Hans Ulrich Scherer

Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin

Autoimmunity is complicated by bone loss. In human rheumatoid arthritis (RA), the most severe inflammatory joint disease, autoantibodies against citrullinated proteins are among the strongest risk factors for bone destruction. We therefore hypothesized that these autoantibodies directly influence bone metabolism. Here, we found a strong and specific association between autoantibodies against citrullinated proteins and serum markers for osteoclast-mediated bone resorption in RA patients. Moreover, human osteoclasts expressed enzymes eliciting protein citrullination, and specific N-terminal citrullination of vimentin was induced during osteoclast differentiation. Affinity-purified human autoantibodies against mutated citrullinated vimentin (MCV) not only bound to osteoclast surfaces, but also led to robust induction of osteoclastogenesis and bone-resorptive activity. Adoptive transfer of purified human MCV autoantibodies into mice induced osteopenia and increased osteoclastogenesis. This effect was based on the inducible release of TNF-α from osteoclast precursors and the subsequent increase of osteoclast precursor cell numbers with enhanced expression of activation and growth factor receptors. Our data thus suggest that autoantibody formation in response to citrullinated vimentin directly induces bone loss, providing a link between the adaptive immune system and bone.

Glycan profiling of anti–citrullinated protein antibodies isolated from human serum and synovial fluid

OBJECTIVE Anti-citrullinated protein antibodies (ACPA) exhibit unique specificity for rheumatoid arthritis. However, it is incompletely understood whether and how ACPA contribute to disease pathogenesis. The Fc part of human IgG carries 2 N-linked glycan moieties that are crucial for the structural stability of the antibody and that modulate both its binding affinity to Fcgamma receptors and its ability to activate complement. We undertook this study to analyze Fc glycosylation of IgG1 ACPA in serum and synovial fluid (SF) in order to further characterize the immune response to citrullinated antigens. METHODS ACPA were isolated by affinity purification using cyclic citrullinated peptides as antigen. IgG1 Fc glycosylation was analyzed by mass spectrometry. ACPA IgG1 glycan profiles were compared with glycan profiles of total serum IgG1 obtained from 85 well-characterized patients. Glycan profiles of paired SF and serum samples were available from 11 additional patients. RESULTS Compared with the pool of serum IgG1, ACPA IgG1 lacked terminal sialic acid residues. In SF, ACPA were highly agalactosylated and lacked sialic acid residues, a feature that was not detected for total SF IgG1. Moreover, differential ACPA glycan profiles were detected in rheumatoid factor (RF)-positive and RF-negative patients. CONCLUSION ACPA IgG1 exhibit a specific Fc-linked glycan profile that is distinct from that of total serum IgG1. Moreover, Fc glycosylation of ACPA differs markedly between SF and serum. Since Fc glycosylation directly affects the recruitment of Fc-mediated effector mechanisms, these data could further our understanding of the contribution of ACPA to disease pathogenesis.

Effect of Sildenafil on digital ulcers in systemic sclerosis – analysis from a single centre pilot study

Objective In this pilot study, the effect of sildenafil on digital ulcer (DU) healing and related clinical symptoms was analysed. Methods A total of 19 patients with systemic sclerosis (SSc) were treated with maximally tolerated sildenafil doses up to 6 months. Primary outcome was the healing of DUs. Changes in other clinical symptoms were also evaluated. Results In all, 49 DUs were present at baseline; this decreased to 17 ulcers (p<0.001) at the end of sildenafil treatment. Furthermore, the visual analogue scale (VAS) score for Raynauds phenomenon (RP), pain and activity improved (p=0.003, p=0.002 and p=0.05, respectively). A total of 9 patients developed 12 new DUs during sildenafil treatment. Conclusions This study indicates an effect of sildenafil on DU healing in patients with SSc and an improvement of RP and associated symptoms that should be validated in controlled studies.

Anti-citrullinated protein antibodies acquire a pro-inflammatory Fc glycosylation phenotype prior to the onset of rheumatoid arthritis

