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1.  TNF receptor gene therapy results in suppression of IgG2a anticollagen antibody in collagen induced arthritis 
Annals of the Rheumatic Diseases  2003;62(8):707-714.
Background: Therapeutic strategies to block tumour necrosis factor α (TNFα) activity in experimental autoimmune arthritis models and rheumatoid arthritis (RA) have proved highly successful, and provide sustained beneficial effects.
Objective: To examine whether TNFα inhibition has immunological activity beyond the reduction of inflammation in collagen induced arthritis (CIA), an established experimental model of RA.
Methods: Arthritic DBA/1 mice received single periarticular injections of retroviral constructs encoding human TNF receptor (TNF-R) into the affected arthritic paw, at the onset of arthritis. Severity of arthritis, antibodies to collagen type II (CII), and extent of pathological joint damage of arthritic paws were compared between TNF-R and media treated (control) animals 3, 7, 14, 21, and 49 days after disease onset.
Results: Severity of CIA was significantly decreased in TNF-R treated animals compared with controls, 14–34 days after disease onset. Joint destruction was reduced in TNF-R injected joints and in the uninjected contralateral and ipsilateral paws of TNF-R treated animals. Seven days after disease onset, TNF-R treated mice had lower levels of inflammatory Th1 driven IgG2a antibodies to CII (p<0.05) than controls. This altered the anticollagen IgG2a:IgG1 ratio towards Th2 driven IgG1.
Conclusions: Local TNF-R gene therapy in CIA appears to have systemic effects on the anti-CII antibodies. The overall influence of TNF-R gene therapy is that it inhibits the progression of CIA mainly by suppressing the inflammatory Th1 response rather than by stimulating a Th2 response. Therefore, periarticular TNF-R gene therapy may have excellent therapeutic potential in RA.
doi:10.1136/ard.62.8.707
PMCID: PMC1754640  PMID: 12860724
2.  Effect of sclerostin-neutralising antibody on periarticular and systemic bone in a murine model of rheumatoid arthritis: a microCT study 
Arthritis Research & Therapy  2013;15(5):R125.
Introduction
Patients with chronic inflammatory diseases have increased bone loss and bone fragility and are at increased risk of fracture. Although anti-resorptive drugs are effective in blocking inflammation-induced bone loss, they are less effective at rebuilding bone. We have previously shown that treatment with sclerostin antibody (Scl-AbI) builds bone and can prevent or restore bone loss in a murine model of inflammatory bowel disease. In this study, we tested the effect of Scl-AbI in a murine model of rheumatoid arthritis (the collagen-induced arthritis model, CIA). We hypothesised that sclerostin blockade can protect and restore bone both locally and systemically without affecting progression of inflammation.
Methods
CIA was induced in male DBA/1 mice, which were treated with either PBS or Scl-AbI (10 mg/kg, weekly) prophylactically for 55 days or therapeutically for 21 days (starting 14 days post onset of arthritis). Systemic inflammation was assessed by measuring the serum concentration of anti-CII IgG1, IgG2a and IgG2b by ELISA. Changes in bone mass and structure, either at sites remote from the joints or at periarticular sites, were measured using DEXA and microCT. Bone focal erosion was assessed in microCT scans of ankle and knee joints.
Results
Circulating anti-CII immunoglobulins were significantly elevated in mice with CIA and there were no significant differences in the levels of anti-CII immunoglobulins in mice treated with PBS or Scl-ABI. Prophylactic Scl-AbI treatment prevented the decrease in whole body bone mineral density (BMD) and in the bone volume fraction at axial (vertebral body) and appendicular (tibial proximal metaphysis trabecular and mid-diaphysis cortical bone) sites seen in PBS-treated CIA mice, but did not prevent the formation of focal bone erosions on the periarticular bone in the knee and ankle joints. In the therapeutic study, Scl-AbI restored BMD and bone volume fraction at all assessed sites but was unable to repair focal erosions.
Conclusions
Sclerostin blockade prevented or reversed the decrease in axial and appendicular bone mass in the murine model of rheumatoid arthritis, but did not affect systemic inflammation and was unable to prevent or repair local focal erosion.
doi:10.1186/ar4305
PMCID: PMC3979059  PMID: 24432364
Sclerostin; sclerostin antibody; inflammation; collagen-induced arthritis; rheumatoid arthritis; bone loss; focal erosion; micro computed tomography; microCT
3.  Local Gene Transfer of OPG Prevents Joint Damage and Disease Progression in Collagen-Induced Arthritis 
The Scientific World Journal  2013;2013:718061.
