Antigen-induced Arthritis Research Articles

The Immune-Modulating Cytokine and Endogenous Alarmin Interleukin-33 Is Upregulated in Skin Exposed to Inflammatory UVB Radiation

The cellular and molecular mechanisms by which UV radiation modulates inflammation and immunity while simultaneously maintaining skin homeostasis is complex and not completely understood. Similar to the effects of UV, IL-33 has potent immune-modulating properties that are mediated by the downstream induction of cytokines and chemokines. We have discovered that exposure of mice in vivo or human skin samples ex vivo to inflammatory doses of UVB induced IL-33 expression within the epidermal and dermal skin layers. Using a combination of murine cell lines and primary human cells, we demonstrate that both UV and the oxidized lipid platelet activating factor induce IL-33 expression in keratinocytes and dermal fibroblasts. Highlighting the significance of these results, we found that administering IL-33 to mice in vivo suppressed the induction of Th1-mediated contact hypersensitivity responses. This may have consequences for skin cancer growth because UV-induced squamous cell carcinomas that evade immunological destruction were found to express significantly higher levels of IL-33. Finally, we demonstrate that dermal mast cells and skin-infiltrating neutrophils closely associate with UV-induced IL-33-expressing fibroblasts. Our results therefore identify and support a role for IL-33 as an important early danger signal produced in response to inflammation-inducing UV radiation.

S100A8 enhances osteoclastic bone resorption in vitro through activation of Toll‐like receptor 4: Implications for bone destruction in murine antigen‐induced arthritis

Rheumatoid arthritis, which is associated with elevated levels of S100A8 and S100A9, is characterized by severe bone erosions caused by enhanced osteoclast formation and activity. The aim of the present study was to investigate the role of S100A8 and S100A9 in osteoclastic bone destruction in murine antigen-induced arthritis (AIA). Bone destruction was analyzed in the arthritic knee joints of S100A9-deficient mice in which S100A8 protein expression was also lacking, and in wild-type (WT) controls. Osteoclast precursors from S100A9-deficient and WT mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with S100A8, S100A9, or S100A8/A9 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced glycation end products (anti-RAGE)-blocking antibody, soluble RAGE, or Toll-like receptor 4 (TLR-4)-deficient osteoclast precursors. The formation of osteoclasts and actin rings, the regulation of osteoclast markers, and bone resorption were analyzed. Bone erosions and cathepsin K staining were significantly suppressed in S100A9-deficient mice after AIA induction. However, osteoclast precursors from S100A9-deficient mice developed normally into functional osteoclasts, which excludes a role for intrinsic S100A8/A9. In contrast to the results observed with S100A9 and S100A8/A9, the addition of S100A8 during osteoclastogenesis resulted in stimulation of osteoclast formation in conjunction with enhanced actin ring formation and increased bone resorption. Analysis of the putative receptor for S100A8 in osteoclastogenesis revealed that osteoclast differentiation and function could not be inhibited by blocking RAGE, whereas the increase in osteoclast numbers and enhanced bone resorption were completely abrogated using TLR-4-deficient osteoclast precursors. These results demonstrate that S100A8 stimulated osteoclast formation and activity and suggest that both S100A8 and TLR-4 are important factors in mediating osteoclastic bone destruction in experimental arthritis.

Treatment of antigen-induced arthritis in rabbits by the intra-articular injection of methylprednisolone, 90Y or chlorambucil.

Rabbits with a bilateral antigen-induced arthritis were injected intra-articularly (i.a.) in one joint with methylprednisolone (1 mg), 90Y (18.5 MBq) or chlorambucil (1 mg) as a single dose. The severity of arthritis was determined by measuring joint swelling and skin surface temperature, macroscopic and histological changes in the joint being assessed 8 weeks after induction of arthritis when the rabbits were killed. Methylprednisolone injected at the time of antigen challenge or 3 weeks later caused a reduction in joint swelling and temperature (P < 0.05) for 1 to 6 weeks after injection. 90Y had an initial proinflammatory effect lasting several days, but later caused a modest reduction in joint swelling for up to 4 weeks (P < 0.05). Eight weeks after induction of arthritis, neither methylprednisolone nor 90Y-treated joints showed any significant reduction in erosion or histopathology compared with control arthritic joints. Chlorambucil injected 1 week after antigen challenge caused a rapid reduction in joint swelling which was maintained for the duration of the study. Joint surface temperature was reduced to a lesser extent. Eight weeks after induction of arthritis, chlorambucil-treated joints showed a decrease (P < 0.05) in all of the parameters of disease pathology assessed. Treatment with chlorambucil intra-articularly was clearly more effective than with methylprednisolone or 90Y at the doses employed and deserves further study as a potential treatment for chronic synovitis.

