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Objective: Disease‐associated, differentially hypermethylated regions have been reported in rheumatoid arthritis (RA), but no DNA methyltransferase inhibitors have been evaluated in either RA or any animal models of RA. The present study was conducted to evaluate the therapeutic potential of 5′‐azacytidine (5′‐azaC), a DNA methyltransferase inhibitor, and explore the cellular and gene regulatory networks involved in the context of autoimmune arthritis. Methods: A disease‐associated genome‐wide DNA methylation profile was explored by methylated CpG island recovery assay–chromatin immunoprecipitation (ChIP) in arthritic B cells. Mice with proteoglycan‐induced arthritis (PGIA) were treated with 5′‐azaC. The effect of 5′‐azaC on the pathogenesis of PGIA was explored by measuring serum IgM and IgG1 antibody levels using enzyme‐linked immunosorbent assay, investigating the efficiency of class‐switch recombination (CSR) and Aicda gene expression using real‐time quantitative polymerase chain reaction, monitoring germinal center (GC) formation by immunohistochemistry, and determining alterations in B cell subpopulations by flow cytometry. The 5′‐azaC–induced regulation of the Aicda gene was explored using RNA interference, ChIP, and luciferase assays. Results: We explored arthritis‐associated hypermethylated regions in mouse B cells and demonstrated that DNA demethylation had a beneficial effect on autoimmune arthritis. The 5′‐azaC–mediated demethylation of the epigenetically inactivated Ahr gene resulted in suppressed expression of the Aicda gene, reduced CSR, and compromised GC formation. Ultimately, this process led to diminished IgG1 antibody production and amelioration of autoimmune arthritis in mice. Conclusion: DNA hypermethylation plays a leading role in the pathogenesis of autoimmune arthritis and its targeted inhibition has therapeutic potential in arthritis management. [ABSTRACT FROM AUTHOR]

Objective Autologous stem cell transplantation (ASCT) induces long-term drug-free disease remission in patients with juvenile idiopathic arthritis. This study was undertaken to further unravel the immunologic mechanisms underlying ASCT by using a mouse model of proteoglycan-induced arthritis (PGIA). Methods For initiation of PGIA, BALB/c mice received 2 intraperitoneal injections of human PG in a synthetic adjuvant on days 0 and 21. Five weeks after the first immunization, the mice were exposed to total body irradiation (7.5 Gy) and received (un)manipulated bone marrow (BM) grafts from mice with PGIA. Clinical scores, T cell reconstitution, (antigen-specific) T cell cytokine production, and intracellular cytokine expression were determined following autologous BM transplantation (ABMT). Results ABMT resulted in amelioration and stabilization of arthritis scores. BM grafts containing T cells and T cell-depleted grafts provided the same clinical benefit, with similar reductions in PG-induced T cell proliferation and the number of PG-specific autoantibodies. In vivo reexposure to PG did not exacerbate disease. Following ABMT, basal levels of disease-associated proinflammatory cytokines (interferon-γ [IFNγ], interleukin-17 [IL-17], and tumor necrosis factor α [TNFα]) were reduced. In addition, restimulation of T cells with PG induced a strong reduction in disease-associated proinflammatory cytokine production. Finally, although the remaining host T cells displayed a proinflammatory phenotype following ABMT, IFNγ, IL-17, and TNFα production by the newly reconstituted donor-derived T cells was significantly lower. Conclusion Taken together, our data suggest that ABMT restores immune tolerance by renewal and modulation of the Teff cell compartment, leading to a strong reduction in proinflammatory (self antigen-specific) T cell cytokine production. [ABSTRACT FROM AUTHOR]

Objective To identify epigenetic factors that are implicated in the pathogenesis of rheumatoid arthritis (RA), and to explore the therapeutic potential of the targeted inhibition of these factors. Methods Polymerase chain reaction (PCR) arrays were used to investigate the expression profile of genes that encode key epigenetic regulator enzymes. Mononuclear cells from RA patients and mice were monitored for gene expression changes, in association with arthritis development in murine models of RA. Selected genes were further characterized by quantitative reverse transcription-PCR, Western blot, and flow cytometry methods. The targeted inhibition of the up-regulated enzymes was studied in arthritic mice. Results A set of genes with arthritis-specific expression was identified by the PCR arrays. Aurora kinases A and B, both of which were highly expressed in arthritic mice and treatment-naive RA patients, were selected for detailed analysis. Elevated aurora kinase expression was accompanied by increased phosphorylation of histone H3, which promotes proliferation of T lymphocytes. Treatment with VX-680, a pan-aurora kinase inhibitor, promoted B cell apoptosis, provided significant protection against disease onset, and attenuated inflammatory reactions in arthritic mice. Conclusion Arthritis development is accompanied by changes in expression of a number of epigenome-modifying enzymes. Drug-induced down-regulation of the aurora kinases, among other targets, seems to be sufficient to treat experimental arthritis. Development of new therapeutics that target aurora kinases can potentially improve RA management. [ABSTRACT FROM AUTHOR]