This study examined the influence of osteoprotegerin (OPG) gene transfer on a murine collagen-induced arthritis model. A single periarticular injection of AAV-OPG or AAV-LacZ on the arthritic paw successfully incorporated the exogenous gene to the local tissue and resulted in marked transgene expression in the joint homogenate for at least three weeks. Clinical disease scores were significantly improved in OPG treated mice starting at 28-day post-treatment (P < 0.05). Histological assessment demonstrated that OPG gene transfer dramatically protected mice from erosive joint changes compared with LacZ controls (P < 0.05), although treatment appeared less effective on the local inflammatory progress. MicroCT data suggested significant protection against subchondral bone mineral density changes in OPG treated CIA mice. Interestingly, mRNA expressions of IFN-g and MMP3 were noticeably diminished following OPG gene transfer. Overall, gene transfer of OPG effectively inhibited the arthritis-associated periarticular bone erosion and preserved the architecture of arthritic joints, and the study provides evidence that the cartilage protection of the OPG gene therapy may be associated with the down-regulation of MMP3 expression.
doi:10.1155/2013/718061
PMCID: PMC3814078  PMID: 24222748
4.  Enhanced expression of genes involved in coagulation and fibrinolysis in murine arthritis 
Arthritis Research  2000;2(6):504-512.
We have analyzed the pattern of procoagulant and fibrinolytic gene expression in affected joints during the course of arthritis in two murine models. In both models, we found an increased expression of tissue factor, tissue factor pathway inhibitor, urokinase plasminogen activator, and plasminogen activator inhibitor 1, as well as thrombin receptor. The observed pattern of gene expression tended to favor procoagulant activity, and this pattern was confirmed by functional assays. These alterations would account for persistence of fibrin within the inflamed joint, as is seen in rheumatoid arthritis.
Introduction:
Accumulation of fibrin in the joints remains one of the most striking histopathological features of rheumatoid arthritis (RA). Recently, we have provided evidence of the deleterious role of synovial fibrin deposition in arthritic joints in antigen-induced arthritis (AIA), a well-established murine model of RA.
A local imbalance between fibrin formation and fibrin dissolution may result in fibrin deposition in the joints.
On the one hand, fibrin formation is mainly initiated by tissue factor (TF), a transmembrane protein serving as a receptor for factor VII. Under normal conditions, TF expression and activity are tightly regulated. Constitutive TF expression is restricted to perivascular and epithelial cells, and the catalytic activity of the TF/VIIa complex can be inhibited by tissue factor pathway inhibitor (TFPI). Pathological conditions can perturb the cell-type-restricted pattern of TF expression. In particular, recent reports have shown that transcriptional activation of TF can be mediated by molecular mechanisms involving induction of the early growth response gene 1 (EGR1) or of the protease-activated receptor (PAR1) or vascular endothelial growth factor (VEGF) genes.
On the other hand, fibrin degradation is mediated primarily by plasmin, which is the active form of the zymogen plasminogen. Conversion of plasminogen to plasmin is under the control of serine protease plasminogen activators, such as the urokinase plasminogen activator (uPA), and their inhibitors, such as the plasminogen activator inhibitor (PAI-1).
Aims:
We hypothesized that the deposition of fibrin in the joints may result from an imbalance in the local expression of key genes involved in coagulation and fibrinolytic pathways. To test this hypothesis, we investigated mRNA levels in arthritic versus nonarthritic joint tissues from two murine models of RA: AIA and collagen-induced arthritis (CIA). Genes that are directly implicated in coagulation (TF, TFPI) and fibrinolysis (UPA, PAI1), and other genes that may influence the expression of TF (EGR1, PAR1, VEGF), were investigated using a novel multiprobe RNase protection assay (RPA). Furthermore, we evaluated coagulation activity in arthritic and nonarthritic mice.
Methods:
Mice with AIA or CIA were sacrificed at different time points: 2, 4, and 16 h and 3, 7, and 14 d after intra-articular antigen injection for AIA; 42 d after the first immunization for CIA. Total RNA was prepared from arthritic and nonarthritic knees for AIA, or arthritic and nonarthritic hind paws for CIA. Messenger RNA (mRNA) levels of the genes described above were determined by RPA and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. Coagulation assays were performed on joint tissue extracts and concentrations of thrombin-antithrombin III (TAT) complex were measured in plasma.
Results:
In AIA, all the genes studied except VEGF were upmodulated as early as 2 h. PAR1, TFPI, EGR1, and UPA expression decreased to control levels by 16 h, whereas the expression of TF and PAI1 remained elevated. At later times, only TF, PAI1, and UPA showed sustained overexpression. In CIA, gene expression was assayed at only one time point (42 d after immunization) and all genes showed higher mRNA levels in the affected paws than in control paws. In AIA mice, procoagulant activity and TF activity were significantly increased in arthritic joints, and in CIA mice, plasma TAT levels were significantly enhanced.
