Rat Model Of Antigen-induced Arthritis Research Articles

Fcγ Receptor I-Coupled Signaling in Peripheral Nociceptors Mediates Joint Pain in a Rat Model of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is often accompanied by joint pain and inflammation. Previous studies have demonstrated that functional Fcγ receptor I (FcγRI) is expressed in dorsal root ganglion (DRG) neurons and might contribute to pain in rodent models of antigen-induced arthritis (AIA). This study was undertaken to elucidate the roles of nociceptive neuronal FcγRI-coupled signaling in the development of joint pain in AIA. RNA sequencing was used to investigate the transcriptome profile changes in the DRG in a rat model of AIA. A primary sensory neuron-specific Fcgr1a conditional-knockout (CKO) rat was established by crossing rats carrying a loxP-flanked Fcgr1a with a Pirt-specific Cre line. Behavioral, morphologic, and molecular studies were conducted to evaluate the differences between wild-type (WT) and CKO rats after AIA. We first showed that AIA induced a transcriptome profile change in the DRG, involving a number of key proteins downstream of the FcγRI-related signaling pathway. Compared to the WT rats, both the IgG immune complex-induced acute pain and AIA-induced pain were alleviated in CKO rats. Moreover, the AIA-induced activation of FcγRI-related signaling in DRGs was significantly reduced in CKO rats. In addition, CKO rats showed attenuated joint swelling after AIA. These results indicate that activation of FcγRI-coupled signaling in DRG neurons plays an important role in the development of joint pain in AIA. Our findings may provide novel insights into the interactions between the peripheral nervous system and the immune system in pathologic conditions and might suggest potential biotargets for the treatment of pain in RA.

Nociceptive neuronal Fc-gamma receptor I is involved in IgG immune complex induced pain in the rat

Antigen-specific immune diseases such as rheumatoid arthritis are often accompanied by pain and hyperalgesia. Our previous studies have demonstrated that Fc-gamma-receptor type I (FcγRI) is expressed in a subpopulation of rat dorsal root ganglion (DRG) neurons and can be directly activated by IgG immune complex (IgG-IC). In this study we investigated whether neuronal FcγRI contributes to antigen-specific pain in the naïve and rheumatoid arthritis model rats. In vitro calcium imaging and whole-cell patch clamp recordings in dissociated DRG neurons revealed that only the small-, but not medium- or large-sized DRG neurons responded to IgG-IC. Accordingly, in vivo electrophysiological recordings showed that intradermal injection of IgG-IC into the peripheral receptive field could sensitize only the C- (but not A-) type sensory neurons and evoke action potential discharges. Pain-related behavioral tests showed that intradermal injection of IgG-IC dose-dependently produced mechanical and thermal hyperalgesia in the hindpaw of rats. These behavioral effects could be alleviated by localized administration of non-specific IgG or an FcγRI antibody, but not by mast cell stabilizer or histamine antagonist. In a rat model of antigen-induced arthritis (AIA) produced by methylated bovine serum albumin, FcγRI were found upregulated exclusively in the small-sized DRG neurons. In vitro calcium imaging revealed that significantly more small-sized DRG neurons responded to IgG-IC in the AIA rats, although there was no significant difference between the AIA and control rats in the magnitude of calcium changes in the DRG neurons. Moreover, in vivo electrophysiological recordings showed that C-nociceptive neurons in the AIA rats exhibited a greater incidence of action potential discharges and stronger responses to mechanical stimuli after IgG-IC was injected to the receptive fields. These results suggest that FcγRI expressed in the peripheral nociceptors might be directly activated by IgG-IC and contribute to antigen-specific pain in pathological conditions.

Effects of Oral Administration of D-002 (Beeswax Alcohols) on Histological and Functional Outcomes in a Rat Model of Antigen-Induced Arthritis: Preliminary Study

D-002, a mixture of higher aliphatic beeswax alcohols, has been shown to display anti-inflammatory effects associated with the dual inhibition of ciclooxygenase and 5-lipoxygenase. Oral D-002 supplementation has been effective in experimental osteoarthritis, ameliorating all features of joint histopathology. Clinical studies have demonstrated that D-002 reduces osteoarthritis symptoms. However, D-002 effects on experimental models of rheumatoid arthritis (RA) have not been evaluated. To investigate whether D-002 improves histopathological and functional outcomes in a rat model of antigen-induced arthritis. First experiment. Rats were randomized into a negative vehicle-control (sham) and four groups injected with complete Freund's adjuvant (CFA): a positive vehicle-control, three treated with D-002 (50, 200 and 400 mg/kg/day) for 21 days. Second experiment. Rats were randomized into a sham and four CFA-injected groups: a positive vehicle-control, two treated with D-002 (25 and 100 mg/kg/day), one with methorexate (MTX) (0.3 mg/kg) for 28 days. Arthritis severity was evaluated by bodyweight loss, decreased exploratory activity and histological changes of tarsal joint and spleen samples in both experiments, except the exploratory activity, assessed only in the first one. CFA injection decreased the bodyweight and the exploratory activity, and induced infiltration of mononuclear cells, pannus formation and vascularity in the tarsal joint of positive control rats. These changes were significantly and markedly ameliorated by D-002 as compared to the positive control. MTX also reversed CFA-induced changes. The reduction of the infiltration of mononuclear cell with D-002 400 mg/kg was greater (80.9%) than with MTX (66.8%), but effects on other variables were similar. No abnormalities in spleen samples of D-002-treated groups were detected. This is the first report demonstrating the efficacy of oral treatment with D-002 in a rat model of antigen-induced arthritis. Results suggest that D-002 could help manage RA, but confirmation of such potential benefit requires extensive further research.