Objective To determine whether myeloid cells (such as granulocytes) present in the synovial fluid (SF) of arthritic joints have an impact on adaptive immunity. Specifically, we investigated the effects of SF cells harvested from the joints of mice with proteoglycan-induced arthritis (PGIA), on dendritic cell (DC) maturation and antigen-specific T cell proliferation. Methods We monitored DC maturation (MHCII and CD86 expression) by flow cytometry upon coculture of DCs with SF cells or spleen myeloid cells from mice with PGIA. The effects of these myeloid cells on T cell proliferation were studied using T cells purified from PG-specific T cell receptor (TCR)-transgenic (Tg) mice. Phenotype analysis of myeloid cells was performed by immunostaining, reverse transcription-polymerase chain reaction, Western blotting, and biochemical assays. Results Inflammatory SF cells significantly suppressed the maturation of DCs upon coculture. PG-TCR-Tg mouse T cells cultured with antigen-loaded DCs showed dramatic decreases in proliferation in the presence of SF cells. Spleen myeloid cells from arthritic mice did not have suppressive effects. SF cells were unable to suppress CD3/CD28-stimulated proliferation of the same T cells, suggesting a DC-dependent mechanism. SF cells exhibited all of the characteristics of myeloid-derived suppressor cells (MDSCs) and exerted suppression primarily through the production of nitric oxide and reactive oxygen species by granulocyte-like cells. Conclusion SF in the joints of mice with PGIA contains a population of granulocytic MDSCs that potently suppress DC maturation and T cell proliferation. These MDSCs have the potential to limit the expansion of autoreactive T cells, thus breaking the vicious cycle of autoimmunity and inflammation. [ABSTRACT FROM AUTHOR]

Objective Synovial inflammation, a feature of both osteoarthritis (OA) and meniscal injury, is hypothesized to be triggered in part via stimulation of Toll-like receptors (TLRs). We undertook this study to test whether a TLR-2- or TLR-4-stimulating factor in synovial fluid (SF) from patients with early knee OA with meniscal injury could lead to inflammatory activation of synoviocytes. Methods SF was obtained from patients with early OA cartilage damage undergoing arthroscopic meniscal procedures. SF was used to stimulate primary cultures of fibroblast-like synoviocytes (FLS) and cell lines transfected with TLR-2 or TLR-4. SF was used either alone or in combination with a TLR-2 stimulus (palmitoyl-3-cysteine-serine-lysine-4 [Pam3CSK4]) or a TLR-4 stimulus (lipopolysaccharide [LPS]). In blocking experiments, SF was preincubated with anti-CD14 antibody. Results SF from these patients did not stimulate interleukin-8 (IL-8) release from TLR transfectants. Compared with SF on its own, SF (at concentrations of 0.09-25%) in combination with TLR-2 or TLR-4 ligands resulted in significant augmentation of IL-8 release from both transfectants and primary FLS. Soluble CD14 (sCD14), a coreceptor for TLRs, was measured in SF from patients with early OA at levels comparable to those in patients with advanced OA and patients with rheumatoid arthritis. Blockade with anti-CD14 antibody abolished the ability of SF to augment IL-8 production in response to LPS, and diminished Pam3CSK4 responses. Conclusion SF augments FLS responses to TLR-2 and TLR-4 ligands. This effect was largely due to sCD14. Our results demonstrate that sCD14 in the setting of OA and meniscal injury sensitizes FLS to respond to inflammatory stimuli such as TLR ligands. [ABSTRACT FROM AUTHOR]

Objective The spondylarthritides (such as ankylosing spondylitis) are multisystem inflammatory diseases that frequently result in uveitis. Despite the common co-occurrence of uveitis with arthritis, there has been no explanation for the susceptibility of the eye to inflammation. Using an innovative intravital videomicroscopic approach, we discovered the coexistence of uveitis with axial and peripheral joint inflammation in mice immunized with cartilage proteoglycan (PG). The aim of this study was to elucidate the characteristics of uveitis and test the impact of interferon-γ (IFNγ) deficiency on the eye versus the joint and spine. Methods Female T cell receptor (TCR)-transgenic mice or IFNγ-knockout mice crossed to TCR-transgenic mice were immunized with PG. Uveitis was assessed by intravital videomicroscopy and histology. The clinical and histopathologic severity of arthritis and spondylitis were evaluated. The bone remodeling process within the spine was assessed by whole-body near-infrared imaging. Immunoblotting and immunofluorescence staining were used to examine the expression of PG and ADAMTS-5 and to examine the cellular composition of eyes with uveitis. Results PG neoepitopes along with the aggrecanase ADAMTS-5 were present in the eye, as they were the joint. Anterior uveitis developed in response to PG immunization. The cellular infiltrate consisted mainly of neutrophils and eosinophils. Unexpectedly, IFNγ deficiency markedly exacerbated uveitis while ameliorating joint and spine disease, indicating divergent mechanisms that drive diseases in the eye versus the joints and spine. Conclusion This study provides the first detailed description of a murine disease model in which uveitis coincides with arthritis and spondylitis. Our observations provide a great opportunity for understanding the pathogenesis of a relatively common but poorly understood disease. [ABSTRACT FROM AUTHOR]

The article discusses a study by Tibor T. Glant and colleagues who evaluated the extent of replication of an autoimmunity model of rheumatoid arthritis (RA) with the disease parameters of proteoglycan–induced arthritis (PGIA) and RA. It is inferred that GIA and PGIA models may represent 2 subtypes of seropositive RA, wherein more than 1 type of autoantibody could monitor disease severity and response to treatment.

