cells, high levels induce the generation of Tregs that secrete TGF-b and IL-10 and thereby inhibit osteoclastogenesis [12]. The contribution of B cells to bone erosion in RA is still a matter of debate. Some authors have demonstrated that B cells enhance osteoclast maturation via expression of RANKL [13] or via an elevated population of CD5+ B cells that, within the synovium of RA patients [14], produce considerable amounts of IL-6 [15]. Conversely, others have shown that B cells inhibit osteoclastogenesis by secretion of TGF-b [16] or by regulatory B cells characterized by the expression of IL-10 [17]. Moreover, B cells in RA produce not only cytokines but also autoantibodies like RF and ACPA. The latter are highly specific for RA and have recently been shown to induce osteoclast differentiation by direct binding to osteoclast precursors [18]. We set out to investigate which immune cells and which humoral factors impact on bone resorption in RA. We focused on CTX-1 as a serum marker for ongoing bone turnover as opposed to the accumulated bone erosion assessable on radiographs. CTX-1 is the carboxyterminal cross-linked telopeptide generated during the degradation of collagen type I via cathepsin K and thus directly mirrors osteoclast activity as well as predicts future radiological progression [1922]. Performing a cross-sectional study including serum parameters and cellular composition, we identified the CD5+ B cell population as the major factor associated with bone resorption in RA. Methods Patients Sixty-one consecutive ACPA-positive RA patients were enrolled at the Clinics Südstadt Rostock, Germany. Our patient cohort consisted of 72% females and had a mean age of 63.1 years (range 3087). The patients showed a mean disease duration of 13.7 years (range 0.440.1), a mean treatment duration of 4.7 years (range 025.1), a mean CRP of 14.7 mg/l (range 0144; normal range 03) and a mean 28-joint DAS (DAS28) of 3.1 (range 0.97.1). Treatment regimes were heterogeneous, including DMARDs alone (n = 34), anti-B cell therapy (n = 2), anti-TNF-a therapy (n = 12), anti-IL6 receptor (n = 7), modulation of T cell co-stimulation (n = 4) and no therapy at all (n = 2). The majority of patients (82%) included in the present study were treated with vitamin D supplementation, but none with bisphosphonates or other known bone-modifying treatments. This study was approved by the ethics committee of the Rostock University Medical Center (A2011-134). All patients fulfilled the 1987 ACR classification criteria for RA and gave informed written consent prior to blood sampling according to the Declaration of Helsinki. Serum samples were collected between 8:30 and 11:00 a.m. Food intake prior to serum sampling was not specifically controlled for. 546 Flow cytometry Peripheral blood mononuclear cells (PBMCs) were isolated from 7.5 ml of EDTA blood by Ficoll density gradient centrifugation. First, aqua fluorescent reactive dye (Invitrogen, Darmstadt, Germany) was used for live/dead discrimination according to the manufacturer’s instructions. Next, cells were stained for surface markers in ice-cold PBS pH 7.4, 0.5% BSA and 0.1% sodium azide. Appropriate isotype controls were used to quantify CD157, glucocorticoid-induced TNF receptorrelated protein, CD80 and CD86. All cells were fixed after the surface staining with either 4% paraformaldehyde for 10 min at room temperature or prior intracellular staining for Foxp3 and helios using the FoxP3 Staining Buffer Set (Miltenyi, Bergisch Gladbach, Germany) according to manufacturer’s instructions. Data were acquired on a FACS Aria II machine (BD, Heidelberg, Germany) and analysed using FACS Diva Software (BD). For normal control, B cell values refer to previously published data [23, 24]. The MiFlowCyt-conform documentation of our flow cytometry experiments, including, for example. the gating strategies, can be found in supplementary 1-MiFlowCyt documentation S1, available at Rheumatology Online. ACPA ELISA IgG1 and IgG4 antibody levels for CCP and mutated citrullinated vimentin (MCV) were determined by a direct ELISA as