Intra-articular Injection Of mBSA Research Articles

Local administration of 4-Thiouridine, a novel molecule with potent anti-inflammatory properties, protects against experimental colitis and arthritis

Previous studies in a rat model of Sephadex induced lung inflammation showed that 4-Thiouridine (4SU), a thiol substituted nucleoside, was very effective in reducing edema, leukocyte influx and TNF levels in bronchoalvelolar lavage fluid. However, little is known about the factors and mechanisms underlying these effects. In the present study, we have used two separate mouse models of chronic inflammation, a model of dextran sulphate sodium (DSS) induced colitis and a model of antigen induced arthritis, to evaluate the anti-inflammatory effect of 4-thiouridine. We have analyzed a broad spectrum of inflammatory mediators in order to delineate the mechanisms behind a potential anti-inflammatory effect of 4SU. Colitis was induced in C57BL/6 mice by administration of 3.5% DSS in drinking water for 5 days and the potential anti-colitic effect of 4SU was assessed by monitoring the disease activity index (DAI), measurement of colon length and histopathological analysis of colon tissue. We analyzed tissue myeloperoxidase (MPO) activity, serum pro-inflammatory cytokines (IL-1β, IL-6 and TNF), mRNA and protein expression of pro-inflammatory cytokines, COX-2, and NF-κB activity in colitis tissue. Intracolonic administration of 4SU (5 mg/kg & 10 mg/kg.) significantly inhibited MPO activity and reduced the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF) as well as COX-2. Further, NF-κB activation was also blocked by attenuating the phosphorylation of IkB kinase (IKK α/β) in DSS-induced colitis tissues. Arthritis was induced by intra-articular injection of mBSA in the knee of NMRI mice pre-immunized with mBSA and 4SU was administered locally by direct injection into the knee joint. The antiarthritic potential of 4SU was calculated by histopathological scores and histochemical analysis of joint tissue. Further, immunohistochemistry was used to study inflammatory cell infiltration and expression of cytokines and adhesion molecules in the synovium. Local administration of 50–100 mg/kg 4SU at the time of arthritis onset clearly prevented development of joint inflammation and efficiently inhibited synovial expression of CD18, local cytokine production and recruitment of leukocytes to the synovium. Taken together, our data clearly demonstrates a potent anti-inflammatory effect of 4SU in two experimental models. In conclusion 4SU could be a new promising candidate for therapeutic modulation of chronic inflammatory diseases like ulcerative colitis and arthritis.

Antigen‐Induced Arthritis Leads to Depression‐Like Behavior in Mice Mediated by Central TNFα

Rheumatoid arthritis (RA) is a relapsing inflammatory condition of the joints. There are many debilitating neurological and psychological manifestations associated with RA, including chronic pain, and depression. These symptoms contribute to a reduction of quality of life, particularly because they are often present when the peripheral disease is in remission. While progress has been made in the treatment of joint inflammation, mechanisms that underlie the central neural manifestations of RA have yet to be characterized and treatment options identified.Using a mouse model of RA, we sought to demonstrate depression‐like behaviour in the absence of joint inflammation and determine a mechanism for this condition. We hypothesized that inflammation would lead to depression‐like behaviour mediated by central cytokines. To test the hypothesis, we used the antigen‐induced arthritis (AIA) model in mice. C57Bl/6 male mice (8–10 wk) were sensitized with methylated bovine serum albumin (mBSA) 14 days prior to the immune challenge. Knee intra‐articular injection of mBSA was performed to induce joint inflammation. After 1 or 13 days, mice were subjected to the tail suspension test to evaluate the presence of depression‐like behavior and pain was assessed with a Von Frey apparatus. To evaluate the inflammatory state of the brain, multiplex cytokine analysis (32 cytokines) were performed in hippocampal specimens and microglial staining was performed using anti‐Iba‐1 antibodies. Anti‐TNFα injections were performed intracerebroventricularly (icv) to evaluate to role of this cytokine on behaviour.One day after immune challenge, joint inflammation was observed in the knee. After 13 days, no neutrophil infiltration was observed, suggesting a basal, non‐inflamed state. We observed high levels of pain on day 1, which persisted until day 13. Depression‐like behaviour was only seen on day 13, suggesting an alteration in behavior in the absence of knee inflammation. To validate this result, mice with AIA were treated with the antidepressant imipramine (30 mg/kg). We saw a reduction in the depression‐like behavior previously observed. Moreover, to rule out joint pain on the behavior reported, we treated AIA mice locally with the non‐opioid analgesic dipyrone. Reducing joint pain did not influence the depression‐like behavior. Microglia analysis on the hippocampus showed no overt activation of microglia at either time point. Cytokine analysis showed an increase in KC, MCP‐1, and TNFα (p=0.08) in the hippocampus of AIA mice after day 1, but no changes at day 13. In order to test if TNFα may be involved in the depression behaviour, we injected anti‐TNFα icv on day 0. At day 13, we observed a reduction of depression‐like behaviour in AIA mice, suggesting a role for TNFα on this outcome.We conclude that depression‐like behaviour in a mouse model of RA is mediated by central cytokines. The mechanism of how TNFα is modulating this behavior remains to be determined.Support or Funding InformationThis work was supported by Canadian Institutes of Health Research (CIHR) and National Counsel of Technological and Scientific Development (CNPq – Brazil).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The P2X7 Receptor, Cathepsin S and Fractalkine in the Trigeminal Subnucleus Caudalis Signal Persistent Hypernociception in Temporomandibular Rat Joints