OBJECTIVE: To investigate whether genetic preponderance of a T cell receptor (TCR) recognizing an arthritogenic peptide of human cartilage proteoglycan (PG) is sufficient for development of arthritis. METHODS: We performed a longitudinal study using BALB/c mice expressing a TCR that recognizes the arthritogenic ATEGRVRVNSAYQDK peptide of human cartilage PG. PG-specific TCR-transgenic (PG-TCR-Tg) mice were inspected weekly for peripheral arthritis until 12 months of age. Peripheral joints were examined histologically, and T cell responses, T cell activation markers, serum cytokines, and autoantibodies were measured. Apoptosis and signaling studies were performed in vitro on T cells from aged PG-TCR-Tg mice. RESULTS: Spontaneous arthritis developed as early as 5-6 months of age, and the incidence increased to 40-50% by 12 months of age. Progressive inflammation began with cartilage and bone erosions in the interphalangeal joints, and later expanded to the proximal joints of the front and hind paws. Spontaneous arthritis was associated with a high proportion of activated CD4+ T cells, enhanced interferon-[gamma] and interleukin-17 (IL-17) production, and elevated levels of serum autoantibodies. PG-TCR-Tg mice lacking IL-4 developed arthritis earlier and at a higher incidence than IL-4-sufficient mice. Antigen-specific activation-induced cell death was diminished in vitro in CD4+ T cells of PG-TCR-Tg mice with spontaneous arthritis, especially in those lacking IL-4. CONCLUSION: The presence of CD4+ T cells expressing a TCR specific for an arthritogenic PG epitope is sufficient to trigger spontaneous autoimmune inflammation in the joints of BALB/c mice. IL-4 appears to be a negative regulator of this disease, through attenuation of activation-induced cell death. [ABSTRACT FROM AUTHOR]

This article discusses a study on the effects of proinflammatory cytokine treatment or the complete absence of select cytokines on the expression of RANKL and osteoprotegerin (OPG) in synovial fibroblasts. The study used proinflammatory cytokines or antiosteoclastogenic cytokines in stimulating fibrolasts. It showed higher levels of RANKL and OPG expressed by fibroblasts from rheumatoid and arthritic joints in mice.

The periprosthetic granulomatous soft tissue [designated iterfacial membrane (IFM) in this study] exhibits heterogeneous histopathological features, in which highly vascularized areas with dense cellularity alternate with fibrotic and pseudocapsule-like tissue structures. Although macrophage/monocyte activation is a prominent event in the periprosthetic environment, fibroblasts also phagocytose particulate wear debris both in vivo and in vitro. Particulate wear debris and/or cytokines/growth factors alone or in combination (e.g., in conditioned media of explant cultures of IFMs) stimulated normal synovial and IFM fibroblasts to express inflammatory mediators and growth factors such as interleukin (IL)-1β, IL-6, IL-8, three isoforms of vascular endothelial growth factor (VEGF), monocyte/macrophage chemoattractant protein-1 (MCP-1), macrophage-colony-stimulating factor (M-CSF), cycloxygenases (Cox-1 and Cox-2), acid- and basic-fibroblast growth factors (FGF-1 and FGF-2), leukemia inhibitory factor-1 (LIF-1), transforming growth factor β-1 (TGF-β1), receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG). Thus, the fibroblast is capable of expressing a wide array of angiogenic and osteoclastogenic factors which are involved in the detrimental processes of the periprosthetic osteolysis. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1378-1388, 2007 [ABSTRACT FROM AUTHOR]

The article presents a study which investigated the role of interleukin-4 (IL-4) in the innate immune response versus the adaptive immune response in mice. Results showed that the severity of proteoglycan-induced arthritis (PGIA) was exacerbated in IL-4α and lysozyme M gene mice. According to the authors, the findings indicate that IL-4 functions as a major antiinflammatory cytokine in PGIA by governing the activity of macrophages/neutrophils.

Abstract: Chemokines, or chemotactic cytokines, are major regulators of the inflammatory response and have been identified as pathogenic factors in the periprosthetic soft tissue. Particulate wear debris induced NF-κB activation, the major transcriptional regulator of IL-8 and MCP-1 pro-inflammatory genes and, indeed, both IL-8 and MCP-1 chemokine gene expressions were upregulated in titanium particulate-stimulated human osteoblasts. Here, we demonstrate that phagocytosed particles activate the IL-8 gene promoter via a NF-κB-mediated mechanism. Transfection of a dominant negative mutant IκBα protein that cannot be serine phosphorylated led to suppression of IL-8 promoter activity. The p65/RelA NF-κB subunit activity was affected in both a time- and titanium particle concentration-dependent fashion. Titanium particles led to increased ERK, JNK, and p38 activation in MG-63 osteoblast cells, and IL-8 protein release was suppressed by specific inhibitors of the ERK and p38 MAPK pathways. Together, our results suggest that wear debris particles induce chemokine expression in osteoblasts via NF-κB-mediated transcriptional activation, which is controlled by the MAPK signal transduction pathway. [Copyright &y& Elsevier]