previously described [25]. Briefly, we combined CCPcoated (Euroimmun, Lübeck, Germany) and MCV-coated (Orgentec, Mainz, Germany) ELISA plates with detection antibodies specific for IgG1 (Binding Site, Birmingham, UK) or IgG4 (AbD Serotec, Puchheim, Germany). In the first step, the sera were applied in dilutions of 1:400 and 1:20 for IgG1 and IgG4, respectively. Thereafter the plates were incubated with IgG1-specific (1:15 000) or IgG4-specific (1:25 000) horseradish peroxidase (HRP)coupled detection antibodies. Finally, colour reaction was performed using 3,30 ,5,50 -tetramethylbenzidine (TMB) substrate (BioLegend, Fell, Germany) and the optical density (OD) was determined by an automated plate reader (Millenia Kinetic Analyser; DPC, Los Angeles, CA, USA). IgM MCV serum levels were determined after depletion of IgG in order to remove potential background originating from IgG-specific IgM RF. IgG was depleted using protein-G agarose (Sigma Aldrich, Munich, Germany). The resin was washed five times with 1% BSA in PBS pH 7.4 prior to incubation with 1:62.5 diluted serum in an oscillatory shaker for 1 h. The resin was cleaned after use by washing five times with 100 mM glycine-HCl buffer pH 2.7 and five times with PBS pH 7.4 containing 0.05% sodium azide for storage at 4 C. Depleted samples (100 ml/well) were applied to a MCV-coated plate (Orgentec) for 1.5 h. After a washing step, plate-bound IgG and IgM were detected using 100 ml of HRP-coupled anti-human IgM (1:3000; AbD Serotec) or anti-human IgG antibodies (1:5000; Biomol, Hamburg, Germany). Finally, the plates were developed with TMB substrate and www.rheumatology.oxfordjournals.org Bone resorption and CD5+ B cells ODs were determined by an automated plate reader (Millenia Kinetic Analyser; DPC). Avidity index of ACPA The avidity of ACPA was determined using an elution ELISA system as previously described [26]. In brief, sera were first titrated (1:251:3200) to determine the dilution showing a midrange OD for the CCP2 assay in order to allow the binding of low-avidity antibodies, if present. To determine the avidity, diluted patient serum was applied to a CCP2 plate and the initial OD (without elution) was measured as well as the OD of the remaining platebound antibodies after elution with 2 200 ml glycine-HCl pH 2.7. An HRP-coupled anti-human IgG antibody (1:5000; Biomol) was used for detection. The plates were developed with TMB substrate and the ODs were determined by an automated plate reader (Millenia Kinetic Analyser; DPC). An avidity index (AI) was calculated as described elsewhere [27]. CTX-1 and serum cytokine levels Serum CTX-1 was determined by using a competitive ELISA (USCN Life Science, Houston, TX, USA) and serum TNF-a by using a sandwich ELISA (Diaclone SAS, Besancon Cedex, France) according to the manufacturer’s instructions. The CTX-1 ELISA has an intra-assay coefficient of variation (CV) of 19.7% (our own measurements performed in duplicate) and an inter-assay CV of <12% (according to the manufacturer’s information), the minimal detectable dose is 46.7 pg/ml and the detection range is given as 0.123510 ng/ml (according to the manufacturer’s information). To minimize interassay variation, we ran the CTX-1 ELISA on three consecutive days using one plate each day. IL-6 and IL-10 serum levels were determined using DuoSet ELISA antibody pairs (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. In brief, Medisorb ELISA plates (Thermo Scientific, Waltham, MA, USA) were coated with 2 mg/ml capture antibody in carbonatebicarbonate buffer pH 9.4 (Thermo Scientific) at 4 C overnight. After blocking with 1% BSA in PBS pH 7.4 (Thermo Scientific), serial dilution of standard protein (500 pg/ml to 4.7 pg/ml) and undiluted sera were applied for 2 h. Biotinylated detection antibodies were incubated at concentrations of 50 and 150 ng/ml for IL-6 and IL-10, respectively. StreptavidinHRP included within the kit was diluted 1:200 and incubated for 30 min. The subsequent colour reaction was performed using TMB solution (BioLegend) and the OD was determined by an automated plate