Temporomandibular joint (TMJ) is frequently involved with rheumatoid arthritis with a high prevalence that could result in a chronic pain state. Once the disease is established in the joint, the antigen-specific immune reaction initiates a neuro-immune cascade of events that causes sensitization of the central nervous system. This study establishes animal experimental models that evaluate the chronicity of albumin-induced arthritis hypernociception in the TMJ. Antigen-induced arthritis was generated in rats with methylated bovine serum albumin (mBSA) diluted in complete Freund’s. Intra-articular injection of mBSA (10 µg/TMJ/week) during 3 weeks resulted in a persistent inflammatory hypernociception which was characterized by an inflammatory episode characterized by the increased of lymphocytes, macrophages and pro-inflammatory interleukins IL-12 and IL-18. The persistent model of inflammatory hypernociception induced by arthritis in the TMJ elicited protein levels of P2X7 receptors, cathepsin S and fractalkine in the trigeminal subnucleus caudalis. Overall, the results of the present work suggest that a persistent inflammatory hypernociception of albumin-induced arthritis in the TMJ leads to the activation of the central nervous system signaling by P2X7/cathepsin S/fractalkine pathway.

Local but Not Systemic Administration of Uridine Prevents Development of Antigen-Induced Arthritis.

ObjectiveUridine has earlier been show to down modulate inflammation in models of lung inflammation. The aim of this study was to evaluate the anti-inflammatory effect of uridine in arthritis.MethodsArthritis was induced by intra-articular injection of mBSA in the knee of NMRI mice pre-immunized with mBSA. Uridine was either administered locally by direct injection into the knee joint or systemically. Systemic treatment included repeated injections or implantation of a pellet continuously releasing uridine during the entire experimental procedure. Anti-mBSA specific immune responses were determined by ELISA and cell proliferation and serum cytokine levels were determined by Luminex. Immunohistochemistry was used to identify cells, study expression of cytokines and adhesion molecules in the joint.ResultsLocal administration of 25–100 mg/kg uridine at the time of arthritis onset clearly prevented development of joint inflammation. In contrast, systemic administration of uridine (max 1.5 mg uridine per day) did not prevent development of arthritis. Protection against arthritis by local administration of uridine did not affect the anti-mBSA specific immune response and did not prevent the rise in serum levels of pro-inflammatory cytokines associated with the triggering of arthritis. In contrast, local uridine treatment efficiently inhibited synovial expression of ICAM-1 and CD18, local cytokine production and recruitment of leukocytes to the synovium.ConclusionLocal, but not systemic administration of uridine efficiently prevented development of antigen-induced arthritis. The protective effect did not involve alteration of systemic immunity to mBSA but clearly involved inhibition of synovial expression of adhesion molecules, decreased TNF and IL-6 production and prevention of leukocyte extravasation. Further, uridine is a small, inexpensive molecule and may thus be a new therapeutic option to treat joint inflammation in RA.

AB0135 Immunomodulatory and Antiinflammatory Properties of Antigen B, A Protein Secreted by Echinococcus Granulosus Larval Stage, in Experimental Arthritis

BackgroundAntigen B (AgB) is a protein secreted by Echinococcus granulosus larval stage (1) and seems to be responsible for immuneregulation via Th2 response (2). response to promotes survival of the...