Gonda A, Gál I, Szántó S, Sarraj B, Glant TT, Hunyadi J, Mikecz K. CD44, but not l-selectin, is critically involved in leucocyte migration into the skin in a murine model of allergic dermatitis. CD44 and l-selectin (CD62L) are major adhesion receptors that mediate leucocyte recruitment at inflammatory sites and lymph nodes, by supporting cell rolling under blood flow. Both CD44 and CD62L have been implicated in inflammatory skin disorders, but their specific involvement in an immediate-type allergic reaction remains uncertain. We used mice deficient in CD44 or CED62L or both in order to determine whether one or both of these molecules were required for leucocyte extravasation in an atopic dermatitis-like allergic response. Wild-type (WT) mice and mice deficient in CD44, CD62L or both were immunized with ovalbumin (OVA). Inflammatory reaction in the ear was elicited once by means of intradermal injection of OVA. Effective sensitization of CD62L knockout (KO) mice required intraperitoneal antigen injection; however, OVA-specific T helper 2 (Th2)-type immune responses and IgE production in mice lacking CD44, CD62L or both were comparable to those in WT mice following intraperitoneal immunization. We employed intravital videomicroscopy to monitor the recruitment of fluorescence-labelled leucocytes to the ear tissue following challenge with OVA. The number of adherent leucocytes was significantly reduced in CD44 KO and CD44/CD62L double KO mice, indicating that CD44 was involved in firm adhesion, the committed step of leucocyte extravasation. Histology of the OVA-challenged ears showed a diminished leucocyte infiltration in the ears of CD44 KO and double KO mice. The results of our study demonstrate that CD44, but not CD62L, is required for leucocyte extravasation during a Th2-type inflammatory response. [ABSTRACT FROM AUTHOR]

The unilateral canine model is the most commonly used model of experimental osteoarthritis (OA). In this model, the anterior cruciate ligament (ACL) of one knee is transected and the contralateral joint is usually used as a control. However, dogs, similar to humans, can develop OA spontaneously with old age. Additionally, certain breeds of dogs are genetically predisposed to OA and can develop symptoms at a young age. The goal of this study was to compare the pathological changes of proteoglycans in OA cartilage from dogs that developed OA spontaneously to those that underwent ACL transection. For this reason, biglycan, decorin and fibromodulin levels and degradation patterns were compared by Western blot hybridization, and aggrecan contents were quantified by dimethylmethylene blue assay. The changes in proteoglycan levels in the cartilage of dogs with spontaneous OA, regardless of their age, were very similar to those published for human OA cartilage. However, when OA developed as a result of ACL-surgery, the changes in proteoglycans were different from those of slowly developing spontaneous OA. Therefore, these differences should be taken into consideration when the ACL-transection model is used. [Copyright &y& Elsevier]

Exposure of human osteoblasts to ultrafine titanium (Ti) particles has been shown to alter osteoblast gene expression. We previously reported that Ti particles can increase IL-6 release and suppress the gene expression of procollagens α1[I] and α1[III] in human osteoblasts. In this study, we now demonstrate that Ti particles can rapidly induce the chemotactic cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), two immediate early stress responsive chemokines important for the activation and chemotaxis of neutrophils and macrophages, respectively. In MG-63 osteosarcoma cells and bone marrow derived primary osteoblasts Ti particles selectively increased the steady state levels of IL-8 and MCP-1 mRNA in a time and concentration dependent manner. The increased chemokine mRNA correlated with increased secretion of IL-8 and MCP-1 protein. Actinomycin D, a potent RNA polymerase II inhibitor, blocked the Ti particle induction of IL-8 and MCP-1 mRNA expression, whereas cycloheximide, which inhibits protein synthesis, failed to inhibit chemokine gene expression suggesting Ti particles directly target activation of chemokine gene transcription. Consistent with a transcriptional mechanism not involving new protein synthesis, we demonstrate that Ti particles induce the binding of the p65 and p50 subunits of the latent transcription factor NF-κB to the IL-8 gene promoter. Taken together, these data demonstrate that Ti particles can activate transcription of the stress responsive chemokine genes IL-8 and MCP-1 in human osteoblasts. [Copyright &y& Elsevier]