reader (Millenia Kinetic Analyser; DPC). Statistics Statistical analyses were performed in R (version 3.0.0; R Project for Statistical Computing, Vienna, Austria). The mean (S.D.) is given for all cell populations. Differences between two groups were tested by MannWhitney U-test and correlations were calculated by Spearman’s rank correlation. To proceed with a hypothesis-free www.rheumatology.oxfordjournals.org approach, we utilized importance scores from a random forest analysis. Prior to random forest model fitting, missing values (1.1% of all values) were imputed using the pcaMethods package. For factor analysis we first calculated a correlation matrix using Spearman’s rank correlation. The R Sweave documentation (pdf) of our statistical analyses can be found in supplementary 2-Rsweave statistical documentation S2, available at Rheumatology Online. Original fcs files are available from the corresponding author upon request. Results The bone resorption marker CTX-1 does not correlate with clinical parameters To screen for clinical parameters and possible confounders impacting on bone resorption in RA patients, we performed correlation analyses between serum CTX-1 levels and various patient data. We could thus show that serum CTX-1 titres correlate with neither age (P = 0.95, R = 0) nor sex (P = 0.44) and therefore rule out postmenopausal osteoporosis in the elderly female patient as a confounder for bone resorption in RA (Fig. 1A and B). CTX-1 levels also did not correlate with different treatment regimens (Fig. 1C), DAS28 (P = 0.84, R = 0.03) or CRP (P = 0.38, R = 0.12) (Fig. 1D and 1E). These results suggest that bone resorption in RA is not merely a function of disease activity or inflammation. We next investigated whether bone resorption in RA patients correlates with serum ACPA levels, and in particular with the IgG1, IgG4 and IgM isotypes. However, we did not find any correlation between the CTX-1 levels and IgG1 CCP (P = 0.78, R = 0.03), IgG1 MCV (P = 0.58, R = 0.07), IgG4 CCP (P = 0.89, R = 0.02) or IgG4 MCV (P = 0.97, R = 0) in our cohort (Fig. 1F and G). Moreover, we did not see any differences in IgM MCV levels comparing patients with high CTX-1 and patients with low CTX-1 levels (P = 0.87; Fig. 1H). Adaptive immune cells in the peripheral blood of RA patients significantly correlate with CTX-1 We continued to analyse whether peripheral blood cells that represent the adaptive immune system were somehow linked to bone resorption in RA patients. To that extent we determined the percentages of Tregs [CD25+Foxp3+helios+CD127 among live CD4+; 2.49% (S.D. 2.64)], transitional B cells [CD24highCD38high among live CD19+; 4.58% (S.D. 4.74)], naive B cells [CD24+CD38+ among live CD19+; 46.37% (S.D. 12.81)], memory B cells [CD24+CD38 among live CD19+; 39.4% (S.D. 13.89)] and CD5+ B cells [CD5+ among live CD19+; 18.1% (S.D. 11.32)] by flow cytometry and correlated these data with the respective CTX-1 levels in the patients’ sera (Fig. 2; for reasons of clarity, the scaling is provide in supplementary Fig. S2, available at Rheumatology Online). We found significant correlations between CTX-1 levels and all five cell populations. The strongest associations were found for the percentages of transitional B cells (P = 0.0004, R = 0.44), memory B cells (P = 0.0008, R = 0.42) and 547 Robby Engelmann et al. FIG. 1 Clinical parameters are not associated with serum CTX-1 level Serum CTX-1 does not correlate with (A) the age or (B) sex of RA patients. (C) CTX-1 levels do not vary between patients receiving different treatments. CTX-1 serum levels are independent of (D) the DAS28 and (E) CRP level. Serum ACPA levels [(F) CCP IgG1 and (G) IgG4] do not correlate with CTX-1. (H) No differences in IgM mutated citrullinated vimentin levels were detected when comparing patients with high and low CTX-1. Each dot represents one patient. Horizontal lines indicate medians. Differences between groups were tested by the MannWhitney U-test and correlations were calculated using Spearman’s test. DAS28: 28-joint DAS. CD5+ B cells (P = 0.002, R = 0.41). Importantly, the cell populations showed strong correlations between each other. For example, the percentages of memory and naive B cells were strongly correlated (P < 0.001, R = 0.90), as were the percentages of CD5+ B cells and transitional B cells (P < 0.001, R = 0.61). We therefore performed multivariate analyses to define the relevant predictors for CTX-1 levels. 