Objective To determine whether anticitrullinated protein antibodies (ACPA) exhibit specific changes in Fc glycosylation prior to the onset of arthritis. Methods Serum samples of patients with ACPA-positive arthralgia (n=183) were collected at baseline and at various time points of follow-up. 105 patients developed arthritis after a median of 12 months (IQR 6–24) and were classified as having either rheumatoid arthritis (RA, n=48) or undifferentiated arthritis (UA, n=57) based on the 1987 American College of Rheumatology (ACR) criteria. ACPA and total serum IgG were isolated by affinity purification and cleaved by trypsin. ACPA-IgG1 Fc-glycopeptides were subsequently analysed by nano-liquid chromatography mass spectrometry and compared to those of total IgG1. Results At baseline, ACPA-IgG1 and total IgG1 from arthralgia patients displayed similar Fc glycosylation patterns. By contrast, at the onset of arthritis, ACPA exhibited a decrease in galactose residues in RA patients, but not in UA patients. This decrease occurred around 3 months prior to diagnosis and was paralleled by an increase in systemic inflammation (erythrocyte sedimentation rate). Galactosylation of total IgG1 was also decreased in RA, but this did not precede the onset of arthritis. Interestingly, we additionally noted a higher degree of ACPA-IgG1 Fc core fucosylation at baseline as compared with total IgG1, which further increased prior to diagnosis. Conclusions ACPA display significant changes in Fc galactosylation and fucosylation prior to the onset of RA. These changes towards a more pro-inflammatory phenotype could be involved in driving the disease process.

Anti-cyclic citrullinated peptide antibodies are a collection of anti-citrullinated protein antibodies and contain overlapping and non-overlapping reactivities

Objective Anti-citrullinated protein antibodies (ACPA) and anti-cyclic citrullinated peptide (anti-CCP) antibodies are a hallmark of rheumatoid arthritis and are believed to play a role in disease pathogenesis. These antibodies are typically detected in ELISA with citrullinated peptides (eg, CCP2) or proteins as antigens. The absolute concentration of anti-CCP antibodies in serum is unknown. Although antibodies to several citrullinated proteins can mainly be detected within anti-CCP-positive sera, it is currently unknown whether anti-CCP antibodies are in fact ACPA. Likewise, it is unknown to what extent antibody responses to different citrullinated antigens are crossreactive. Methods An affinity purification method was established in which citrullinated antigen-specific antibodies were eluted from ELISA plates and then used for detection of other citrullinated antigens in ELISA or western blot. For additional crossreactivity studies, ELISA-based inhibition assays were performed with citrullinated or control peptides as inhibitors. Results The concentration of anti-CCP IgG antibodies was estimated to be at least 30 μg/ml in patients with high anti-CCP levels (>1600 μg/ml). Affinity-purified anti-CCP antibodies were able to recognise citrullinated fibrinogen (cit-fib) and citrullinated myelin basic protein (cit-MBP) on western blot. Furthermore, antibodies specific for cit-fib and cit-MBP were crossreactive. However, additional crossreactivity studies indicated that non-overlapping antibody responses to citrullinated peptides can also exist in patients. Conclusions This report shows for the first time that anti-CCP antibodies recognise multiple citrullinated proteins and are thus a collection of ACPA. More importantly, the data indicate that different ACPA responses are crossreactive, but that crossreactivity is not complete, as distinct non-crossreactive responses can also be detected in patients with RA.

Fc-Glycosylation of IgG1 is Modulated by B-cell Stimuli

We have recently shown that IgG1 directed against antigens thought to be involved in the pathogenesis of rheumatoid arthritis harbor different glycan moieties on their Fc-tail, as compared with total sera IgG1. Given the crucial roles of Fc-linked N-glycans for the structure and biological activity of IgG, Fc-glycosylation of antibodies is receiving considerable interest. However, so far little is known about the signals and factors that could influence the composition of these carbohydrate structures on secreted IgG produced by B lymphocytes. Here we show that both “environmental” factors, such as all-trans retinoic acid (a natural metabolite of vitamin A), as well as factors stimulating the innate immune system (i.e. CpG oligodeoxynucleotide, a ligand for toll-like receptor 9) or coming from the adaptive immune system (i.e. interleukin-21, a T-cell derived cytokine) can modulate IgG1 Fc-glycosylation. These factors affect Fc-glycan profiles in different ways. CpG oligodeoxynucleotide and interleukin-21 increase Fc-linked galactosylation and reduce bisecting N-acetylglucosamine levels, whereas all-trans retinoic acid significantly decreases galactosylation and sialylation levels. Moreover, these effects appeared to be stable and specific for secreted IgG1 as no parallel changes of the corresponding glycans in the cellular glycan pool were observed. Interestingly, several other cytokines and molecules known to affect B-cell biology and antibody production did not have an impact on IgG1 Fc-coupled glycan profiles. Together, these data indicate that different stimuli received by B cells during their activation and differentiation can modulate the Fc-linked glycosylation of secreted IgG1 without affecting the general cellular glycosylation machinery. Our study, therefore, furthers our understanding of the regulation of IgG1 glycosylation at the cellular level.