Objective: This study was undertaken to investigate how fibroblasts respond to stimulation with particulate wear debris and/or conditioned media obtained from pathologic tissue, and whether these activated fibroblasts express compounds that are involved in bone resorption., Methods: Conditioned media from explant cultures of synovial tissue, periprosthetic soft tissue (interface membranes), titanium particles, and proinflammatory cytokines were used to stimulate fibroblasts. RNase protection assay was used to measure altered gene expression, and enzyme-linked immunosorbent assay, Western blot hybridization, and flow cytometry were used to determine fibroblast protein expression. Tartrate-resistant acid phosphatase staining was used to identify multinucleated osteoclast-like cells., Results: The most dominant compounds measured in the conditioned media from interface membranes were tumor necrosis factor alpha (TNFalpha), monocyte chemoattractant protein 1 (MCP-1), interleukin-1beta (IL-1beta), IL-6, IL-8, and vascular endothelial growth factor. Fibroblasts phagocytosed particulate wear debris and responded to cytokine/chemokine stimulation. The most prominent up-regulated genes and proteins secreted by fibroblasts in response to stimulation were matrix metalloproteinase 1, MCP-1, IL-1beta, IL-6, IL-8, cyclooxygenase 1 (COX-1), COX-2, leukemia inhibitory factor 1, transforming growth factor beta1 (TGFbeta1), and TGFbeta receptor type I. In addition, interface membrane fibroblasts expressed RANKL and osteoprotegerin in response to stimulation with conditioned media, TNFalpha, or IL-1beta. Stimulated fibroblasts cocultured with bone marrow cells in the presence of macrophage colony-stimulating factor induced osteoclastogenesis., Conclusion: Interface membrane fibroblasts respond directly to particulate wear debris, possibly via phagocytosis, expressing proinflammatory cytokines and RANKL. Thus, these cells may be actively involved in osteoclastogenesis and pathologic (periprosthetic) bone resorption.

Objective: To better understand the role of antigen (arthritogenic epitope)-specific T cells in the development of autoimmune arthritis., Methods: A transgenic (Tg) mouse expressing the T cell receptor (TCR) Valpha1.1 and V(beta)4 chains specific for a dominant arthritogenic epitope (designated 5/4E8) of human cartilage proteoglycan (HuPG) aggrecan was generated. This TCR-Tg mouse strain was backcrossed into the PG-induced arthritis (PGIA)-susceptible BALB/c strain and tested for arthritis incidence and severity., Results: CD4+ TCR-Tg T cells carried functionally active TCR specific for a dominant arthritogenic epitope of HuPG (5/4E8). T cells of naive TCR-Tg mice were in an activated stage, since the in vitro response to HuPG or to peptide stimulation induced interferon-gamma and interleukin-4 production. TCR-Tg mice uniformly, without exception, developed severe and progressive polyarthritis, even without adjuvant. Inflamed joints showed extensive cartilage degradation and bone erosions, similar to that seen in the arthritic joints of wild-type BALB/c mice with PGIA. Spleen cells from both naive and HuPG-immunized arthritic TCR-Tg mice could adoptively transfer arthritis when injected into syngeneic BALB/c.SCID recipient mice., Conclusion: TCR-Tg BALB/c mice display increased arthritis susceptibility and develop aggravated disease upon in vivo antigen stimulation. This model using TCR-Tg mice is a novel and valuable research tool for studying mechanisms of antigen (arthritogenic epitope)-driven regulation of arthritis and understanding how T cells recognize autoantigen in the joints. This type of mouse could also be used to develop new immunomodulatory strategies in T cell-mediated autoimmune diseases.

Objective: To describe the kinetics of leukocyte migration into a distal joint during the development of chronic inflammation in a murine model of rheumatoid arthritis (RA), to identify leukocyte subpopulations recruited in the synovial vessels, and to test in real time the effects of an antiinflammatory compound on leukocyte-endothelial cell interactions in the arthritic joint., Methods: We used intravital video microscopy (IVM), which was adapted to the microcirculation of the mouse ankle, to monitor the kinetics of leukocyte-endothelium interactions (rolling and firm adhesion) during the onset and progression of proteoglycan-induced arthritis (PGIA), a chronic autoimmune model of RA. Subpopulations of rolling and adherent leukocytes were identified by in vivo immunostaining. Leukocyte extravasation into the ankle joint was verified histologically., Results: Between the onset of arthritis and the beginning of the destructive phase of PGIA, we found a steady increase in the number of leukocytes that exhibited firm adherence to the endothelium of synovial vessels, which clearly underscores the chronic, self-perpetuating character of joint inflammation in this autoimmune model. We showed, however, that granulocytes, and not T cells, constituted the major cell population that was continuously recruited to the inflamed ankle. Using IVM, we could detect instant changes in leukocyte adhesion behavior in the synovial vessels of the arthritic joint upon administration of a compound that antagonizes leukocyte rolling., Conclusion: IVM of the microcirculation of the mouse ankle could become an essential tool for investigating the mechanisms that regulate leukocyte migration to the joint in systemic models of RA as well as for preclinical testing of antiinflammatory therapies.