OP0235 Pet high affinity translocator protein ligands as a novel modality for macrophage targeting in arthritis

BackgroundActivated macrophages infiltrate synovial tissue in early stages of rheumatoid arthritis (RA). Therefore, macrophage targeting is attractive for early diagnostics of RA. The expression of translocator protein (TSPO) on (activated)...

AB0085 Murine antigen-induced arthritis is not affected by transgenic disruption of osteoblastic glucocorticoid signaling

BackgroundThe role of endogenous glucocorticoids (GC) in the initiation and maintenance of rheumatoid arthritis (RA) remains unclear. We demonstrated previously that disruption of GC signaling in osteoblasts results in a...

FRI0011 Type I IFN inhibits arthritis via down-modulation of arthritogenic T-cell proliferation and cytokine production but independently of the enzymatic activity of IDO

BackgroundThe cytokine interferon-alpha (IFNa) may induce adaptive-immunity independent arthritis if present in the joint (1) but may in contrast prevent antigen-induced arthritis if activated during immunisations (2). Here we investigate...

Interferon alpha inhibits antigen-specific production of proinflammatory cytokines and enhances antigen-specific transforming growth factor beta production in antigen-induced arthritis

IntroductionInterferon alpha (IFN-α) has a complex role in autoimmunity, in that it may both enhance and prevent inflammation. We have previously shown that the presence of IFN-α at sensitization protects against subsequent antigen-triggered arthritis. To understand this tolerogenic mechanism, we performed a descriptive, hypothesis-generating study of cellular and humoral responses associated with IFN-α-mediated protection against arthritis.MethodsArthritis was evaluated at day 28 in mice given a subcutaneous injection of methylated bovine serum albumin (mBSA), together with Freund adjuvant and 0 to 5,000 U IFN-α at days 1 and 7, followed by intraarticular injection of mBSA alone at day 21. The effect of IFN-α on mBSA-specific IgG1, IgG2a, IgG2b, IgA, and IgE was evaluated by enzyme-linked immunosorbent assay (ELISA). Cytokines in circulation and in ex vivo cultures on mBSA restimulation was evaluated with ELISA and Luminex, and the identity of cytokine-producing cells by fluorescence-activated cell sorting (FACS) analysis.ResultsAdministration of IFN-α protected mice from arthritis in a dose-dependent manner but had no effect on antigen-specific antibody levels. However, IFN-α did inhibit the initial increase of IL-6, IL-10, IL-12, and TNF, and the recall response induced by intraarticular mBSA challenge of IL-1β, IL-10, IL-12, TNF, IFN-γ, and IL-17 in serum. IFN-α decreased both macrophage and CD4+ T cell-derived IFN-γ production, whereas IL-17 was decreased only in CD4+ T cells. Ex vivo, in mBSA-restimulated spleen and lymph node cell cultures, the inhibitory effect of in vivo administration of IFN-α on proinflammatory cytokine production was clearly apparent, but had a time limit. An earlier macrophage-derived, and stronger activation of the antiinflammatory cytokine transforming growth factor beta (TGF-β) was observed in IFN-α-treated animals, combined with an increase in CD4+ T cells producing TGF-β when arthritis was triggered by mBSA (day 21). Presence of IFN-α at immunizations also prevented the reduction in TGF-β production, which was induced by the intraarticular mBSA injection triggering arthritis in control animals.ConclusionsAdministration of IFN-α has a profound effect on the cellular response to mBSA plus adjuvant, but does not affect antigen-specific Ig production. By including IFN-α at immunizations, spleen and lymph node cells inhibit their repertoire of antigen-induced proinflammatory cytokines while enhancing antiinflammatory TGF-β production, first in macrophages, and later also in CD4+ T cells. On intraarticular antigen challenge, this antiinflammatory state is reenforced, manifested as inhibition of proinflammatory recall responses and preservation of TGF-β levels. This may explain why IFN-α protects against antigen-induced arthritis.