548 CD5+ B cells are most intriguingly associated with CTX-1 serum levels in RA patients We performed a random forest analysis to calculate importance scores for each of the variables (i.e. cell populations). These scores reflect the gain of information each predictor added to the modelling of CTX-1 serum levels as the outcome. Interestingly, we found the percentages of CD5+ B cells, the CD86 expression level on transitional B www.rheumatology.oxfordjournals.org Bone resorption and CD5+ B cells FIG. 2 Serum bone resorption marker CTX-1 correlates with B cell populations in RA patients The correlation matrix provides P- and R-values (upper right) and original data (lower left) for the correlation of CTX-1 serum levels with the percentages of regulatory T cells (CD25+Foxp3+helios+CD127 cells among live CD4+), transitional B cells (CD24highCD38high among live CD19+), naive B cells (CD24+CD38+ among live CD19+), memory B cells (CD24+CD38 among live CD19+) and CD5+ cells among live CD19+ B cells. Correlations were tested using Spearman’s test. Each dot represents one patient. Dashed lines indicate correlations. *P < 0.05, **P < 0.01, ***P < 0.001. For detailed scaling of the graphs see supplementary Fig. S2, available at Rheumatology Online. cells and the percentages of CD5+ cells among the transitional B cell subsets most strongly associated with CTX-1 serum levels (Fig. 3A). Likewise, we performed factor analyses whereby serum CTX-1 levels, the percentages of transitional B cells as well as the percentage of CD5+ B cells aggregated into one factor with loadings of 0.48, 0.65 and 0.96, respectively. We therefore concluded that bone resorption in RA patients—as measured via CTX-1 serum levels—is associated with CD5+ B cells. To narrow down the specific CD5+ B cell subset correlating with CTX-1 levels, we determined the percentages of CD5+ cells among the transitional, naive and memory B www.rheumatology.oxfordjournals.org cell compartments. We thus found significant correlations between CTX-1 levels and the percentages of CD5+ cells among naive (P = 0.014, R = 0.34) and transitional B cell populations (P = 0.003, R = 0.39), but not among memory B cells (P = 0.19, R = 0.18). Some authors consider CD5 expression a marker for B cell activation [28]. However, our data demonstrated that the expression of the co-stimulatory molecules CD80 (P = 0.18, median MFI CD5+ vs all B cells: 64 vs 73) and CD86 (P = 0.22, median MFI CD5+ vs all B cells: 89 vs 84) were not significantly elevated on CD5+ B cells compared with all B cells. We therefore concluded that in RA patients CD5 expression is not merely a result of B cell activation. 549 Robby Engelmann et al. FIG. 3 Multivariate analyses show an association between CD5+ B cells and serum CTX-1 in RA patients (A) Random forest importance scores indicate an association of CD5+ B cells with CTX-1 serum levels. Nine parameters featuring the highest importance scores are shown. The percentage of CD5+ B cells showed the highest importance score and was set to 100%. (B) The avidity index is similar between patients with high and low CTX-1 levels and/or the percentage of CD5+ B cells. (C) Plots show no significant correlation between serum CTX-1 and IL-6 or IL-10 level. Each dot represents one patient. Horizontal lines indicate medians. The strong association with CD5+ transitional B cells led us to investigate CD1d expression on B cell populations as an additional marker for regulatory B cells. Our data showed that the expression of CD1d was independent of CD5 and comparable between naive and memory B cells. However, CD1d expression was significantly elevated in the transitional B cell population (P < 0.001) and did not correlate with CTX-1 