Objective: To characterize experimentally induced spondylarthropathy (SpA) in arthritis-susceptible inbred mice and in their F(1) and F(2) hybrid generations of susceptible and resistant mouse strains., Methods: SpA was induced in susceptible BALB/c and C3H/HeJCr (C3H) strains of mice, and in their F(1) and F(2) generations derived from intercrosses with arthritis- and/or spondylitis-resistant DBA/2 and DBA/1 parent strains, by systemic immunization with cartilage proteoglycan (PG) aggrecan. The incidence and severity of PG-induced spondylitis (PGIS) were scored histologically, and these scores for spine involvement were correlated with serum antibody and cytokine levels and with in vitro T cell responses to cartilage PG., Results: PGIS was induced by systemic immunization with cartilage PG in adjuvant, and approximately 60-70% of susceptible mouse strains and their F(2) hybrids developed spondylitis either with or without arthritis. Adjuvants, particularly those activating the innate immune system and enforcing the Th1 dominance, had significant effects on the outcome and progression of SpA. The DBA/1 strain appeared to carry genes protecting this strain and its F(1) and F(2) hybrids from spondylitis, whereas the DBA/2 strain, although resistant to PGIS, harbored genes permitting PGIS in its hybrid generations. Arthritis- and/or spondylitis-susceptible BALB/c and C3H parent strains and their F(2) hybrids exhibited the highest incidence and severity of spondylitis., Conclusion: PGIS, a murine model of autoimmune spondylitis, shows similarities to ankylosing spondylitis. Segregation of susceptibility to PG-induced arthritis (PGIA) from that to PGIS in different genetic crosses suggests that PGIA and PGIS are separate diseases. Therefore, this model allows for the elucidation of genetic components involved in the etiology of SpA, independent of those controlling the susceptibility to PGIA.

Objective: To gain insight into the mechanisms of the antiinflammatory effect of tumor necrosis factor alpha (TNFalpha)-induced protein 6 (Tnfip6) in arthritis, using Tnfip6-deficient animals., Methods: TNFalpha-stimulated gene 6 (TSG-6) coding for Tnfip6 was disrupted. Tnfip6-deficient mice were backcrossed into proteoglycan-induced arthritis (PGIA)-susceptible BALB/c mice, and arthritis was induced by systemic immunization with cartilage proteoglycan (PG). Thioglycollate-induced sterile peritonitis was also assessed, to monitor the early events of neutrophil extravasation in wild-type and Tnfip6-deficient mice in the presence or absence of treatment with recombinant murine Tnfip6., Results: The onset of PGIA was similar, but progression and severity were significantly greater, in Tnfip6-deficient mice compared with wild-type BALB/c mice. However, this was not associated with enhanced T or B cell responses to cartilage PGs, but rather, an early and more extensive infiltration of the synovium with neutrophil leukocytes was the most prominent histopathologic feature of PGIA in Tnfip6-deficient mice. This was accompanied by elevated serum levels of interleukin-6 and amyloid A, and significantly increased activities of the enzymes plasmin, myeloperoxidase, and neutrophil elastase in the inflamed paw joints of Tnfip6-null mice, when compared with that of the wild-type littermates. Loss of control over several components of inflammation resulted in extensive and rapid cartilage degradation, bone erosion, joint ankylosis, and deformities in Tnfip6-null animals. In support of the antiinflammatory effect of Tnfip6 via the inhibition of polymorphonuclear (PMN) cell efflux, neutrophil invasion during thioglycollate-induced peritonitis was 2-fold higher in Tnfip6-deficient animals than in wild-type animals, but was dramatically suppressed by intravenous injection of recombinant murine Tnfip6., Conclusion: Tnfip6 is a multifunctional antiinflammatory protein that is produced at the site of inflammation and can be retained by the hyaluronan-rich extracellular matrix. A major effect of Tnfip6 is the inhibition of the extravasation of PMN cells, predominantly neutrophils, into the site of inflammation, most likely via a CD44/hyaluronan/Tnfip6-mediated blocking mechanism.

Objective: To determine whether the rheumatoid arthritis (RA)-predisposing class II molecules of the major histocompatibility complex (MHC) can present cartilage proteoglycan (PG) aggrecan, and if so, to determine the epitope repertoire of the human cartilage PG in HLA-transgenic mice and determine whether HLA-transgenic mice develop arthritis in response to immunization with human cartilage PG., Methods: Mice transgenic for HLA-DR2.Ab(0), DR3.Ab(0), DR4.Ab(0), and DQ8.Ab(0), lacking their own (mouse) class II antigens (Ab(0)), on the original (arthritis-resistant) and the arthritis-susceptible BALB/c backgrounds, were immunized with human cartilage PG. The T cell epitope repertoire presented by these class II MHC alleles was determined using a synthetic peptide library (143 peptides of the core protein of human cartilage PG), and arthritis development was monitored and compared in wild-type and HLA-transgenic/congenic BALB/c mice., Results: Mice of the 4 HLA-transgenic lines, either on the original mixed, arthritis-resistant background or DR4.Ab(0)- and DQ8.Ab(0)-transgenic/congenic mice on the arthritis-susceptible BALB/c genetic background, responded well to PG immunization (as assessed by T cell responses and antibody and cytokine production), and a number of T cell epitopes along the core protein of human cartilage PG were identified. DR4.Ab(0)- and DQ8.Ab(0)-transgenic mice immunized with human cartilage PG developed arthritis, but only when these class II MHC molecules were present on the arthritis-susceptible (BALB/c) genetic background., Conclusion: A number of human cartilage PG epitopes can be presented by HLA alleles that predispose to the development of RA, but the epitopes of the cartilage PG presented by HLA-DR4 or HLA-DQ8 can induce arthritis only in the presence of an appropriate genetic (non-MHC) background.