P088 Type I interferon protects against antigen induced arthritis independently of indolediamine 2, 3 dioxygenase

Our earlier data have shown that type I interferon, when given at the time of antigen immunization can protect against mBSA induced arthritis (Type I IFN protects against arthritis, Eur J Immunol June 2011;41(6):1687–95). This protection ismanifested by inhibition of antigen specific proliferation of lymphocytes and decreased production of inflammatory cytokines, possibly resulting in immune tolerance. Indoleamine 2, 3 dioxygenase (IDO) is an important mediator of immune tolerance. Here we investigated if IFNa mediated arthritis protection and inhibition of antigen specific proliferation is dependent on IDO by inhibiting the enzymatic activity of IDO by 1 Methyl Tryptophan (1MT) at different time points during arthritis induction. Arthritis was induced by immunization with mBSA with or without 1000 U Interferon alpha (IFNα) at day 1 and 7 followed by intra-articular injection of mBSA at day 21. Inhibition of IDO activity, (10 mg 1-MT/30 g mice/day), either throughout the experiment from day −1 or from day 20, had no significant effect on IFNa mediated protection. Furthermore, in line with earlier data, the IFNa mediated arthritis protection in the IDO-inhibited group was also accompanied by lower antigen specific proliferation of lymph node cells and splenocytes restimulated with mBSA ex vivo at day 28. These results show that the tolerogenic mechanism underlying the IFNa mediated arthritis protection is independent of the enzymatic activity of IDO.

P096 IFN-A protects against antigen induced arthritis through induction of antigen specific cellular tolerance

Type I interferons are pleiotropic cytokines having an important regulatory role in autoimmune diseases including rheumatoid arthritis. Our earlier data show that IFN α protects against mBSA induced arthritis (Type I IFN protects against arthritis. Eur J Immunol June 2011;41(6):1687–95) . This IFN α mediated protection was manifested by inhibition of antigen specific proliferation but antigen specific total IgG levels remained largely unaffected. To further define the altered cellular response associated with IFNa-mediated protection against arthritis, the circulating cytokines as well as cytokines produced upon antigen restimulation of leukocytes ex vivo during the development of arthritis (day 14, day 21 and on the day of termination, day 28) was determined. We also assessed the effect of IFN α on antigen-specific IgG isotypes, which may differ depending on the dominant T helper cell response. Arthritis was induced by two injections (day 1 and day 7) of mBSA and adjuvant, with or without 100–5000 U of IFNa. Arthritis was triggered by intra-articular injection of mBSA on day 21 and arthritis was scored on day 28. Presence of IFNaat the time of antigen-sensitization protected mice from arthritis in a dose-dependent manner. No significant differences were found on antigen specific serum IgG1, IgG2a and IgG2b levels regardless of IFN α treatment. The circulating cytokine analysis as measured by Luminex showed that IFN- α decreases the production of cytokines IL-6 and IL-13 in the initial phase (day 14) of arthritis induction. Furthermore, this inhibitory effect of IFNawas also apparent in antigen-specific cytokine release ex-vivo at day 14. In the arthritis phase (day 28), IFN α significantly reduced the levels of circulating, pro-inflammatory cytokines (IL-1 β , IL-17, TNF- α , IFN γ , and IL-12), which were clearly up-regulated in the control group. However, antigen-specific release of cytokines (IFN γ , IL-6, IL-17 and IL-10) after re-stimulation ex-vivo at the day of termination (day 28) showed that splenocytes from IFNatreated mice responded with higher cytokine levels than controls. Conclusion: The ability of IFNato prevent arthritis development is most likely due to its effects on cellular rather than humoral immunity. Presence of IFNaat the time of antigen sensitization (day 1 and day 7) inhibits both in vivo and ex vivo release of pro-inflammatory cytokines throughout the arthritis induction. This results in clearly lower serum concentrations of pro-inflammatory cytokines after intra-articular challenge (day 28) and likely explains the protective effect of IFNa. The inhibition of cytokine release at the time of arthritis manifestation in the IFN α treated group is in line with our earlier published data of inhibited antigen specific re-call responses upon rechallange with mBSA. The increase in ex-vivo release of cytokines from splenocytes at this time point in the IFNa-treated group may indicate that the inhibitory effect on cytokine release by IFNaadministrated on day 1 and day 7 has a time limit.