Treatment of antigen-induced arthritis in rabbits with liposome-entrapped methotrexate injected intra-articularly.

Rabbits with a bilateral antigen-induced arthritis were injected intra-articularly in one joint with methotrexate as the free drug or entrapped in liposomes. Free methotrexate (1 mg) injected as a single dose at the time of antigen challenge, suppressed the development of joint swelling and the rise in skin surface temperature of treated joints by 20-30% compared with contralateral control arthritic joints. The beneficial effect of methotrexate occurred within 24 h of injection and was maintained for at least 56 days. However, methotrexate was ineffective in suppressing arthritis when injected 7 days after antigen challenge. Liposomal methotrexate suppressed the development of arthritis at a dose one-tenth that of the free drug and it was also effective in suppressing arthritis of 7 days duration, although significant beneficial effects of liposomal methotrexate were delayed for 10 to 14 days after injection. Neither free nor liposomal methotrexate was effective in suppressing an established arthritis, having no significant effect on joint swelling or skin surface temperature when injected 21 and 35 days after antigen challenge. At the end of the study, 8 or 9 weeks after induction of arthritis, the joints were examined morphologically and histologically. Free methotrexate generally had no significant effect on joint pathology. However, liposomal methotrexate suppressed the development of synovial hyperplasia, cellular infiltration and the erosion of cartilage and bone when injected at the time of antigen challenge or 7 days later, but affected none of these parameters in an established arthritis of 3 weeks duration.

Proteoglycan biosynthesis as a determinant of patella damage in the murine antigen-induced arthritis model.

The incorporation of 35SO4 into cartilage proteoglycan has been employed as a measure of patella damage in the murine antigen-induced arthritis model. In a preliminary set of experiments, where both the proteoglycan concentration and 35SO4 incorporation were determined in control and arthritic patella over a 2 day to 6 day week period, the arthritic joint contained significantly higher levels of radioactivity compared with the controls. A subsequent study over an extended period of 10 weeks confirmed the earlier results, and indicated that the 6 week samples showed the greatest difference (71%) in 35SO4 incorporation between the arthritic and control patella.

Comparative efficacy of a secretory phospholipase A2 inhibitor with conventional anti-inflammatory agents in a rat model of antigen-induced arthritis

IntroductionPreviously, secretory phospholipase A2 (sPLA2) inhibition has been used as an adjunct to conventional rheumatoid arthritis therapy in human clinical trials without significant improvement of arthritic pathology. In this study, we compared the efficacy of a potent and orally active group IIa secretory phospholipase A2 inhibitor (sPLA2I) to conventional anti-arthritic agents; infliximab, leflunomide and prednisolone, in a rat model of antigen-induced arthritis.MethodsInitially, to establish efficacy and dose-response, rats were orally dosed with the sPLA2I (1 and 5 mg/kg) two days prior to arthritis induction, and then daily throughout the 14-day study period. In the second trial, rats were orally dosed with the sPLA2I (5 and 10 mg/kg/day) beginning two days after the induction of arthritis, at the peak of joint swelling. Separate groups of rats were also dosed with the tumour necrosis factor-alpha (TNF-α) inhibitor infliximab (single 3 mg/kg i.v. injection), leflunomide (10 mg/kg/day, oral) or prednisolone (1 mg/kg/day, oral) at this same time point and used as comparative treatments.ResultsIn the pathology prevention trial, both 1 and 5 mg/kg dose groups of sPLA2I demonstrated a significant reduction in joint swelling and gait disturbances; however, only the higher 5 mg/kg dose resulted in significantly reduced histopathology scores. In the post-induction trial, rats dosed with sPLA2I showed a significant improvement in joint swelling and gait scoring, whereas none of the conventional therapeutics achieved a significant decrease in both of these two disease markers. Histopathological scoring at the end-point of the study demonstrated significantly reduced median scores in rats treated with 10 mg/kg sPLA2I and leflunomide.ConclusionsThe results from this study suggest a pathogenic role for sPLA2 enzymes in this model of arthritis in rats, and the potential clinical utility of sPLA2 inhibition as a safer, and more effective, alternative to conventional anti-arthritic therapeutics.