levels. In conclusion, our analyses suggest that CD5+ but not CD1dhigh B cells are positively associated with serum CTX-1 level in RA patients. How could CD5+ B cells promote CTX-1 levels? Finally, we addressed two CD5+ B cell-associated mechanisms for their potential impact on bone resorption as measured via CTX-1 levels in the serum. First, CD5+ B1 cells undergo very limited affinity maturation and therefore produce low-affinity IgG antibodies, among them selfreactive ones [18]. Interestingly, Suwannalai et al. [29] proposed a population of low-avidity ACPA associated with radiographic progression in RA. These authors defined 550 an AI reflecting the ratio of ACPA bound to a CCP2coated plate after elution with a chaotropic reagent to the amount of ACPA initially bound to the plates. We similarly determined the AI for 22 RA patients, who featured either high or low CTX-1 levels and covered a range of CD5+ percentages. Again, we did not find any correlation between CTX-1 levels and ACPA avidity, suggesting that in our cohort the avidity of self-reactive antibodies does not contribute to CTX-1 levels (Fig. 3B). Secondly, CD5+ B cells produce IL-6, which in turn promotes osteoclast maturation and bone resorption. Moreover, CD5+ B cells are the main producers of B cell-derived IL-10, which drives autoimmunity via numerous pleiotropic effects [30]. We therefore determined IL-6 and IL-10 levels in the serum of our RA patients and correlated them with both serum CTX-1 levels and percentages of CD5+ B cells. Unfortunately we did not find any association between CTX-1 and IL-6 (P = 0.33), CTX-1 and IL-10 (P = 0.56) (Fig. 3C) or between the percentages of CD5+ B cells and IL-6 (P = 0.64) or IL-10 (P = 0.25). www.rheumatology.oxfordjournals.org Bone resorption and CD5+ B cells Factor analysis showed that IL-6 (loading 0.3) as a major pro-inflammatory cytokine aggregated into one factor with CRP, DAS28 and steroid treatment dosage (loadings 0.87, 0.65 and 0.74, respectively). Moreover, IL-6 correlated with therapy duration (P = 0.003, R = 0.38). We therefore concluded that CD5+ B cells contribute to elevated CTX-1 serum levels in RA by an as yet unknown mechanism. Discussion The present study investigates reasons for bone resorption in RA patients and focuses on clinical parameters, serum markers and immune cell populations. We aimed to evaluate ongoing bone resorption and erosive progression instead of established erosions and we therefore assessed CTX-1 levels in the serum [22]. Indeed, serum CTX-1 levels have been shown to be elevated in RA patients and to predict subsequent radiographic progression [21]. We here confirm these findings as our cohort exhibited elevated CTX-1 levels [1.8 ng/ml (S.D. 0.81)] compared with a healthy control group [1.13 ng/ml (S.D. 0.81)] whose serum was analysed using exactly the same assay [31]. We refrained from determining our own reference range in a healthy control cohort since our focus was on correlations between bone erosion and peripheral blood parameters among RA patients. Bone resorption results from the uncoupling of osteoblastic bone formation and osteoclastic bone resorption [32]. In RA, this uncoupling can occur in three ways: directly via RANKL expressed on Th17 cells; indirectly via soluble TNF-a and IL-6, which impact on the osteoblasts to induce further osteoclast maturation; or via ACPA [18, 33, 34]. It is therefore fair to assume that patients with high disease activity will show elevated CTX-1 levels. This, however, was not the case in our study (Fig. 1C). The more effective therapies for the prevention of bone erosion combine DMARDs—to contain inflammation—with biologics neutralizing TNF-a, IL-6 or B cells, which produce autoantibodies [32]. And indeed, our patients with a high DAS28 received anti-TNF-a, anti-IL-6R or anti-CD19 so that immune cellmediated bone resorption was possibly compensated for by the neutralization of osteoblasts stimulating cytokines and autoantibodies [35, 36]. Interestingly, we could not confirm dose dependency between serum ACPA levels and bone resorption as previously