Discussion:
Fibrin deposition in synovia is prominent in both RA and experimental arthritis, suggesting that this protein may play a role in the pathogenesis of chronic inflammation. In this study, we have tried to shed some light on the molecular mechanisms leading to extravascular fibrin deposition, using two well-established mouse models of RA: AIA and CIA. The kinetics of gene expression was first analyzed in mice with AIA, because this model allows for an accurate, temporally controlled sampling of synovial inflammation. We then extended our observations by analyzing one time point in CIA, 42 d after immunization, when chronic inflammation is present. We found that in both models, coagulation and fibrinolysis in arthritic joints were significantly increased, and that the most significant increases were in TF and PAI-1.
Although the molecular mechanism or mechanisms responsible for the transcriptional changes observed are not completely understood, the increases in TF, PAI-1, and uPA are probably due to the production of proinflammatory cytokines such as IL-1 and TGF-α. These cytokines, whose presence in the inflamed synovium is well documented, are known to induce these genes through the activation of nuclear factor κB (NF-κB), a transcription factor. TF induction is also under the control of a proximal enhancer containing a binding site for the inducible transcription factor EGR1. Indeed, the early rise of EGR1 expression in AIA is consistent with its classification as immediate-early gene and may be responsible for the induction of early expression of TF. Early TF stimulation in AIA can also be accounted for by the transient overexpression of PAR1. Contrary to what has been shown in RA, VEGF expression remained essentially unchanged throughout the progression of AIA, probably reflecting a peculiarity of this murine model.
The alteration of the patterns of gene expression was accompanied by increased functional coagulation activity, which was more marked in AIA than in CIA.
Conclusion:
Prominent fibrin deposition in two different animal models of RA – AIA and CIA – can be attributed to modulations in key regulatory genes for coagulation and fibrinolysis.
PMCID: PMC17822  PMID: 11056680
arthritis; coagulation; fibrinolysis; mice; RNase protection
5.  Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis 
Arthritis Research  1999;1(1):81-91.
Destruction of cartilage and bone are hallmarks of human rheumatoid arthritis (RA), and controlling these erosive processes is the most challenging objective in the treatment of RA. Systemic interleukin-4 treatment of established murine collagen-induced arthritis suppressed disease activity and protected against cartilage and bone destruction. Reduced cartilage pathology was confirmed by both decreased serum cartilage oligomeric matrix protein (COMP) and histological examination. In addition, radiological analysis revealed that bone destruction was also partially prevented. Improved suppression of joint swelling was achieved when interleukin-4 treatment was combined with low-dose prednisolone treatment. Interestingly, synergistic reduction of both serum COMP and inflammatory parameters was noted when low-dose interleukin-4 was combined with prednisolone. Systemic treatment with interleukin-4 appeared to be a protective therapy for cartilage and bone in arthritis, and in combination with prednisolone at low dosages may offer an alternative therapy in RA.
Introduction:
Rheumatoid arthritis (RA) is associated with an increased production of a range of cytokines including tumour necrosis factor (TNF)-α and interleukin (IL)-1, which display potent proinflammatory actions that are thought to contribute to the pathogenesis of the disease. Although TNF-α seems to be the major cytokine in the inflammatory process, IL-1 is the key mediator with regard to cartilage and bone destruction. Apart from direct blockade of IL-1/TNF, regulation can be exerted at the level of modulatory cytokines such as IL-4 and IL-10. IL-4 is a pleiotropic T-cell derived cytokine that can exert either suppressive or stimulatory effects on different cell types, and was originally identified as a B-cell growth factor and regulator of humoral immune pathways. IL-4 is produced by activated CD4+ T cells and it promotes the maturation of Th2 cells. IL-4 stimulates proliferation, differentiation and activation of several cell types, including fibroblasts, endothelial cells and epithelial cells. IL-4 is also known to be a potent anti-inflammatory cytokine that acts by inhibiting the synthesis of proinflammatory cytokines such as IL-1, TNF-α, IL-6, IL-8 and IL-12 by macrophages and monocytes. Moreover, IL-4 stimulates the synthesis of several cytokine inhibitors such as interleukin-1 receptor antagonist (IL-1Ra), soluble IL-1-receptor type II and TNF receptors IL-4 suppresses metalloproteinase production and stimulates tissue inhibitor of metalloproteinase-1 production in human mononuclear phagocytes and cartilage explants, indicating a protective effect of IL-4 towards extracellular matrix degradation. Furthermore, IL-4 inhibits both osteoclast activity and survival, and thereby blocks bone resorption in vitro. Of great importance is that IL-4 could not be detected in synovial fluid or in tissues. This absence of IL-4 in the joint probably contributes to the disturbance in the Th1/Th2 balance in chronic RA.