Intratympanic treatment of acute acoustic trauma with a cell-permeable JNK ligand: a prospective randomized phase I/II study

Objectives. Intratympanic administration of a cell-permeable JNK ligand has been shown to prevent hearing loss after acute acoustic trauma in animal models. Conclusions. Functional and morphological analysis of the treated ears revealed that AM-111 had an excellent otoprotective effect, even when administered hours after the noise exposure. Blocking the signal pathway with D-JNKI-1 is therefore a promising way to protect the morphological integrity and physiological function of the inner ear in various conditions involving acute sensorineural hearing loss. Subjects and methods. For the first application of AM-111 in humans, we organized a clinical phase I/II trial in patients with acute acoustic trauma after exposure to firecrackers in Berlin and Munich on New Years Eve 2005/2006. We randomly selected 11 patients for intratympanic treatment with AM-111 at a concentration of 0.4 mg/ml or 2 mg/ml within 24 h after noise exposure. Pure tone audiometry and otoacoustic emissions were assessed before treatment and on days 3 and 30 thereafter. Results. Based on clinical experience and on a calculation using an empirically derived exponential hearing recovery function AM-111 seems to have had a therapeutic effect. A total of 13 adverse events were reported in 5 study participants. None of the adverse events were serious or severe.

Cutting Edge: TNFR-Shedding by CD4 + CD25 + Regulatory T Cells Inhibits the Induction of Inflammatory Mediators

CD4+CD25+ regulatory T (Treg) cells play an essential role in maintaining tolerance to self and nonself. In several models of T cell-mediated (auto) immunity, Treg cells exert protective effects by the inhibition of pathogenic T cell responses. In addition, Treg cells can modulate T cell-independent inflammation. We now show that CD4+CD25+ Treg cells are able to shed large amounts of TNFRII. This is paralleled by their ability to inhibit the action of TNF-α both in vitro and in vivo. In vivo, Treg cells suppressed IL-6 production in response to LPS injection in mice. In contrast, Treg cells from TNFRII-deficient mice were unable to do so despite their unhampered capacity to suppress T cell proliferation in a conventional in vitro suppression assay. Thus, shedding of TNFRII represents a novel mechanism by which Treg cells can inhibit the action of TNF, a pivotal cytokine driving inflammation.

Glycosylation of immunoglobulin G determines osteoclast differentiation and bone loss

Immunglobulin G (IgG) sialylation represents a key checkpoint that determines the engagement of pro- or anti-inflammatory Fcγ receptors (FcγR) and the direction of the immune response. Whether IgG sialylation influences osteoclast differentiation and subsequently bone architecture has not been determined yet, but may represent an important link between immune activation and bone loss. Here we demonstrate that desialylated, but not sialylated, immune complexes enhance osteoclastogenesis in vitro and in vivo. Furthermore, we find that the Fc sialylation state of random IgG and specific IgG autoantibodies determines bone architecture in patients with rheumatoid arthritis. In accordance with these findings, mice treated with the sialic acid precursor N-acetylmannosamine (ManNAc), which results in increased IgG sialylation, are less susceptible to inflammatory bone loss. Taken together, our findings provide a novel mechanism by which immune responses influence the human skeleton and an innovative treatment approach to inhibit immune-mediated bone loss.

The heat shock protein gp96: a receptor-targeted cross-priming carrier and activator of dendritic cells

Abstract Heat shock proteins like gp96 (grp94) are able to induce specific cytotoxic T-cell (CTL) responses against cells from which they originate and are currently studied in clinical trials for use in immunotherapy of tumors. We have recently demonstrated that gp96 binds to at least one yet unidentified receptor restricted to antigen-presenting cells (APCs) like dendritic cells (DCs) but not to T cells. Moreover we have shown, that for CTL activation by gp96-chaperoned peptides receptor-mediated uptake of gp96 by APCs is required. Lately, we have discovered a second function of gp96 when interacting with professional APCs. Gp96 is able to mediate maturation of DCs as determined by up-regulation of MHC class II, CD86 and CD83 molecules, secretion of pro-inflammatory cytokines IL-12 and TNF-α and enhanced T-cell simulatory capacity. Furthermore, the gp96 receptor(s) are down-regulated on mature DCs, suggesting that the gp96 receptor(s) behave similar to other endocytic receptors like CD36, mannose receptor etc. Our findings now provide additional evidence for the remarkable immunogenicity of gp96: first, the existence of specific gp96 receptors on APCs and second, the capacity to activate dendritic cells which is strictly required to enable these highly sophisticated APCs to prime CTL responses.