described [18]. However, we argue that by enrolling consecutive ACPA-positive patients into a crosssectional study we accepted the influence of treatment and disease activity on bone resorption. This is in contrast to the previous selection of newly diagnosed and untreated patients matched for age, sex and disease activity [18]. We found peripheral CD5+ B cells associated with bone resorption in RA patients. The origin and the exact immunological features of CD5+ B cells are still a matter of debate. Some authors have suggested that the expression of CD5 is merely a marker for activation [28]. Our finding that co-stimulatory molecules like CD80 and CD86 are expressed independently of CD5 argues against this notion. Furthermore, CD5+ B cells were formerly thought to be a population prone to produce www.rheumatology.oxfordjournals.org autoantibodies and to thus promote autoimmunity. And indeed, CD5+ B cells have been described to produce RF [37, 38] and to be elevated in RA patients [39]. Yet it became clear that CD5+ B cells are not the main producers of autoantibodies [40]. This is in line with our study, as we did not see a correlation between ACPA and the percentage of CD5+ B cells. More recently, CD5 has been suggested to be a negative regulator of B cell receptor activation and thus may serve to control autoimmunity [41]. Along the same lines, a population of regulatory B cells characterized by the marker combination CD19+CD24highCD38highCD5+CD1dhigh has been described [42]. These regulatory B cells are capable of producing high levels of IL-10 and suppress Th1 and Th17 differentiation of naive Th cells dependent on CD80 and CD86 co-stimulation [43]. However, the CD5+ B cell population in our study that correlates with bone resorption is unlikely to belong to the regulatory B cells. In our study the regulatory B cell-associated CD1d expression is only elevated on CD19+CD24highCD38high transitional B cells. Most importantly, these transitional B cells showed lower importance scores and loadings than the CD5+ B cells in our multivariate analyses. CD5+ B cells are known to produce IL-6 [44, 45] and IL-6 in turn supports osteoclast differentiation [33, 34], leading to an increase in bone resorption. We therefore analysed IL-6 serum levels yet did not find a correlation between serum IL-6 level and CTX-1. However, we cannot exclude that CD5+ B cells contribute locally to elevated IL-6 levels within the joints. Moreover, CD5+ B cells undergo limited germinal centre reactions and therefore lack somatic hypermutations, which in turn results in low-affinity antibodies [46]. Suwannalai et al. [29] recently demonstrated that lowavidity ACPA is somehow associated with more severe radiological progression in RA. We therefore determined the amount of low-avidity ACPA, but again were unable to find any differences comparing patients with either high or low CTX-1 and CD5+ cells. In summary, we identified the CD5+ B cell population to be significantly associated with bone resorption in a cross-sectional cohort of RA patients. However, a possible functional link between CD5+ cells and bone resorption in RA still needs to be determined. Rheumatology key messages The frequency of CD5+ B cells is associated with bone resorption in RA patients. . Neither systemic IL-6 nor low-affinity ACPA is associated with bone resorption in RA patients. + . A possible link between CD5 cells and bone resorption in RA patients needs to be determined. . Acknowledgements We thank Dr Carsten Wiethe for his help in setting up the panels for the flow cytometry and Birgit Ahrens for collecting the patient data. 551 Robby Engelmann et al. Funding: This study was funded by the German Research Foundation (DFG; Mu 844/13-1), an intramural grant (FORUN), as well as an Investigator Initiated Research Grant from Pfizer (WI170970). Disclosure statement: B.M.-H. received grant support from Pfizer. C.K. has provided consultancy services for AbbVie, Chugai, Pfizer and Roche; has received research support from Pfizer and has served on speakers’ bureaus for AbbVie, Berlin Chemie, Chugai, MSD, Pfizer, Roche and UCB. All other authors have declared no conflicts of interest. 