Collagen-induced arthritis (CIA) is a widely used model of arthritis that displays several features of human RA. Recently it was demonstrated that the onset of CIA is under stringent control of IL-4 and IL-10. Furthermore, it was demonstrated that exposure to IL-4 during the immunization stage reduced onset and severity of CIA. However, after cessation of IL-4 treatment disease expression increased to control values.
Aims:
Because it was reported that IL-4 suppresses several proinflammatory cytokines and matrix degrading enzymes and upregulates inhibitors of both cytokines and catabolic enzymes, we investigated the tissue protective effect of systemic IL-4 treatment using established murine CIA as a model. Potential synergy of low dosages of anti-inflammatory glucocorticosteroids and IL-4 was also evaluated.
Methods:
DBA-1J/Bom mice were immunized with bovine type II collagen and boosted at day 21. Mice with established CIA were selected at day 28 after immunization and treated for days with IL-4, prednisolone, or combinations of prednisolone and IL-4. Arthritis score was monitored visually. Joint pathology was evaluated by histology, radiology and serum cartilage oligomeric matrix protein (COMP). In addition, serum levels of IL-1Ra and anticollagen antibodies were determined.
Results:
Treatment of established CIA with IL-4 (1 μg/day) resulted in suppression of disease activity as depicted in Figure 1. Of great interest is that, although 1 μg/day IL-4 had only a moderate effect on the inflammatory component of the disease activity, it strongly reduced cartilage pathology, as determined by histological examination (Fig. 1). Moreover, serum COMP levels were significantly reduced, confirming decreased cartilage involvement. In addition, both histological and radiological analysis showed that bone destruction was prevented (Fig. 1). Systemic IL-4 administration increased serum IL-1Ra levels and reduced anticollagen type II antibody levels. Treatment with low-dose IL-4 (0.1 μg/day) was ineffective in suppressing disease score, serum COMP or joint destruction. Synergistic suppression of both arthritis severity and COMP levels was noted when low-dose IL-4 was combined with prednisolone (0.05 mg/kg/day), however, which in itself was not effective.
Discussion:
In the present study, we demonstrate that systemic IL-4 treatment ameliorates disease progression of established CIA. Although clinical disease progression was only arrested and not reversed, clear protection against cartilage and bone destruction was noted. This is in accord with findings in both human RA and animal models of RA that show that inflammation and tissue destruction sometimes are uncoupled processes. Of great importance is that, although inflammation was still present, strong reduction in serum COMP was found after exposure to IL-4. This indicated that serum COMP levels reflected cartilage damage, although a limited contribution of the inflamed synovium cannot be excluded.
Increased serum IL-1Ra level (twofold) was found after systemic treatment with IL-4, but it is not likely that this could explain the suppression of CIA. We and others have reported that high dosages of IL-1Ra are needed for marked suppression of CIA. As reported previously, lower dosages of IL-4 did not reduce clinical disease severity of established CIA. Of importance is that combined treatment of low dosages of IL-4 and IL-10 appeared to have more potent anti-inflammatory effects, and markedly protected against cartilage destruction. Improved anti-inflammatory effect was achieved with IL-4/prednisolone treatment. In addition, synergistic effects were found for the reduction of cartilage and bone destruction. This indicates that systemic IL-4/prednisolone treatment may provide a cartilage and bone protective therapy for human RA.
Effects in mice of treatment with interleukin-4 or control on disease activity, cartilage damage and bone destruction. Mice were treated intraperitoneally for 7 days with either vehicle (control) or 1 μg/day interleukin-4 (IL-4). CIA, collagen-induced arthritis. *P < 0.05, versus control, by Mann-Whitney U test.
PMCID: PMC17779  PMID: 11056663
bone destruction; cartilage oligomeric matrix protein levels; collagen-induced arthritis; interleukin-4; prednisolone
6.  IL-4 gene therapy for collagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand and prevents bone erosion 
Journal of Clinical Investigation  2000;105(12):1697-1710.
Bone destruction is the most difficult target in the treatment of rheumatoid arthritis (RA). Here, we report that local overexpression of IL-4, introduced by a recombinant human type 5 adenovirus vector (Ad5E1mIL-4) prevents joint damage and bone erosion in the knees of mice with collagen arthritis (CIA). No difference was noted in the course of CIA in the injected knee joints between Ad5E1mIL-4 and the control vector, but radiographic analysis revealed impressive reduction of joint erosion and more compact bone structure in the Ad5E1mIL-4 group. Although severe inflammation persisted in treated mice, Ad5E1mIL-4 prevented bone erosion and diminished tartrate-resistant acid phosphatase (TRAP) activity, indicating that local IL-4 inhibits the formation of osteoclast-like cells. Messenger RNA levels of IL-17, IL-12, and cathepsin K in the synovial tissue were suppressed, as were IL-6 and IL-12 protein production. Osteoprotegerin ligand (OPGL) expression was markedly suppressed by local IL-4, but no loss of OPG expression was noted with Ad5E1mIL-4 treatment. Finally, in in vitro studies, bone samples of patients with arthritis revealed consistent suppression by IL-4 of type I collagen breakdown. IL-4 also enhanced synthesis of type I procollagen, suggesting that it promoted tissue repair. These findings may have significant implications for the prevention of bone erosion in arthritis.