Intravenously delivered glucocorticoid liposomes inhibit osteoclast activity and bone erosion in murine antigen-induced arthritis

The objective of this study was to determine the effect of systemic delivery of prednisolone phosphate (PLP) encapsulated within long circulating ‘stealth’ liposomes on bone erosion and osteoclast activity during experimental antigen-induced arthritis (AIA). Liposomal PLP strongly suppressed knee joint swelling, synovial infiltrate and bone erosion in antigen-induced arthritis. The number of active osteoclasts was not only suppressed in bone lesions near inflamed synovium, but also within the trabecular bone of the tibia, suggesting a systemic suppression of osteoclast activation. Furthermore, liposomal PLP directly blocked osteoclast differentiation and bone resorption in vitro while it also suppressed expression of osteoclast differentiation factors M-CSF and RANKL in the synovium. Targeting studies showed that liposomes are most efficiently phagocytosed by macrophages and early precursors of osteoclasts in the bone marrow rather than by mature osteoclasts, indicating a possible inhibition of osteoclast differentiation from an early stage. Conclusion: Liposomal glucocorticoid delivery rather than free PLP offers a more efficacious way to inhibit both inflammation and bone erosion in rheumatoid arthritis.

Safety of glucocorticoids can be improved by lower yet still effective dosages of liposomal steroid formulations in murine antigen-induced arthritis: Comparison of prednisolone with budesonide

The goal of this study was to compare the effects of liposomal and free glucocorticoid formulations on joint inflammation and activity of the hypothalamic-pituitary-adrenal (HPA) axis during experimental antigen-induced arthritis (AIA). A dose of 10 mg/kg liposomal prednisolone phosphate (PLP) gave a suppression of the HPA-axis, as measured by plasma corticosterone levels in mice with AIA and in naïve mice. In a subsequent dose-response study, we found that a single dose of 1 mg/kg liposomal prednisolone phosphate (PLP) was still equally effective in suppressing joint inflammation as 4 repeated once-daily injections of 10 mg/kg free PLP. Moreover, the 1 mg/kg liposomal PLP dose gave 22% less suppression of corticosterone levels than 10 mg/kg of liposomal PLP at day 14 of the AIA. In order to further optimize liposomal glucocorticoids, we compared liposomal PLP with liposomal budesonide phosphate (BUP) (1 mg/kg). At 1 day after treatment, liposomal BUP gave a significantly stronger suppression of joint swelling than liposomal PLP (lip. BUP 98% vs. lip. PLP 79%). Both formulations also gave a strong and lasting suppression of synovial infiltration in equal amounts. However, at day 21 after AIA, liposomal PLP still significantly suppressed corticosterone levels, whereas this suppression was not longer statistically significant for liposomal BUP. Conclusion: Liposomal delivery improves the safety of glucocorticoids by allowing for lower effective dosing. The safety of liposomal glucocorticoid may be further improved by encapsulating BUP rather than PLP.

Liposomal targeting of glucocorticoids to the inflamed synovium inhibits cartilage matrix destruction during murine antigen-induced arthritis

