H Erlandsson Harris

Characteristics of synovial fluid effusion in collagen-induced arthritis (CIA) in the DA rat; a comparison of histology and antibody reactivities in an experimental chronic arthritis model and rheumatoid arthritis (RA).

We have characterized the cellular content and some antibody reactivities in synovial fluid (SF) from DA rats with CIA. Since CIA is widely used as a model for RA, in which many studies concerning immune responses are performed on SF samples, we considered it important to describe the local, disease‐causing immune reactions in CIA. At the peak of disease (day 22 after immunization), the major cell population in CIA SF was granulocytes (72%), but macrophages (17.9%), plasma cells (2.6%) and lymphocytes (7.7%) were also present. The CIA synovial membrane (SM) obtained at the same time was mainly infiltrated by monocytes, with granulocytes, lymphocytes and plasma cells also present. Cell populations in blood did not differ between arthritic and normal DA rats. Equally, high anti‐collagen type II (CII) and rheumatoid factor (RF) levels could be detected both in SF and in sera. Notably, RF levels were also increased in normal DA rats. Moderate levels of anti‐heat shock protein 65 kD (hsp) antibodies were recorded systemically in both normal and diseased animals. In conclusion, the cellular composition in SF and in SM are similar in rat CIA and in RA. The morphological differences between SF and SM that are characteristic for RA could also be demonstrated in CIA. The antibody data indicate systemic production of anti‐CII and anti‐hsp antibodies as well as RF, but they give no support for local production of these antibodies in the joints, which is the case in RA.

Arthritogenicity of collagen type II is increased by chlorination

During inflammation, activated neutrophils, monocytes and macrophages produce and release myeloperoxidase (MPO). MPO converts hydrogen peroxide to hypochlorous acid, a highly reactive and oxidizing agent. Proteins subjected to hypochlorous acid become chlorinated. We analysed how chlorination of the cartilage antigen collagen type II (CII) affects its immunogenic and arthritogenic properties by studying immune responses to chlorinated CII in comparison to immune responses to CII and by studying the development of arthritis in rats immunized with CII–Cl. CII–Cl immunization of LEW.1AV1 rats caused a 100% incidence of arthritis with a mean maximum score of 9·2 (maximal score possible 16). The same dose of non‐chlorinated CII did not induce arthritis at all. Rats immunized with CII–Cl developed high anti‐CII–Cl IgG titres and also developed IgG antibodies recognizing the non‐chlorinated form of CII. Analysis of cytokine mRNA expression in lymph nodes 10 days after immunzation revealed an increased expression of interferon (IFN)‐γ mRNA and interleukin (IL)‐1β mRNA in CII–Cl‐immunized rats compared to CII‐immunized rats. Thus, chlorination of CII increased its immunogenicity as well as its arthritogenicity. As neutrophils, monocytes and macrophages are abundant cells in arthritic joints of patients with rheumatoid arthritis, chlorination might be a mechanism by which immunoreactivity to CII is induced and by which chronic joint inflammation is supported.

Antigen inhibition of collagen-induced arthritis is associated with up-regulation of IL-4 mRNA and induction of Ox40 on T cells in draining lymph nodes.

The addition of a foreign antigen to an inoculum completely inhibits the development of collagen‐induced arthritis (CIA). However, the mechanism of this phenomenon, antigen ‐inhibition, is incompletely understood. Previous studies have demonstrated that the inhibition of arthritis is not mediated through suppression of the antibody response to cartilage antigens. In this paper we investigated cytokine mRNA levels in lymph nodes cells recovered 3, 7 or 16 days from animals immunized with either collagen II in IFA or OVA + collagen II in IFA. At day 7, but not at other time‐points, IL‐4 mRNA was up‐regulated in the lymph nodes of OVA‐inhibited non‐arthritic animals compared to control animals which all developed arthritis. No significant differences between the two groups could be detected when expression of IFN‐γ, IL‐2, TNF‐α, IL‐1β or IL‐10 mRNA was analysed. Flow cytometry analysis of draining lymph node cells demonstrated that the T cell marker Ox40 was up‐regulated in the OVA‐inhibited group. Our results indicate that the complete inhibition of CIA caused by addition of OVA to the collagen II inoculum is due to the presence of a TH2 environment resulting from an increased production of IL‐4 mRNA and a parallel increase in Ox40+ T cells.