PMCID: PMC378501  PMID: 10862785
7.  Gene expression profiling and functional analysis of angiogenic markers in murine collagen-induced arthritis 
Arthritis Research & Therapy  2012;14(4):R169.
Introduction
Dysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA). To provide a more profound understanding of arthritis-associated angiogenesis, we evaluated the expression of angiogenesis-modulating genes at onset, peak and declining phases of collagen-induced arthritis (CIA), a well-established mouse model for RA.
Methods
CIA was induced in DBA/1 mice with type II collagen. Functional capillary density in synovial tissue of knee joints was determined by intravital fluorescence microscopy. To assess the ability of arthritic joint homogenates to induce angiogenesis, an endothelial chemotaxis assay and an in vivo matrigel plug assay were employed. The temporal expression profile of angiogenesis-related genes in arthritic paws was analysed by quantitative real-time RT-PCR using an angiogenesis focused array as well as gene specific PCR. Finally, we investigated the therapeutic effect of a monoclonal antibody specifically blocking the binding of VEGF to neuropilin (NRP)-1.
Results
Although arthritic paw homogenates displayed angiogenic activity in vitro and in vivo, and synovia of arthritic paws appeared highly vascularised on histological examination, the functional capillary density in arthritic knee synovia was significantly decreased, whereas capillary diameter was increased. Of the 84 genes analysed, 41 displayed a differential expression in arthritic paws as compared to control paws. Most significant alterations were seen at the peak of clinical arthritis. Increased mRNA expression could be observed for VEGF receptors (Flt-1, Flk-1, Nrp-1, Nrp-2), as well as for midkine, hepatocyte growth factor, insulin-like growth factor-1 and angiopoietin-1. Signalling through NRP-1 accounted in part for the chemotactic activity for endothelial cells observed in arthritic paw homogenates. Importantly, therapeutic administration of anti-NRP1B antibody significantly reduced disease severity and progression in CIA mice.
Conclusions
Our findings confirm that the arthritic synovium in murine CIA is a site of active angiogenesis, but an altered balance in the expression of angiogenic factors seems to favour the formation of non-functional and dilated capillaries. Furthermore, our results validate NRP-1 as a key player in the pathogenesis of CIA, and support the VEGF/VEGF receptor pathway as a potential therapeutic target in RA.
doi:10.1186/ar3922
PMCID: PMC3580563  PMID: 22817681
8.  AAV2-Mediated Subretinal Gene Transfer of mIL-27p28 Attenuates Experimental Autoimmune Uveoretinitis in Mice 
PLoS ONE  2012;7(5):e37773.
Background
Advances in gene transfer techniques have provided long-term, safe and stable transduction of retinal cells following subretinal injection with adeno-associated viral (AAV) vectors. In this study we investigated whether subretinal injection of AAV2-murine IL-27p28 vector was effective in inhibiting experimental autoimmune uveoretinitis (EAU) induced in B10RIII mice.
Methodology/Principal Findings
An AAV2 vector encoding the murine IL-27p28 gene (rAAV2-CMV-mIL-27p28) was prepared and subretinally injected into B10RIII mice (4.35×108 vector genome (v.g.)). AAV2 vector mediating green fluorescent protein (rAAV2-CMV-GFP) served as a control (5×108 v.g.). The concentration of mIL-27p28 in homogenized eyes and serum was assayed by enzyme linked immunosorbent assay (ELISA) after subretinal injection. Human IRBP161–180 peptide and Complete Freund’s Adjuvant were injected into mice receiving either the rAAV2-CMV-mIL-27p28 or rAAV2-CMV-GFP vector. EAU was evaluated clinically and pathologically. The level of IL-17 and IL-10 in homogenized eyes was measured on day 12 and day 21 following immunization. Delayed type hypersensitivity (DTH) and IRBP161–180–specific proliferation of lymphocytes from the spleen and lymph nodes were assayed to examine the influence of the subretinal delivery of rAAV2-CMV-mIL-27p28 on the systemic immune response. IL-27p28 was detectable by ELISA within the eyes from two weeks following subretinal injection of the rAAV2-CMV-mIL-27p28 vector and showed a sustained high expression from day 14 to 9 months with a highest expression at 5 months. Subretinal injection of the vector significantly attenuated the severity of EAU disease clinically and pathologically in association with a significantly decreased IL-17 expression and an increased IL-10 expression. The IL-27p28 vector did not affect the systemic immune response, as determined by DTH and IRBP161–180–specific lymphocyte proliferation.