13 Manabe N, Kawaguchi H, Chikuda H et al. Connection between B lymphocyte and osteoclast differentiation pathways. J Immunol 2001;167:262531. 14 Sowden JA, Roberts-Thomson PJ, Zola H. Evaluation of CD5-positive B cells in blood and synovial fluid of patients with rheumatic diseases. Rheumatol Int 1987;7:2559. 15 Sigal LH. Basic science for the clinician 54: CD5. J Clin Rheumatol 2012;18:838. 16 Weitzmann MN, Cenci S, Haug J et al. B lymphocytes inhibit human osteoclastogenesis by secretion of TGFb. J Cell Biochem 2000;78:31824. 17 Mauri C, Bosma A. Immune regulatory function of B cells. Annu Rev Immunol 2012;30:22141. Supplementary data Supplementary data are available at Rheumatology Online. References 1 Gravallese EM, Manning C, Tsay A et al. Synovial tissue in rheumatoid arthritis is a source of osteoclast differentiation factor. Arthritis Rheum 2000;43:25058. 2 Pettit AR, Walsh NC, Manning C, Goldring SR, Gravallese EM. RANKL protein is expressed at the pannus-bone interface at sites of articular bone erosion in rheumatoid arthritis. Rheumatology 2006;45:106876. 3 Redlich K, Hayer S, Ricci R et al. Osteoclasts are essential for TNF-a-mediated joint destruction. J Clin Invest 2002; 110:141927. 4 Jimenez-Boj E, Redlich K, Türk B et al. Interaction between synovial inflammatory tissue and bone marrow in rheumatoid arthritis. J Immunol 2005;175:25792588. 5 Müller B. Cytokine imbalance in non-immunological chronic disease. Cytokine 2002;18:3349. 6 Okamoto K, Takayanagi H. Regulation of bone by the adaptive immune system in arthritis. Arthritis Res Ther 2011;13:219. 7 Kotake S, Udagawa N, Takahashi N et al. IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis. J Clin Invest 1999;103: 134552. 8 Sato K, Suematsu A, Okamoto K et al. Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction. J Exp Med 2006;203: 267382. 9 Lacey DL, Boyle WJ, Simonet WS et al. Bench to bedside: elucidation of the OPGRANKRANKL pathway and the development of denosumab. Nat Rev Drug Discov 2012; 11:40119. 10 Romas E, Gillespie MT, Martin TJ. Involvement of receptor activator of NFkappaB ligand and tumor necrosis factoralpha in bone destruction in rheumatoid arthritis. Bone 2002;30:3406. 11 Kasagi S, Chen W. TGF-beta1 on osteoimmunology and the bone component cells. Cell Biosci 2013;3:4. 12 Luo CY, Wang L, Sun C, Li DJ. Estrogen enhances the functions of CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress osteoclast differentiation and bone resorption in vitro. Cell Mol Immunol 2011;8:508. 552 18 Harre U, Georgess D, Bang H et al. Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. J Clin Invest 2012;122: 1791802. 19 Garnero P, Ferreras M, Karsdal M et al. The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation. J Bone Miner Res 2003;18:85967. 20 Garnero P, Geusens P, Landewé R. Biochemical markers of joint tissue turnover in early rheumatoid arthritis. Clin Exp Rheumatol 2003;21(Suppl 31):S548. 21 Syversen SW, Goll GL, van der Heijde D et al. Cartilage and bone biomarkers in rheumatoid arthritis: prediction of 10-year radiographic progression. J Rheumatol 2009; 36:26672. 22 Garnero P, Thompson E, Woodworth T, Smolen JS. Rapid and sustained improvement in bone and cartilage turnover markers with the anti-interleukin-6 receptor inhibitor tocilizumab plus methotrexate in rheumatoid arthritis patients with an inadequate response to methotrexate: results from a substudy of the multicenter doubleblind, placebo-controlled trial of tocilizumab in inadequate responders to methotrexate alone. Arthritis Rheum 2010; 62:3343. 23 Kaminski DA, Wei C, Qian Y, Rosenberg AF, Sanz I. Advances in human B cell phenotypic profiling. Front Immunol 2012;3:302. 24 Moura RA, Weinmann P, Pereira PA et al. Alterations on peripheral blood B-cell subpopulations in very early arthritis patients. Rheumatology 2010;49:108292. 25 Engelmann R, Brandt J, Eggert M et al. IgG1 and IgG4 are the predominant subclasses among auto-antibodies against two citrullinated antigens in RA. Rheumatology 2008;47:148992. 