The indirect antinociceptive mechanism of 15d‐PGJ2 on rheumatoid arthritis‐induced TMJ inflammatory pain in rats

Inflammation of the temporomandibular joint (TMJ) induced by rheumatoid arthritis (RA) have resulted in persistent pain and caused distress to many patients. Considering that not all patients respond to traditional drugs therapy to RA and it has demonstrated that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) into TMJ has a potential peripheral antinociceptive effect, the aim of this study was to evaluate the peripheral effect of 15d-PGJ2 in RA-induced TMJ inflammatory hypernociception. Antigen-induced arthritis (AIA) was generated in rats with methylated bovine serum albumin (mBSA). RA-induced TMJ hypernociception was assessed by measuring the behavioural nociceptive responses. After behavioural experiments, the animals were terminally anaesthetized and periarticular tissues were removed and homogenized. The supernatants were used to evaluate the levels of tumour necrosis factor (TNF)-α, interleukin (IL)-1β and keratinocyte-derived chemokine (KC) by enzyme-linked immunosorbent assay as well the expression of PKCε and PKA by western blotting analysis. The intra-articular injection of mBSA, but not phosphate buffered saline (control), in immunized rats induced dose- and time-dependent behavioural nociceptive responses in which the peak of nociceptive responses were obtained by using 10 μg/TMJ of mBSA after 24 h. Pretreatment with 15d-PGJ2 (30, 100 and 300 ng/TMJ) inhibited the RA-induced TMJ inflammatory hypernociception. In addition, 15d-PGJ2 reduced the RA-induced release of TNF-α, IL-1β and KC (p < 0.05) as well the expression of PKA and PKCε (p < 0.05). In the present study, we demonstrated that 15d-PGJ2 was able to reduce the RA-induced TMJ inflammatory hypernociception by an indirect mechanism. This antinociceptive effect is in part due to decrease of TNF-α, IL-1β and KC levels and PKA/PKCε expression in the TMJ.

JNK-1 deficiency limits macrophage-mediated antigen-induced arthritis

To elucidate the nonredundant roles of JNK-1 and JNK-2 in antigen-induced arthritis (AIA). Mice that were genetically disrupted in Jnk1 or Jnk2 were primed by injection of methylated bovine serum albumin (mBSA) in Freund's complete adjuvant and then challenged on day 21 by intraarticular injection of mBSA into the right knee. Bone marrow chimeras were generated and similarly treated. Joints were harvested and prepared for histologic assessment. T cell responses were verified by cytokine and proliferation responses, and relative immunoglobulin responses were measured by enzyme-linked immunosorbent assay. Cytokine messenger RNA expression levels were measured by quantitative polymerase chain reaction analysis. Thioglycollate-elicited and zymosan A-elicited macrophage recruitment was tested in vivo, and cell migration was tested in vitro. The peptide inhibitor D-JNKi was injected daily starting 4 days after intraarticular injection of mBSA into wild-type (WT) mice, and inflammation was scored histologically. JNK-1-deficient, but not JNK-2-deficient, mice had a reduction in inflammatory cell infiltration and joint damage. This effect was primarily restricted to hematopoietic cells, but B and T cell responses were preserved in mBSA-injected mice. JNK-1-deficient macrophages produced cytokines and chemokines at a level comparable to that in their WT counterparts. However, macrophage migration was impaired in vivo and in vitro. Targeting JNK with the peptide inhibitor D-JNKi dramatically reduced inflammation and joint destruction in WT mice. AIA is dependent on JNK-1, but not JNK-2. JNK-1 is a promising molecular target for reducing autoimmune inflammation, since its inhibition impairs macrophage migration.

Double-stranded RNA protects against antigen-induced arthritis via the type I interferon receptor (99.23)

Abstract Viral infections may contribute to different forms of arthritis. Both viral components, e.g. dsRNA and host-derived anti-viral agents, e.g. type I interferons (IFN), can induce arthritis if present in a joint. This joint inflammation is transient and mediated by antigen-independent (innate) immunity. In order to study the impact viral components may have on the development of antigen-dependent arthritis, dsRNA was administered systemically during the activation of antigen-induced arthritis. The methylated bovine serum (mBSA)-induced arthritis model used consists of two s.c. immunizations with mBSA in Freund's incomplete adjuvant one week apart, followed by intraarticular injection of mBSA alone three weeks after the first immunization. The importance of IFN-signalling in the onset of mBSA-induced arthritis was studied (1) by using mice deficient for the IFN receptor (IFNAR ko, strain 129) and (2) by including the IFN-activator viral dsRNA in the immunizations. Administration of dsRNA during the immunizations with mBSA inhibited arthritis development and severity in wt, but not in IFNAR ko mice (p&amp;lt;0.05). This protection was accompanied by lower levels of anti-mBSA-specific IgG in serum (p&amp;lt;0.05). This anti-inflammatory effect of IFN-signalling contrasts the potent arthritogenic properties dsRNA and IFN have if present in the joint and underscores the opposing effects IFN signalling may have locally/systemically and on innate/adaptive immunity. Grant resources for this work includes The King Gustav V 80-years foundation and the Swedish Physicians Society