Conclusions/Significance
A high and stable expression of IL-27p28 was observed for at least 9 months following subretinal delivery of rAAV2-CMV-mIL-27p28. The amelioration of EAU disease severity was associated with a decreased IL-17 expression and an increased IL-10 expression.
doi:10.1371/journal.pone.0037773
PMCID: PMC3358269  PMID: 22629453
9.  Tumour necrosis factor receptor gene therapy affects cellular immune responses in collagen induced arthritis in mice 
Annals of the Rheumatic Diseases  2005;64(11):1550-1556.
Background: Collagen induced arthritis (CIA) is an animal model of rheumatoid arthritis (RA) amenable to immunotherapy directed against tumour necrosis factor α (TNFα).
Objective: To evaluate whether local TNF receptor (TNF-R) gene therapy in DBA/1 mice exerts an influence beyond anti-inflammatory effects. Two measures of CIA pathogenesis were investigated—namely, immunity to collagen II (CII) 245–270 peptide (the major immunodominant epitope within bovine CII) and the preferential activation of T cell Vß8.2 variable region receptors in arthritic DBA/1 mice.
Methods: DBA/1 mice received single periarticular injections of media or retroviral vectors containing LacZ or human TNF-R into affected arthritic paws at disease onset. Disease severity was monitored, immune responses towards the immunodominant bovine CII 245–270 and subdominant CII 334–360 peptide epitopes were assessed by ELISA, and T cell Vß usage was analysed by real time polymerase chain reaction for the LacZ transduced, TNF-R, and viral-free media treated control animals. The therapeutic influence of TNF-R gene transduction was compared with other groups at different times after treatment.
Results: Reduced disease severity was seen 15–35 days after treatment, with a concomitant increase in immunity towards the subdominant CII 334–360 peptide epitope rather than the immunodominant CII 245–270 peptide in TNF-R treated animals. Early in the disease, TNF-R treated animals demonstrated a reduction of bias towards the otherwise predominant Vß8.2 T cell subset.
Conclusions: TNF-R gene therapy influences cellular immunity in CIA, leading to overall disease amelioration, thus suggesting that TNF inhibition may have therapeutic potential beyond the control of inflammation in RA.
doi:10.1136/ard.2004.025619
PMCID: PMC1755256  PMID: 15860512
10.  FcgammaR expression on macrophages is related to severity and chronicity of synovial inflammation and cartilage destruction during experimental immune-complex-mediated arthritis (ICA) 
Arthritis Research  2000;2(6):489-503.
We investigated the role of Fcγ receptors (FcγRs) on synovial macrophages in immune-complex-mediated arthritis (ICA). ICA elicited in knee joints of C57BL/6 mice caused a short-lasting, florid inflammation and reversible loss of proteoglycans (PGs), moderate chondrocyte death, and minor erosion of the cartilage. In contrast, when ICA was induced in knee joints of Fc receptor (FcR) γ-chain-/- C57BL/6 mice, which lack functional FcγRI and RIII, inflammation and cartilage destruction were prevented. When ICA was elicited in DBA/1 mice, a very severe, chronic inflammation was observed, and significantly more chondrocyte death and cartilage erosion than in arthritic C57BL/6 mice. The synovial lining and peritoneal macrophages of naïve DBA/1 mice expressed a significantly higher level of FcγRs than was seen in C57BL/6 mice. Moreover, elevated and prolonged expression of IL-1 was found after stimulation of these cells with immune complexes. Zymosan or streptococcal cell walls caused comparable inflammation and only mild cartilage destruction in all strains. We conclude that FcγR expression on synovial macrophages may be related to the severity of synovial inflammation and cartilage destruction during ICA.
Introduction:
Fcγ receptors (FcγRs) present on cells of the haematopoietic lineage communicate with IgG-containing immune complexes that are abundant in the synovial tissue of patients with rheumatoid arthritis (RA). In mice, three classes of FcγR (RI, RII, and RIII) have been described. Binding of these receptors leads to either activation (FcγRI and RIII) or deactivation (FcγRII) of intracellular transduction pathways. Together, the expression of activating and inhibitory receptors is thought to drive immune-complex-mediated diseases.