26 Engelmann R, Brandt J, Eggert M et al. The anti-mutated citrullinated vimentin response classifies patients with rheumatoid arthritis into broad and narrow responders. J Rheumatol 2009;36:26704. 27 Suwannalai P, Scherer HU, van der Woude D et al. Anti-citrullinated protein antibodies have a low avidity compared with antibodies against recall antigens. Ann Rheum Dis 2011;70:3739. 28 Wortis HH, Teutsch M, Higer M, Zheng J, Parker DC. B-cell activation by crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways and results in different B-cell phenotypes. Proc Natl Acad Sci USA 1995;92:334852. www.rheumatology.oxfordjournals.org Bone resorption and CD5+ B cells 29 Suwannalai P, Britsemmer K, Knevel R et al. Low-avidity anticitrullinated protein antibodies (ACPA) are associated with a higher rate of joint destruction in rheumatoid arthritis. Ann Rheum Dis 2014;73:2706. 38 Mantovani L, Wilder RL, Casali P. Human rheumatoid B-1a (CD5+ B) cells make somatically hypermutated high affinity IgM rheumatoid factors. J Immunol 1993;151: 47388. 30 Youinou P, Jamin C, Saraux A. B-cell: a logical target for treatment of rheumatoid arthritis. Clin Exp Rheumatol 2007;25:31828. 39 Youinou P, Mackenzie L, Jouquan J, Le Goff P, Lydyard PM. CD5 positive B cells in patients with rheumatoid arthritis: phorbol ester mediated enhancement of detection. Ann Rheum Dis 1987;46: 1722. 31 Fernandez-Real JM, Bullo M, Moreno-Navarrete JM et al. A Mediterranean diet enriched with olive oil is associated with higher serum total osteocalcin levels in elderly men at high cardiovascular risk. J Clin Endocrinol Metab 2012;97: 37928. 32 Villeneuve E, Haraoui B. Uncoupling of disease activity and structural damage. Does it matter clinically? Ann Rheum Dis 2013;72:12. 33 Kotake S, Sato K, Kim KJ. Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation. J Bone Miner Res 1996;11:8895. 34 Hashizume M, Mihara M. The roles of interleukin-6 in the pathogenesis of rheumatoid arthritis. Arthritis 2011;2011. 35 Smolen JS, Han C, van der Heijde DMFM. Radiographic changes in rheumatoid arthritis patients attaining different disease activity states with methotrexate monotherapy and infliximab plus methotrexate: the impacts of remission and tumour necrosis factor blockade. Ann Rheum Dis 2009;68:8237. 36 Landewé R, van der Heijde D, Klareskog L, van Vollenhoven R, Fatenejad S. Disconnect between inflammation and joint destruction after treatment with etanercept plus methotrexate: results from the trial of etanercept and methotrexate with radiographic and patient outcomes. Arthritis Rheum 2006;54:311925. 37 Burastero SE, Casali P, Wilder RL, Notkins AL. Monoreactive high affinity and polyreactive low affinity rheumatoid factors are produced by CD5+ B cells from patients with rheumatoid arthritis. J Exp Med 1988;168: 197992. www.rheumatology.oxfordjournals.org 40 Reap EA, Sobel ES, Cohen PL, Eisenberg RA. Conventional B cells, not B-1 cells, are responsible for producing autoantibodies in lpr mice. J Exp Med 1993; 177:6978. 41 Hippen KL, Tze LE, Behrens TW. Cd5 maintains tolerance in anergic B Cells. J Exp Med 2000;191: 88390. 42 Blair PA, Noreña LY, Flores-Borja F et al. CD19+CD24hiCD38hi B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic lupus erythematosus patients. Immunity 2010;32: 12940. 43 Lemoine S, Morva A, Youinou P, Jamin C. Human T cells induce their own regulation through activation of B cells. J Autoimmun 2011;36:22838. 44 Cantaert T, Doorenspleet ME, Francosalinas G et al. Increased numbers of CD5+ B lymphocytes with a regulatory phenotype in spondylarthritis. Arthritis Rheum 2012; 64:185968. 45 Spencer NF, Daynes RA. IL-12 directly stimulates expression of IL-10 by CD5+ B cells and IL-6 by both CD5+ and CD5- B cells: possible involvement in ageassociated cytokine dysregulation. Int Immunol 1997;9: 74554. 46 Fischer M, Klein U, Küppers R. Molecular single-cell analysis reveals that CD5-positive peripheral blood B cells in healthy humans are characterized by rearranged Vkappa genes lacking somatic mutation. J Clin Invest 1997;100:166776. 553