Encapsulation of glucocorticoids into long-circulating liposomes provides targeting of these drugs to the inflamed synovium in experimental arthritis and thereby strongly improves their therapeutic index. The goal of this study was to evaluate the effect and mechanisms of intravenous liposomal delivery of prednisolone phosphate (Lip-PLP) on protease mediated cartilage destruction during murine antigen-induced arthritis (AIA). Mice treated with a single injection of Lip-PLP showed a pronounced suppression of synovial immune cell infiltration compared to control, PBS-treated mice. Liposomal PLP also significantly suppressed interleukin 1β (3.6 fold) in the synovium, but not in the blood serum. Furthermore, expression of the proteases MMP-3, -9, -13 and -14 and ADAMTS-4 and -5 was suppressed by Lip-PLP in the synovium, but not within the articular cartilage of the femur and tibia, demonstrating the specific action of Lip-PLP on the synovium. Lip-PLP is phagocytosed by macrophages in vitro and suppresses their expression of IL-1β and MMPs after LPS activation. Moreover, Lip-PLP suppresses destruction of the cartilage matrix during AIA mediated by active MMPs and ADAMTS, as assessed by immunostaining of their respective neoepitopes VDIPEN and NITEGE in various cartilage layers of the knee joint. Conclusion: liposomal delivery of glucocorticoids protects against cartilage matrix destruction during experimental arthritis by inhibiting protease expression and activity in the inflamed synovium.

Impaired B cell immunity in IL-22 knock-out mice in collagen induced arthritis

BackgroundThe role of IL-22, a cytokine produced by Th17 cells, in autoimmunity is not fully understood. Previous research has shown that IL-22 is important in the development of collagen induced...

Inhibition of M1 macrophage activation in favour of M2 differentiation by liposomal targeting of glucocorticoids to the synovial lining during experimental arthritis

Background and objectiveExperimental antigen-induced arthritis (AIA) is highly driven by pro-inflammatory ‘M1’ macrophages which express cytokines such as tumour necrosis factor α (TNFα), interleukin 1β (IL-1β), IL-6 and IL-12. However,...

S100A8 enhances osteoclast-mediated bone resorption in experimental antigen-induced arthritis through activation of toll-like receptor 4

ObjectiveRheumatoid arthritis (RA) is characterised by severe bone erosions caused by enhanced osteoclast formation and activity. Significantly elevated S100A8 and S100A9 levels in serum and synovial fluid of RA patients...

Dual role of IL-21 in experimental arthritis via SOCS regulation and Th17 differentiation

ObjectivesInterleukin-21 (IL-21) is a pleiotropic cytokine that is produced mainly by activated CD4+ Th17 cells. The purpose of this study was to investigate the role of IL-21 in joint pathology...

Gambogic acid and celastrol are two powerful anti-inflammatory drugs in arthritis

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease where the excessive secretion of pro-inflammatory cytokines plays an important role. The authors have previously reported an increase in circulating interleukin 1β...

Antigen-induced arthritis in rabbits: a comparative study between high-resolution ultrasound and contrast-enhanced ultrasound and pathologic findings

The objective of this study was to explore the feasibility and value of high-resolution ultrasound and contrast-enhanced ultrasound in evaluation of synovitis in rabbit knee joint in antigen-induced arthritis (AIA). Thirty-six rabbits were divided into three groups, each injected with different doses of ovalbumin (OVA) into the right knee joint. On week 1 and 4, 6 randomly selected in each group were killed. Each knee joint undergone the high-resolution ultrasound to measure capsule thickness and contrast-enhanced ultrasound to measure synovium thickness. The results from the ultrasound examinations were compared with those of pathologic examinations. Different OVA doses resulted in different modeling success rate, different pathological synovitis score, and different capsule and synovium thickness measured by ultrasound. The diagnostic accuracy of synovitis by ultrasound was high. The ultrasound measurement revealed that the capsule thickness on week 4 was lessened than that on week 1, while the synovial thickness on week 4 was greater than that on week 1. Both the joint capsule and synovium thickness measured by ultrasound were significantly and positively correlated to the pathologic synovitis score (P<0.05). The synovial thickness on week 4 is more correlated to the synovitis score than the capsule thickness, rather than that on week 1. Injection of OVA of different doses results in different modeling success rate and synovitis severity. The high-resolution ultrasound and contrast-enhanced ultrasound can be used to determine whether the AIA was made successfully and evaluate the synovitis severity. In chronic inflammatory phase, the contrast-enhanced ultrasound has better effect.