Earlier studies in our laboratory showed that macrophages of the synovial lining are of utmost importance in the onset and propagation of immune-complex-driven arthritic diseases. Selective depletion of macrophages in the joint downregulated both inflammation and cartilage destruction. As all three classes of FcγR are expressed on synovial macrophages, these cells are among the first that come in contact with immune complexes deposited in the joint. Recently, we observed that when immune complexes were injected into the knee joints of mice, strains susceptible to collagen-type-II arthritis (DBA/1, B10.RIII) developed more severe arthritis than nonsusceptible strains did, or even developed chronic arthritis. One reason why these strains are more susceptible might be their higher levels of FcγRs on macrophage membranes. To test this hypothesis, we investigated the role of FcγRs in inflammation and cartilage damage during immune-complex-mediated arthritis (ICA). First, we studied arthritis and subsequent cartilage damage in mice lacking functional FcγRI and RIII (FcR γ-chain-/- mice). Next, DBA/1 mice, which are prone to develop collagen-type-II arthritis (`collagen-induced arthritis'; CIA) and are hypersensitive to immune complexes, were compared with control C57BL/6 mice as regards cartilage damage and the expression and function of FcγRs on their macrophages.
Aims:
To examine whether FcγR expression on macrophages is related to severity of synovial inflammation and cartilage destruction during immune-complex-mediated joint inflammation.
Methods:
ICA was induced in three strains of mice (FcR γ-chain-/-, C57BL/6, and DBA/1, which have, respectively, no functional FcγRI and RIII, intermediate basal expression of FcγRs, and high basal expression of FcγRs) by passive immunisation using rabbit anti-lysozyme antibodies, followed by poly-L-lysine lysozyme injection into the right knee joint 1 day later. In other experiments, streptococcal-cell-wall (SCW)- or zymosan-induced arthritis was induced by injecting SCW (25 μg) or zymosan (180 μg) directly into the knee joint. At several time points after arthritis induction, knee joints were dissected and studied either histologically (using haematoxylin/eosin or safranin O staining) or immuno-histochemically. The arthritis severity and the cartilage damage were scored separately on an arbitrary scale of 0-3.
FcγRs were immunohistochemically detected using the monoclonal antibody 2.4G2, which detects both FcγRII and RIII. Deposition of IgG and C3c in the arthritic joint tissue was also detected immunohistochemically. Expression of FcγRs by murine peritoneal macrophages was measured using a fluorescence-activated cell sorter (FACS).
Peritoneal macrophages were stimulated using heat-aggregated gamma globulins (HAGGs), and production of IL-1 was measured using a bioassay. To assess the levels of IL-1 and its receptor antagonist (IL-1Ra) during arthritis, tissue was dissected and washed in RPMI medium. Washouts were tested for levels of IL-1 and IL-1Ra using radioimmunoassay and enzyme-linked immunosorbent assay. mRNA was isolated from the tissue, and levels of macrophage inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1, IL-1, and IL-1Ra were determined using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR).
Results:
ICA induced in knee joints of C57BL/6 mice caused a florid inflammation at day 3 after induction. To investigate whether this arthritis was FcγR-mediated, ICA was induced in FcR γ-chain-/- mice, which lack functional FcγRI and RIII. At day3, virtually no inflammatory cells were found in their knee joints. Levels of mRNA of IL-1, IL-1Ra, MCP-1, and MIP-2, which are involved in the onset of this arthritis, were significantly lower in FcR γ-chain-/- mice than in control C57BL/6 mice. Levels of IL-1 protein were also measured. At 6 h after ICA induction, FcR γ-chain-/- mice and control C57BL/6 mice showed similar IL-1 production as measured by protein level. By 24 h after induction, however, IL-1 production in the FcR γ-chain-/- mice was below the detection limit, whereas the controls were still producing a significant amount. To investigate whether the difference in reaction to immune complexes between the DBA/1 and C57BL/6 mice might be due to variable expression of FcγRs in the knee joint, expression in situ of FcγRs in naïve knee joints of these mice was determined. The monoclonal antibody 2.4G2, which detects both FcγRII and RIII, stained macrophages from the synovial lining of DBA/1 mice more intensely than those from C57BL/6 mice. This finding suggests a higher constitutive expression of FcγRs by macrophages of the autoimmune-prone DBA/1 mice. To quantify the difference in FcγR expression on macrophages of the two strains, we determined the occurrence of FcγRs on peritoneal macrophages by FACS analysis. The levels of FcγR expressed by macrophages were twice as high in the DBA/1 mice as in the C57BL/6 mice (mean fluorescence, respectively, 440 ± 50 and 240 ± 30 intensity per cell). When peritoneal macrophages of both strains were stimulated with immune complexes (HAGGs), we found that the difference in basal FcγR expression was functional. The stimulated macrophages from DBA/1 mice had significantly higher IL-1α levels (120 and 135 pg/ml at 24 and 48 h, respectively) than cells from C57BL/6 mice (45 and 50 pg/ml, respectively).