Potent Anti-Inflammatory and Antiproliferative Effects of Gambogic Acid in a Rat Model of Antigen-Induced Arthritis

Background. We have previously reported a continuous activation of caspase-1 and increased interleukin (IL)-1β levels in early rheumatoid arthritis (RA). These observations raised the hypothesis that drugs targeting the IL-1β pathway, in addition to tumour necrosis factor (TNF), may be particularly effective for early RA treatment. We have recently identified gambogic acid as a promising therapeutic candidate to simultaneously block IL-1β and TNF secretion. Our main goal here was to investigate whether gambogic acid administration was able to attenuate inflammation in antigen-induced arthritis (AIA) rats. Methods. Gambogic acid was administered to AIA rats in the early and late phases of arthritis. The inflammatory score, ankle perimeter, and body weight were evaluated during the period of treatment. Rats were sacrificed after 19 days of disease progression and paw samples were collected for histological and immunohistochemical evaluation. Results. We found that inflammation in joints was significantly suppressed following gambogic acid administration. Histological and immunohistochemical evaluation of treated rats revealed normal joint structures with complete abrogation of the inflammatory infiltrate and cellular proliferation. Conclusions. Our results suggest that gambogic acid has significant anti-inflammatory properties and can possibly constitute a prototype anti-inflammatory drug with therapeutic efficacy in the treatment of inflammatory diseases such as RA.

Prevention of Arthritis by Locally Synthesized Recombinant Antibody Neutralizing Complement Component C5

Treatment of patients suffering from chronic diseases such as rheumatoid arthritis with recombinant antibodies is time consuming and fairly expensive and can be associated with side effects due to generalized depletion of the target molecule. We have addressed these issues by developing an alternative approach consisting of the intraarticular injection of a DNA vector encoding for the anti-C5 neutralizing recombinant miniantibody MB12/22. This method allows local production of the antibody in sufficient amount to be effective in preventing joint inflammation in a rat model of antigen-induced arthritis. Injection of the DNA vector in a right knee of normal rats resulted in the production of the minibody detected in the synovial washes by western blot with a strong signal peaking at 3 days after administration. DNA encoding for the minibody was shown for 14 days in the synovial tissue and was undetectable in the controlateral knee and in other organs. The preventive effect of this approach was evaluated in rats receiving a single injection of the vector 3 days before the induction of antigen-induced arthritis and analyzed 3 days later. The treated rats exhibited a lower increase in swelling, associated with a lower number of PMN in the articular washes and reduced deposition of C9 in synovial tissue compared to control rats. These results suggest that treating the inflamed joints with a vector that induces a local production of a neutralizing anti-C5 antibody may represent a useful strategy to inhibit in situ complement activation and to treat patients with monoarthritis. Moreover, this approach may be adopted as a novel therapeutic strategy to prevent monoarthritis as an alternative to local treatment with antibodies commonly used in this form of arthritis, with the advantages of the lower cost and the longer persistence of antibody production.

Treatment of experimental arthritis by targeting synovial endothelium with a neutralizing recombinant antibody to C5

To show that a new recombinant protein (MT07) obtained by fusing a synovial-homing peptide to a neutralizing antibody to C5 can be selectively delivered to inflamed synovium and can effectively control joint inflammation in experimental models of arthritis. Binding of MT07 to human, rat, and mouse synovial tissue was evaluated in vitro by immunofluorescence, and selective localization in the inflamed joints of rats was documented in vivo using time-domain optical imaging. The antiinflammatory effect of MT07 was tested in a rat model of antigen-induced arthritis (AIA) and in a mouse model of collagen antibody-induced arthritis (CAIA). MT07 was able to bind to samples of inflamed synovium from humans, mice, and rats while failing to recognize uninflamed synovium as well as inflamed mouse lung or rat kidney. In vivo analysis of the biodistribution of MT07 confirmed its preferential homing to inflamed joints, with negligible inhibition of circulating C5 levels. MT07 prevented and resolved established inflammation in a rat model of AIA, as demonstrated by changes in joint swelling, polymorphonuclear cell counts in synovial washes, release of interleukin-6 and tumor necrosis factor α, and tissue damage. A similar therapeutic effect was obtained testing MT07 in a CAIA model. Our findings show that the novel recombinant molecule MT07 has the unique ability to selectively target inflamed joints and to exert local control of the inflammatory process by neutralizing the complement system without interfering with circulating C5 levels. We believe that this approach can be extended to other antiinflammatory drugs currently used to treat patients with rheumatoid arthritis.