Galectin 3 aggravates joint inflammation and destruction in antigen‐induced arthritis

Galectin 3, an endogenous β-galactoside-binding lectin, plays an important role in the modulation of immune responses. The finding that galectin 3 is present in the inflamed synovium in patients with rheumatoid arthritis suggests that the protein is associated with the pathogenesis of this disease. We undertook this study to investigate the influence of galectin 3 deficiency in a murine model of arthritis. Wild-type (WT) and galectin 3-deficient (galectin 3(-/-) ) mice were subjected to antigen-induced arthritis (AIA) through immunization with methylated bovine serum albumin. The concentration of serum cytokines (interleukin-6 [IL-6] and tumor necrosis factor α [TNFα]) and antigen-specific antibodies was evaluated using a cytometric bead array platform and enzyme-linked immunosorbent assay (ELISA). Cellular IL-17 responses were examined by flow cytometry, ELISA, and enzyme-linked immunospot assay. The joint inflammation and bone erosion of AIA were markedly suppressed in galectin 3(-/-) mice as compared with WT mice. The reduced arthritis in galectin 3(-/-) mice was accompanied by decreased levels of antigen-specific IgG and proinflammatory cytokines. The frequency of IL-17-producing cells in the spleen was reduced in galectin 3(-/-) mice as compared with WT mice. Exogenously added recombinant galectin 3 could partially restore the reduced arthritis and cytokines in galectin 3(-/-) mice. Our findings show that galectin 3 plays a pathogenic role in the development and progression of AIA and that the disease severity is accompanied by alterations of antigen-specific IgG levels, systemic levels of TNFα and IL-6, and frequency of IL-17-producing T cells. To our knowledge, this is the first report of in vivo evidence that galectin 3 plays a crucial role in the development of arthritis.

Potent anti-inflammatory and antinociceptive activity of the endothelin receptor antagonist bosentan in monoarthritic mice

IntroductionEndothelins are involved in tissue inflammation, pain, edema and cell migration. Our genome-wide microarray analysis revealed that endothelin-1 (ET-1) and endothelin-2 (ET-2) showed a marked up-regulation in dorsal root ganglia during the acute phase of arthritis. We therefore examined the effects of endothelin receptor antagonists on the development of arthritis and inflammatory pain in monoarthritic mice.MethodsGene expression was examined in lumbar dorsal root ganglia two days after induction of antigen-induced arthritis (AIA) using mRNA microarray analysis. Effects of drug treatment were determined by repeated assessment of joint swelling, pain-related behavior, and histopathological manifestations during AIA.ResultsDaily oral administration of the mixed ETA and ETB endothelin receptor antagonist bosentan significantly attenuated knee joint swelling and inflammation to an extent that was comparable to dexamethasone. In addition, bosentan reduced inflammatory mechanical hyperalgesia. Chronic bosentan administration also inhibited joint swelling and protected against inflammation and joint destruction during AIA flare-up reactions. In contrast, the ETA-selective antagonist ambrisentan failed to promote any detectable antiinflammatory or antinociceptive activity.ConclusionsThus, the present study reveals a pivotal role for the endothelin system in the development of arthritis and arthritic pain. We show that endothelin receptor antagonists can effectively control inflammation, pain and joint destruction during the course of arthritis. Our findings suggest that the antiinflammatory and antinociceptive effects of bosentan are predominantly mediated via the ETB receptor.

Videoradiographic analysis of the range of motion in unilateral experimental knee joint arthritis in rats

IntroductionThe translational and predictive value of animal models highly depends on the validity of respective readout parameters. In arthritis research, there has been a shift from sole threshold testing for pain-related behavior, as well as from swelling and histology assessment for inflammation, toward an analysis of joint function as indicated, for instance, by an increasing number of studies on gait abnormalities. Clinically, the range of motion (ROM) of the affected joint plays a major role in diagnosis and the assessment of treatment benefits. This parameter, however, is only insufficiently detected by currently used analytic systems in animals.MethodsHere we used high-resolution videoradiographic analysis to assess ROM in experimental knee joint arthritis in rats. This parameter is described during the 21-day course of antigen-induced arthritis in rats. Furthermore, the therapeutic effects of antinociceptive (morphine) and anti-inflammatory (dexamethasone) treatment on ROM are documented. To obtain additional information on the implications of ROM in animal models, correlations were performed to measure pain-related behavior and inflammation.ResultsThe study animals showed a significant reduction in ROM of the inflamed knee joint in the acute phase of arthritis. This was accompanied by an increase in knee joint movement on the contralateral side, indicating a compensational mechanism. Both morphine and dexamethasone treatment increased and thus normalized ROM. Changes in ROM were further stage-dependently correlated with weight bearing and joint swelling, that is, with both pain-related behavior and signs of inflammation.ConclusionsThe dynamic ROM observed in freely moving rats in our model of knee joint arthritis might serve as a parameter for global disease activity and might thus represent a promising readout parameter for preclinical assessment regarding the overall efficacy not only of antiarthritic but also of antinociceptive compounds.