A8.27 Intraarticular drug delivery of anti-HMGB1 in hyaluronan gels for osteoarthritis treatment

Background and objectives Joint pain due to degeneration of cartilage tissue in osteoarthritis is a major problem affecting people of different ages and there are no long-term solutions available today. During recent years means suppression of inflammation that stops joint destruction have been developed and there is now a need for new approaches to stimulate repair. This should preferably be done without use of complicated cell therapies, and an opportunity to accomplish such development is provided by the application of new, injectable derivatives of hyaluronan. The cross linking chemistry applied here is a very rapid reaction between carbazate moieties on one component and aldehyde groups in the other, where a solid gel is formed within 30 s. HMGB1 is an endogenous molecule released from activated immune cells as well as dying cells with both proinflammatory and osteoclast-activating features. HMGB1 blockade is proven effective in experimental models of RA and the presence of extracellular HMGB1 is reported in OA specimens. The aim of this study was to evaluate the effect of HMGB1 blockade on cartilage repair and evaluate drug delivery into the joint using hyaluronan gels in an experimental osteoarthritis model in mice. Materials and methods OA was induced in mice by surgically destabilising the knee joint through anterior cruciate ligament transection. Anti-HMGB1 encapsulated in the gel was injected intraarticularly. After 8 weeks the animals were sacrificed and knees processed for histology. Sections were stained with Safranin-O and HMGB1 and scored blinded. Results We demonstrated that cartilage could be rescued by treatment with anti-HMGB1 in the OA model. HMGB1 staining in the cartilage specimens showed intra and pericellular expression in chondrocytes of proteoglycan depleted areas in articular cartilage. HMGB1 was also present in ligaments, synovia and osteophytes. A surprising observation was that also the gel alone showed improvement of cartilage. The negative impact of endogenous aldehydes on chondrogenesis have been demonstrated in a number of studies. The gel precursors carry aldehyde and hydrazide functional groups for the gel formation and we believe that interaction and neutralisation of endogenous aldehydes in the joint cavity may have affected cartilage healing. Conclusions The hyaluronan hydrogel has shown promising results for cell free regeneration therapies by local treatment in the joints. We showed that blocking of HMGB1 improved the cartilage regeneration in experimental OA and the hyaluronan gel in itself carried anti-inflammatory properties.

A2.28 Characterisation and potential function of HMGB1 in juvenile idiopathic arthritis

Background and objectives High mobility group box protein 1 (HMGB1) is a prototypic alarmin being released from activated, stressed or dying cells. Extracellular HMGB1 has the ability to induce cell migration as well as cytokine production. Pathogenic effects of HMGB1 have been described in several inflammatory diseases, including arthritis, and HMGB1-specific blockade is beneficial in multiple experimental disease models. Recent reports have underlined the importance of post-translational modifications (PTMs) in determination of HMGB1 function and release mechanisms. We investigated the occurrence of PTMs of HMGB1 obtained from synovial fluid (SF) of juvenile idiopathic arthritis patients (JIA). Materials and methods Synovial fluid was obtained from 17 JIA patients. Total Levels of HMGB1 were determined by ELISA. PTMs of HMGB1 were determined HMGB1-specific immunoprecipitation followed by Liquid chromatography tandem mass-spectrometry (LC-MS/MS). Results Analyses of 17 JIA patients confirmed high HMGB1 levels in SF. Liquid chromatography tandem mass-spectrometry (LC-MS/MS) analyses of PTMs revealed that total HMGB1 levels did not associate with increased LDH activity but strongly correlated with nuclear location sequence 2 (NLS2) hyperacetylation, indicating active release of HMGB1. The correlation between total HMGB1 levels and NLS2 hypoacetylation suggests additional, acetylation-independent release mechanisms. Monomethylation of lysine 43 (K43), a proposed neutrophil-specific PTM, strongly associated with high HMGB1 levels, implying that neutrophils are a source of released HMGB1. Analysis of cysteine redox isoforms: fully reduced HMGB1, disulfide HMGB1 and oxidised HMGB1 revealed that HMGB1 acts as both a chemotactic and a cytokine-inducing mediator. These properties were associated with actively released HMGB1. Conclusions This is the first report that characterises HMGB1-specific PTMs during a chronic inflammatory condition. HMGB1 in SF from JIA patients is actively released through both acetylation- and non-acetylation-dependent manners. The presence of various functional HMGB1 redox isoforms confirms the complexity of its pathogenic role during chronic inflammation. Defining HMGB1 release pathways and redox isoforms is critical for the understanding of the contribution of HMGB1 during inflammatory processes.