Drugs that simultaneously down-regulate IL-1β and TNF secretion significantly reduce inflammation in a rat model of antigen-induced arthritis

We have previously reported an increase in interleukin (IL)-1β levels and a continuous activation of caspase-1 in rheumatoid arthritis (RA) patients in the early phase of the disease. This result suggests that drugs targeting IL-1β regulatory pathways, in addition to tumor necrosis factor (TNF), may constitute promising therapeutic agents in early RA. We have recently used a THP-1 macrophage-like cell line to screen 2320 compounds for those which down-regulate IL-1β and TNF secretion. Gambogic acid, celastrol and pristimerin were three of the most promising therapeutic candidates identified in that study.

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.

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...

Indocyanine green–enhanced imaging of antigen‐induced arthritis with an integrated optical imaging/radiography system

To evaluate a combined indocyanine green-enhanced optical imaging/radiography system for the detection of arthritic joints in a rat model of antigen-induced arthritis. Arthritis of the knee and ankle joints was induced in 6 Harlan rats, using peptidoglycan-polysaccharide polymers. Three rats served as untreated controls. Optical imaging of the knee and ankle joints was done with an integrated optical imaging/radiography system before and up to 24 hours following intravenous injection of 10 mg/kg indocyanine green. The fluorescence signal intensities of arthritic and normal joints were compared for significant differences, using generalized estimating equation models. Specimens of knee and ankle joints were further processed and evaluated by histology. Immediately after administration, indocyanine green provided a significant increase in the fluorescence signal of arthritic joints compared with baseline values (P < 0.05). The fluorescence signal of arthritic joints was significantly higher compared with that of nonarthritic control joints at 1-720 minutes after intravenous injection (P < 0.05). Fusion of indocyanine green-enhanced optical imaging and radiography allowed for anatomic coregistration of the inflamed tissue with the associated joint. Hematoxylin and eosin staining confirmed marked synovial inflammation of arthritic joints and the absence of inflammation in control joints. Indocyanine green-enhanced optical imaging is a clinically applicable tool for detection of arthritic tissue. Using relatively high doses of indocyanine green, long-term enhanced fluorescence of arthritic joints can be achieved. This may facilitate simultaneous evaluations of multiple joints in a clinical setting. Fusion of indocyanine green-enhanced optical imaging scans with radiography increases anatomic resolution.

The role of proinflammatory cytokines in the generation and maintenance of joint pain

The proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) not only promote and maintain inflammation, they also contribute to the generation and maintenance of inflammatory pain by acting at nociceptive nerve cells. A large proportion of dorsal root ganglion (DRG) neurons express TNF receptors and receptor units for stimulation with IL-6. In the rat model of antigen-induced arthritis (AIA), neutralization of TNF-alpha by etanercept and infliximab reduced inflammation-evoked mechanical hyperalgesia at the inflamed knee joint. This treatment also attenuated the infiltration of macrophages into the DRGs usually observed during the acute phase of AIA. Intra-articular application of etanercept reduced the responses of C-fibers to mechanical stimulation of the inflamed joint but did not influence responses to stimulation of the normal joint. Finally, in cultured DRG neurons TNF-alpha increased the proportion of neurons that express the TRPV1 receptor and may thus contribute to the generation of inflammation-evoked thermal hyperalgesia.

The cleavage site of C5 from man and animals as a common target for neutralizing human monoclonal antibodies: in vitro and in vivo studies.

The isolation of an anti-C5 single-chain fragment variable (scFv) antibody, TS-A12/22, from a human phage display library, is described. This antibody inhibits the activation of C5 and the assembly of the terminal complement complex implicated in cell and tissue damage. Using antibody-sensitized sheep erythrocytes and rabbit red cells as target cells in hemolytic assays, we found that TS-A12/22 inhibited the activation of C5 by the convertases of both classical and alternative pathways. Western blot analysis and competition experiments with synthetic peptides showed that TS-A12/22 reacted with the alpha chain of C5 and recognized the cleavage site of this complement component by the C5 convertase. As a result, the antibody prevented splitting of C5 and inhibited the generation of C5a and of the terminal complement complex. The identification of the TS-A12/22 recognition site as a conserved sequence in man, mouse, rat and rabbit enabled the demonstration of in vitro inhibition of complement activity in these species. The scFv TS-A12/22 was tested in a rat model of antigen-induced arthritis and proved to be effective in preventing influx of polymorphonuclear cells into the knee joint and C9 deposition on synovial tissue.