Objective: To apply and analyze the mechanisms of action of dimethyldioctadecylammonium bromide (DDA), a powerful adjuvant that does not have the side effects of the conventionally used Freund's adjuvants, in proteoglycan-induced arthritis (PGIA) and collagen-induced arthritis (CIA)., Methods: PGIA and CIA were generated using standard immunization protocols with cartilage proteoglycan aggrecan (PG) or human type II collagen (CII) emulsified with Freund's complete adjuvant (CFA), and compared with PGIA and CIA generated using immunization protocols in which the same antigens were used in combination with the adjuvant DDA. Immune responses to immunizing and self PGs and CII, and the incidence, severity, and onset of arthritis were monitored throughout the experiments. In addition, a new, inexpensive, and powerful method of inducing arthritis using crude cartilage extracts is described., Results: A significantly reduced onset period and a more severe arthritis were achieved in BALB/c mice immunized with cartilage PGs in DDA. PGs from bovine, ovine, and porcine cartilage, which otherwise have no effect or have only a subarthritogenic effect, and crude extracts of human osteoarthritic cartilage induced a 100% incidence with a very high arthritis score in BALB/c mice. The overall immune responses to either CII or PG were similar in antigen/CFA-immunized and antigen/DDA-immunized animals, but the Th1/Th2 balance shifted significantly toward a Th1 bias in DDA-injected animals with either PGIA or CIA., Conclusion: DDA, which was first used in autoimmune models, is a potent nonirritant adjuvant, which eliminates all undesired side effects of the Freund's adjuvants. DDA exerts a strong stimulatory effect via the activation of nonspecific (innate) immunity and forces the immune regulation toward Th1 dominance. These lines of evidence also suggest the possibility that seemingly innocuous compounds may exert an adjuvant effect in humans and may create the pathophysiologic basis of autoimmunity in susceptible individuals via the activation/stimulation of innate immunity.

Objective: To study the immunologic function and determine the fine epitope structure of a synthetic peptide p135H ((2373)TTYKRRLQKRSSRHP) of the G3 domain of human cartilage proteoglycan (aggrecan), which contains a highly homologous sequence motif of the shared epitope (QKRAA), the most common sequence motif in HLA-DR4 alleles, which predispose humans to the development of rheumatoid arthritis (RA)., Methods: Synthetic p135 peptides with altered sequences were used for (hyper)immunization of arthritis-susceptible BALB/c mice and then challenged with a single dose of cartilage proteoglycan. Human p135 (p135H) and mouse p135 (p135M) synthetic peptides of the G3 domain of aggrecan were used to prime lymphocytes, which were then used for adoptive transfer of arthritis into "presensitized" SCID mice, determining cross-reactivity among p135 peptides and their analogous sequences, and generating T cell hybridomas. T cell hybridomas were also used for arthritis transfer into SCID mice and for characterizing the fine epitope structure of T cell receptor (TCR) and major histo-compatibility complex (MHC) binding sites of the immunogenic/arthritogenic p135H sequence., Results: While p135H peptide-(hyper)immunized mice became sensitized, they developed arthritis only after injection of a single dose of cartilage proteoglycan aggrecan. An altered peptide sequence (p135H-AA) carrying the shared epitope motif (QKRAA) was as effective as the natural peptide p135H sequence for inducing arthritis. Mouse p135M-specific lymphocytes induced arthritis with a lower incidence, but synthetic peptides to Escherichia coli heat-shock protein (DnaJ) or HLA-DR4 allele (both having the shared epitope sequence with different flanking regions) were also positive. Fine epitope sequence recognition of an arthritogenic T cell hybridoma derived from p135H-primed lymphocyte population was determined. Interestingly, in the most central position, a basic amino acid triplet of p135H peptide was found to be the MHC-binding motif, whereas the flanking amino acids bound to the TCR., Conclusion: Peptide p135H, corresponding to the peptide sequence in the G3 domain of human cartilage proteoglycan aggrecan, is immunogenic/arthritogenic in BALB/c mice. Peptide p135H includes a highly homologous motif of the shared epitope, a sequence that is overrepresented in bacterial heat-shock proteins, envelope protein of human JC polyomavirus, and numerous HLA-DR4 alleles. Since the G3 domain of cartilage proteoglycan aggrecan with the p135 sequence is "lost" during the normal metabolic turnover of cartilage proteoglycan or in pathologic conditions, an antigenoriented T cell migration into joints of presensitized (susceptible) individuals may contribute to the organ-specificity of RA.