Anti HMGB1 treatment reduces inflammation in models of experimental autoimmunity

Background and objectives High mobility group box protein 1 (HMGB1) is a nuclear protein present in all cells. HMGB1 is passively released extracellularly during cell death and, similarly, HMGB1 can be actively secreted from activated cells. Extracellular HMGB1 mediates multiple inflammatory processes. High levels of extracellular HMGB1 have been demonstrated in synovial joints of arthritis patients, intraarticular injection of HMGB1 causes synovitis in mice and neutralisation of HMGB1 by peptide inhibitors or polyclonal antibodies ameliorates experimental arthritis. In this study, we investigated and compared the effects of an HMGB1 peptide inhibitor and a mouse monoclonal antibody (mAb) targeting HMGB1 in an experimental model of arthritis and in acute autoimmune hepatitis. Defining a mAb or peptide inhibitor with ameliorative effects will forward the development of HMGB1 blockade suitable for clinical trials. Methods Collagen induced arthritis (CIA) was induced in DBA/1 mice. Arthritis severity was evaluated by clinical grading of inflammation. Mice with established arthritis were treated every other day with mAb 2G7 or irrIgG and monitored for 7 days. Autoimmune hepatitis was induced by ConcavalinA injection in C57/bl6 mice pretreated with Abox, mAb 2G7 or PBS. After 8 h, sera and liver biopsies were collected. Serum ALT levels, along with HTX staining of liver biopsies were assessed. Liver myeloid peroxidase (MPO) were measured as an indication of neutrophil infiltration. Pro-inflammatory mediator levels were quantified by ELISA. HMGB1 levels were evaluated by ELISA and immunohistochemistry. Results mAb 2G7 treatment significantly ameliorated inflammation in CIA mice and also reduced destruction, as assessed by histological analysis. In mice with autoimmune hepatitis, we recorded an upregulated HMGB1 expression damaged liver lobules. Abox treatment minimised liver damage and mAb 2G7 reduced neutrophil infiltration. Reduced interferon γ and CXCL1 levels were demonstrated in both Abox and mAb 2G7 treated mice. Conclusion By these studies, we have for the first time defined a mAb targeting HMGB1 that effectively reduces inflammation and destruction during CIA. Beneficial effects of this mAb were also demonstrated in a model of autoimmune hepatitis. Furthermore, we could confirm the inhibitory effects of the peptide inhibitor A box, previously demonstrated in CIA, in experimental autoimmune hepatitis. Hence, our current work with humanisation of mAb 2G7 and assessements of its therapeutic effects can be performed in the autoimmune hepatitis model. This allows us to rapidly screen multiple humanised mAb clones and also avoids xenoreactivity effects. Hence, we have facilitated the development of HMGB1 blockade for clinical use.

Interleukin 1β and TLR ligands give enhanced cytokine production by their interaction with HMGB1

The proinflammatory protein HMGB1 was recently identified as a mediator of rheumatoid arthritis (RA) and of other inflammatory diseases. This is demonstrated by extracellular HMGB1 expression both in experimental models of arthritis and in synovial biopsies from patients with RA. Furthermore, intra-articular injection of HMGB1 causes destructive synovitis in mice and HMGB1-targeted treatment ameliorates experimental arthritis. Recent data have indicated that HMGB1 alone does not have proinflammatory properties while complexes of HMGB1 and the TLR ligands CpG DNA and lipopolysaccharide …

Dynamics of early synovial cytokine expression in rodent collagen-induced arthritis: a therapeutic study

This study was performed to elucidate pathophysiological events prior and during the course of collagen-induced arthritis (CIA) in DA rats. Kinetic studies of local cytokine responses were determined using immunohistochemisrty and computer-aided image analysis. We also investigated the effect of the macrophage-pacifying compound CNI-1493 on proinflammatory cytokine expressions. Synovial cryosections were analysed at various time points for the presence of IL-1β, TNF and TGF-β. Unexpectedly, an early simultaneous TNF and IL-1β expression was detected in resident cells in the lining layer, preceding disease onset by more than one week. The predominant cytokine synthesis by synovial (ED-1+) macrophages coincided with clinical disease. TNF-production greatly exceeded that of IL-1β. CNI-1493 treatment did not affect the early TNF and IL-1β synthesis, while disease-associated TNF and IL-1β production was greatly reduced. Furthermore, CNI-1493 significantly up-regulated synthesis of the anti-inflammatory cytokine TGF-β and thereby shifted the balance of pro-inflammatory and anti-inflammatory cytokines in the arthritic joint in a beneficial way.