Distinct in vivo roles of CD80 and CD86 in the effector T‐cell responses inducing antigen‐induced arthritis

CD80 and CD86 play a critical role in the initiation of T-cell responses. However, their role in the in vivo effector CD4+ T-cell responses has been less extensively investigated. The current studies have examined the functional relevance of CD80 and CD86 in the effector CD4+ T-cell responses inducing antigen-induced arthritis. Arthritis was induced in C57BL/6 mice by sensitization to methylated bovine serum albumin (mBSA) on day 0, booster immunization (day 7) and intra-articular injection of mBSA (day 21). Control or anti-CD80 and/or anti-CD86 monoclonal antibodies were administered from day 21 to day 28. Arthritis severity and immune responses were assessed on day 28. The development of arthritis was significantly suppressed by inhibition of CD80 or CD86. Blockade of both CD80 and CD86 caused a trend towards reduced disease severity compared to control antibody-treated mice. Neutralization of CD80 attenuated accumulation of CD4+ T cells in joints and enhanced splenocyte production and circulating levels of interleukin-4. Inhibition of CD86 or both CD80 and CD86 reduced T-cell accumulation in joints without affecting T helper type 1/type 2 (Th1/Th2) differentiation or antibody levels. Blockade of CD86, and not CD80, significantly suppressed splenocyte interleukin-17 (IL-17) production. These results provide further in vivo evidence that CD80 and CD86 play important pathogenic roles in effector T-cell responses. CD80 exacerbates arthritis by downregulating systemic levels of IL-4 and increasing T-cell accumulation in joints without affecting IL-17 production. CD86 enhances disease severity by upregulating IL-17 production and increasing the accumulation of effector T cells in joints without affecting Th1/Th2 development.

Interleukin‐6 modulates production of T lymphocyte–derived cytokines in antigen‐induced arthritis and drives inflammation‐induced osteoclastogenesis

To determine the cellular mediators of antigen-induced arthritis (AIA) and the relative contribution of members of the interleukin-6 (IL-6) family and tumor necrosis factor (TNF) in AIA. AIA was induced in mice deficient in T and B lymphocytes, IL-6 (IL-6(-/-)), TNF (TNF(-/-)), IL-11 receptor, and oncostatin M receptor, by immunization with methylated bovine serum albumin (mBSA) followed 7 days later by intraarticular injection of mBSA. Arthritis severity was assessed histologically, and T lymphocyte responses were assessed in vitro. Anti-TNF neutralizing antibody was administered to wild-type mice during AIA. Bone marrow osteoclasts were generated in vitro via culture with RANKL and macrophage colony-stimulating factor. AIA was dependent on CD4+ T lymphocytes, but not CD8+ T lymphocytes or B cells. IL-6(-/-) mice had reduced AIA severity and fewer osteoclasts at sites of bone erosion. This protective effect was not seen with a deficiency of other IL-6 family members and was similar to that in TNF(-/-) mice or wild-type mice receiving TNF blockade treatment. IL-6(-/-) CD4+ T lymphocytes from draining lymph nodes had reduced antigen-induced proliferation and produced less IL-17 and less RANKL, relative to osteoprotegerin, than cells from wild-type mice. Bone marrow from IL-6(-/-) mice generated fewer osteoclasts in vitro than bone marrow from either wild-type or TNF(-/-) mice. AIA is driven by CD4+ T lymphocytes. IL-6 is an important mediator of bone destruction in AIA because it regulates T lymphocyte production of key osteoclastogenic cytokines and inflammation-induced bone marrow osteoclast differentiation. These findings have implications for reducing bone and joint damage in rheumatoid arthritis.

Vegfb gene knockout mice display reduced pathology and synovial angiogenesis in both antigen-induced and collagen-induced models of arthritis.