Objective: To analyze enzymes involved in joint damage by simultaneous investigation of glycosidases and matrix metalloproteinases (MMPs) in patients with various joint diseases., Methods: Activities of glycosidases (beta-D-glucuronidase, beta-D-N-acetyl-glucosaminidase, beta-D-N-acetyl-galactosaminidase, beta-D-galactosidase, and alpha-D-mannosidase) were tested at an acidic pH as well as at the original pH of the synovial fluid (SF) samples in parallel with activities of MMP-1 and MMP-9., Results: Patients with rheumatoid arthritis (RA) were characterized by significantly elevated activities of beta-D-glucuronidase and beta-D-N-acetyl-glucosaminidase in SF compared with patients with osteoarthritis, seronegative spondylarthritis, or acute sports injury. To select the best predictor for distinguishing among patient groups, a stepwise logistic regression analysis was performed; the strongest association was found to be between RA and beta-D-glucuronidase/beta-D-N-acetyl-glucosaminidase activities (measured at the pH of the SF). Further, a significant correlation was observed between the activity of SF beta-D-N-acetyl-glucosaminidase and the level of rheumatoid factor. In vitro digestion of human hyaline cartilage samples revealed that the dominant glycosidases, alone or in combination with MMPs, proved to be effective in depleting glycosaminoglycans (GAGs) from cartilage., Conclusion: These results suggest that exoglycosidases, which are present in the SF of RA patients, may contribute to the depletion of GAGs from cartilage and thereby facilitate the invasion of synovial cells and their attachment to cartilage in RA.

Objective: To explore the effect of sex on clinical and immunologic traits in major histocompatibility complex-matched (H-2d) F(2) hybrid mice with proteoglycan (PG)-induced arthritis and to identify how the quantitative trait locus (QTL) on the X chromosome influences the onset QTL of another chromosome., Methods: (BALB/c x DBA/2)F(2) hybrid mice were immunized with cartilage PG, and a genome-wide linkage analysis was performed using >200 simple sequence-length polymorphic markers. The major clinical traits (susceptibility, onset, and severity) were assessed, and PG-specific T and B cell responses, and the production of proinflammatory and antiinflammatory cytokines (tumor necrosis factor alpha, interleukin-1 [IL-1], IL-6, interferon-gamma, IL-4, IL-10, and IL-12) were measured in 133 arthritic and 426 nonarthritic female and male F(2) hybrid mice. The major clinical and immunologic traits were linked to genetic loci, and potential linkages among these QTLs and the effect of sex were analyzed., Results: Thirteen QTLs reported in previous studies were confirmed. Binary traits (susceptibility to arthritis) and disease onset were female specific and were identified on chromosomes 3, 7, 10, 11, 13, and X. QTLs for disease severity were mostly male specific and were located on chromosomes 1, 4, 5, 8, 14, 15, and 19. In addition, we identified 4 new QTLs for the onset of arthritis on chromosomes 3, 4, and 11, and 1 new QTL for severity on chromosome 14; all showed a strong gender association. A locus on the X chromosome interacted with a QTL on chromosome 10, and these 2 loci together seemed to control disease incidence and onset. Most of the clinical traits (QTLs) shared common regions with the immunologic traits and frequently showed a locus-locus interaction., Conclusion: Numerous immunologic QTLs overlap with clinical QTLs, thus providing information about possible mechanisms underlying QTL function. Disease susceptibility and onset showed predominant linkage with the female sex, under the control of a QTL on the X chromosome, while the severity QTLs were more strongly linked to the male sex.

Objective: To study the chondroprotective effect of constitutively expressed TSG-6 protein (tumor necrosis factor alpha-induced protein 6; Tnfip6) in cartilage, using antigen-induced arthritis (AIA) in mice., Methods: Transgenic mice constitutively expressing TSG-6 protein in cartilage were generated. Cartilage-specific constitutive expression of TSG-6 protein was confirmed by in situ hybridization, Western blot analysis, and immunohistochemistry. Control and transgenic mice were immunized with methylated bovine serum albumin (mBSA), and arthritis was induced by the intraarticular injection of mBSA. Mice were monitored up to day 35 after the challenge, and knee joint sections were examined for loss of cartilage proteoglycan (aggrecan) using Safranin O staining and antibodies to neoepitopes generated by various metalloproteinases (MPs). The loss of aggrecan in Safranin O-stained sections was quantified by morphometric methods., Results: Tsg6/tnfip6 transgenic mice constitutively expressed tsg6/tnfip6 messenger RNA and corresponding TSG-6 protein in cartilage from embryonic life through adulthood, without any phenotypic abnormalities. These mice were used for AIA studies. Intraarticular injection of mBSA uniformly induced severe inflammation both in control (wild-type and an irrelevant transgenic line) mice and in tsg6/tnfip6 transgenic mice. In contrast to the mBSA-injected knee joints of control animals that were heavily damaged from day 5, the cartilage of transgenic mice that constitutively expressed TSG-6 protein remained intact for at least 1 week, and this was followed by a relatively reduced loss of aggrecan. Concomitant with the loss of aggrecan, MP-generated neoepitopes accumulated in unprotected joints. By day 35, the proteoglycan content returned to nearly normal levels in tsg6/tnfip6 transgenic mice, whereas it remained low in MP-damaged knee cartilage of control mice., Conclusion: TSG-6 protein is known to form a complex with inter-alpha-inhibitor (IalphaI), a potent serine protease inhibitor, which may be immobilized via the hyaluronan (HA)-binding domain of TSG-6 protein in the HA-rich extracellular matrix of cartilage. Thus, the local accumulation of TSG-6 protein and TSG-6 protein-bound IalphaI in tsg6/tnfip6 transgenic mice may inhibit serine proteases and subsequent activation of MPs. It is suggested that this mechanism might protect cartilage from extensive degradation even in the presence of acute inflammation.