Noninvasive Quantitative Assessment of Synovial Pannus Angiogenesis by Contrast-enhanced Gray-scale Sonography in Antigen-induced Arthritis in Rabbits

The aim of this study was to evaluate the value of contrast-enhanced gray-scale sonography in quantitative assessment of synovial pannus angiogenesis in antigen-induced arthritis in rabbits. Twenty-four adult New Zealand White rabbits were divided into two groups. Inflammatory arthritis was induced by intra-articular injection of ovalbumin into right knee joints with 4 mg in low-dose group (LD, n = 12) and 8 mg in high-dose group (HD, n = 12). The left side of the knee on each rabbit was used as normal control. Contrast-enhanced gray-scale sonography with time intensity curve (TIC) was performed on the synovia of suprapatellar bursa and posterior capsules 4 weeks after the injection. Immunohistochemical examinations of synovia were applied to assess the microvessel density and the expression of vascular endothelial growth factor. Correlation analysis between sonographic and immunohistochemical findings was performed. Contrast-enhanced gray-scale sonography of all right knees demonstrated intra-articular hypoechoic lesions with enhanced blood flow and no abnormal findings on all left knees. Parameters of TIC, including ascending curve (A), derived peak intensity (DPI), and area under curve (AUC) on arthritis joints were significantly higher in HD group compared to LD group (P < .05). Positive correlation was found between immunohistochemical findings and parameters of A, DPI, and AUC (P < .05). However, no correlation was found between other parameters (slope of descending rate, time to peak, time to rise, and initial intensity) and immunohistochemical findings. Contrast-enhanced gray-scale sonography with TIC measurement could provide reliable method for noninvasive quantitative assessment of synovial pannus angiogenesis of arthritis in clinical settings.

Cooperative role of tumour necrosis factor‐α, interleukin‐1β and neutrophils in a novel behavioural model that concomitantly demonstrates articular inflammation and hypernociception in mice

BACKGROUND AND PURPOSE; Chronic joint inflammation and pain are the hallmarks of disease in patients with inflammatory arthritis, notably rheumatoid arthritis. The aim of the present study was to investigate the relative contribution of tumour necrosis factor (TNF)-α, interleukin (IL)-1β and neutrophil influx for joint inflammation and nociception in a novel murine model of antigen-induced arthritis (AIA). AIA was induced by administration of antigen into knee joint of previously immunized mice. Neutrophil accumulation was determined by counting neutrophils in the joints and assessing myeloperoxidase activity in tissues surrounding the joints. TNF-α, IL-1β and CXCL-1 were measured by elisa. Mechanical hypernociception was assessed in parallel, using an electronic pressure meter. Hypernociception was dependent on antigen dose and the time after its administration; it was prevented by treatment with morphine and associated with neutrophil infiltration and local production of TNF-α, IL-1β and CXCL-1. Administration of a chimeric monoclonal antibody to TNF-α (infliximab) or IL-1receptor antagonist prevented neutrophil influx and hypernociception, and this was comparable to the effects of dexamethasone. Treatment with fucoidin (a leucocyte adhesion inhibitor) greatly suppressed neutrophil influx and local production of TNF-α and IL-1β, and hypernociception. In conclusion, the present study describes a new model that allows for the concomitant evaluation of articular hypernociception and inflammation. Using this system, we demonstrated that a positive feedback loop involving neutrophil influx and the pro-inflammatory cytokines TNF-α and IL-1β is necessary for articular hypernociception after antigen challenge of immunized mice.