To determine the role of vascular endothelial growth factor B (VEGF-B) in 2 mouse models of arthritis, antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA). For AIA studies, monarticular AIA was induced by methylated bovine serum albumin (mBSA) priming of Vegfb gene knockout (Vegfb(-/-)) and wild-type (Vegfb(+/+)) mice, followed by intraarticular injection of mBSA or saline control 8 days later. CIA was induced in Vegfb(-/-) and Vegfb(+/+) mice by intradermal injection of chick type II collagen in adjuvant. Arthritis was monitored in both models using defined criteria (clinical and histologic). Angiogenesis was measured by synovial vessel density in diseased and control joints. In AIA studies, Vegfb(+/+) mice displayed significant knee joint swelling and synovial inflammation 7 days after intraarticular injection of antigen. Synovial inflammation was associated with angiogenesis, since vessel density in AIA synovium was significantly higher in arthritic than in control joints from the same animal. Knee joint swelling, synovial inflammation, and inflammation-associated vessel density in arthritic joints were reduced in Vegfb(-/-) mice compared with arthritic joints from Vegfb(+/+) mice. Similarly, in CIA, both disease incidence and mean clinical severity scores were significantly reduced in Vegfb(-/-) mice compared with Vegfb(+/+) mice. Mean histologic severity scores and mean synovial vessel density were reduced in diseased joints from Vegfb(-/-) mice when compared with diseased joints from Vegfb(+/+) mice. The reduction in inflammation-associated synovial angiogenesis in Vegfb(-/-) mice implicates VEGF-B in pathologic vascular remodeling in inflammatory arthritis. VEGF-B may be an attractive target in the design of anti-angiogenic therapies for rheumatoid arthritis.

Cartilage-specific constitutive expression of TSG-6 protein (product of tumor necrosis factor alpha-stimulated gene 6) provides a chondroprotective, but not antiinflammatory, effect in antigen-induced arthritis.

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. 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. 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. 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.

INTERLEUKIN 6 KNOCK-OUT MICE ARE RESISTANT TO ANTIGEN-INDUCED EXPERIMENTAL ARTHRITIS

In order to assess the potential role of IL-6 in rheumatoid arthritis (RA), we have compared IL-6 deficient (IL-6 ko) mice and their wild-type (wt) counterpart for the capacity to develop methylated bovine serum albumin (mBSA)-induced arthritis. Our data show that IL-6 ko mice are not susceptible to antigen-induced arthritis (AIA). In fact, IL-6 ko mice treated by a standard protocol of immunization with mBSA did not develop joint swelling following intra-articular mBSA injection, nor revealed the characteristic joint lesions by histological examination. Conversely, wt mice treated according to the same protocol developed arthritis about 9 days after intra-articular injection, as detected by knee joint swelling and histological confirmation. We observed that the proliferative response of splenocytes to mBSA was impaired in ko mice following arthritis induction, as compared to the strong response observed in wt mice. Furthermore, anti-mBSA IgG levels were lower in ko mice as compared to wt mice. Finally, we show that sensitivity to AIA can be reconstituted in ko mice by subcutaneous injections of recombinant human IL-6 (rhIL-6). In addition, co-administration of IL-6 with mBSA by intra-articular injection into the joint was only partially effective in conferring sensitivity to AIA, suggesting the importance of a systemic effct for IL-6, but also that an additional role for this cytokine can be envisaged in the local inflammatory reaction during establishment of AIA.

Effect of a Monoclonal Antibody against Interleukin-4 on Suppression of Antigen-Induced Arthritis in Mice by Oral Administration of the Inducing Antigen

We investigated a role for interleukin-4 (IL-4) in suppression of T-cell-mediated antigen-induced arthritis (AIA) in mice by oral administration of the inducing antigen. For this investigation, a monoclonal antibody (11B11 mAb) that specifically neutralizes IL-4 was employed. AIA was induced by immunization with methylated bovine serum albumin (mBSA) (day 0) followed by intraarticular injection of mBSA into the ankle joint. To induce oral tolerance, mBSA was administered orally once a day from days −5 to −1. The 11B11 mAb was injected ip 30 min before each oral administration of mBSA. Oral administration of mBSA resulted in marked suppression of AIA. Suppression of the joint inflammation by the oral antigen was significantly diminished by treatment with 11B11 mAb. The mAb treatment was also followed by blockade of suppression of proliferative responses of lymphoid cells to mBSA by the oral antigen. Furthermore, secretion of IL-4 was significantly increased following oral administration of mBSA and the increased IL-4 secretion was markedly reduced by treatment with 11B11 mAb. There was a decrease in production of IFN-γ in orally tolerized mice that was blocked by the IL-4-neutralizing mAb. Thus, treatment with an anti-IL-4 mAb appears to be effective in blocking suppression of AIA by oral administration of the inducing antigen. The results also suggest that IL-4 may play a role in downregulation of T-cell-mediated inflammation by feeding pathogenic antigens.