When arthritis was induced using other arthritogenic triggers than immune complexes (zymosan, SCW), all the mouse strains tested (DBA/1, FcR γ-chain-/-, and C57BL/6) showed similar inflammation, indicating that the differences described above are found only when immune complexes are used to elicit arthritis.
We next compared articular cartilage damage in arthritic joints of the three mouse strains FcR γ-chain-/-, C57BL/6 (intermediate basal expression of FcγRs), and DBA/1 (high basal expression of FcγRs). Three indicators of cartilage damage were investigated: depletion of PGs, chondrocyte death, and erosion of the cartilage matrix. At day 3 after induction of ICA, there was no PG depletion in FcR γ-chain-/- mice, whereas PG depletion in the matrix of the C57BL/6 mice was marked and that in the arthritic DBA/1 mice was even greater. PG depletion was still massive at days 7 and 14 in the DBA/1 mice, whereas by day 14 the PG content was almost completely restored in knee joints of the C57BL/6 mice. Chondrocyte death and erosion of cartilage matrix, two indicators of more severe cartilage destruction, were significantly higher in the DBA/1 than in the C57BL/6 mice, while both indicators were completely absent in the FcR γ-chain-/- mice. Again, when arthritis was induced using other triggers (SCW, zymosan), all strains showed similar PG depletion and no chondrocyte death or matrix erosion. These findings underline the important role of immune complexes and FcγRs in irreversible cartilage damage.
Discussion:
Our findings indicate that inflammation and subsequent cartilage damage caused by immune complexes may be related to the occurrence of FcγRs on macrophages. The absence of functional FcγRI and RIII prevented inflammation and cartilage destruction after induction of ICA, whereas high basal expression of FcγRs on resident joint macrophages of similarly treated mice susceptible to autoimmune arthritis was correlated with markedly more synovial inflammation and cartilage destruction. The difference in joint inflammation between the three strains was not due to different susceptibilities to inflammation per se, since intra-articular injection of zymosan or SCW caused comparable inflammation. Although extensive inflammatory cell mass was found in the synovium of all strains after intra-articular injection of zymosan, no irreversible cartilage damage (chondrocyte death or matrix erosion) was found. ICA induced in C57BL/6 and DBA/1 mice did cause irreversible cartilage damage at later time points, indicating that immune complexes and FcγRs play an important role in inducing irreversible cartilage damage. Macrophages communicate with immune complexes via Fcγ receptors. Absence of functional activating receptors completely abrogates the synovial inflammation, as was shown after ICA induction in FcR γ-chain-/- mice. However, the γ-chain is essential not only in FcγRI and RIII but also for FcεRI (found on mast cells) and the T cell receptor (TcR)-CD3 (Tcells) complex of γδT cells. However, T, B, or mast cells do not play a role in this arthritis that is induced by passive immunisation. Furthermore, this effect was not caused by a difference in clearance of IgG or complement deposition in the tissue. In this study, DBA/1 mice, which are susceptible to collagen-induced autoimmune arthritis and in a recent study have been shown to react hypersensitively to immune complexes, are shown to express higher levels of FcγRs on both synovial and peritoneal macrophages. Because antibodies directed against the different subclasses of FcγR are not available, no distinction could be made between FcγRII and RIII. Genetic differences in DBA/1 mice in genes coding for or regulating FcγRs may be responsible for altered FcγR expression. If so, these mouse strains would have a heightened risk for immune-complex-mediated diseases.
To provide conclusive evidence for the roles of the various classes of FcγR during ICA, experiments are needed in which FcγRs are blocked with specific antibodies, or in which knockout mice lacking one specific class of FcγR are used. The only available specific antibody to FcγR (2.4G2) has a stimulatory effect on cells once bound to the receptor, and therefore cannot be used in blocking experiments. Experiments using specific knockout mice are now being done in our laboratory.
Macrophages are the dominant type of cell present in chronic inflammation during RA and their number has been shown to correlate well with severe cartilage destruction. Apart from that, in humans, these synovial tissue macrophages express activating FcRs, mainly FcγIIIa, which may lead to activation of these macrophages by IgG-containing immune complexes. The expression of FcRs on the surface of these cells may have important implications for joint inflammation and severe cartilage destruction and therefore FCRs may constitute a new target for therapeutic intervention.
PMCID: PMC17821  PMID: 11056679
autoimmunity; cytokines; Fc receptors; inflammation; macrophages
11.  Establishment and characterization of a sustained delayed-type hypersensitivity model with arthritic manifestations in C57BL/6J mice 
Arthritis Research & Therapy  2012;14(3):R134.
Introduction
Rheumatoid arthritis (RA) is a chronic progressive, inflammatory and destructive autoimmune disease, characterised by synovial joint inflammation and bone erosion. To better understand the pathophysiology and underlying immune mechanisms of RA various models of arthritis have been developed in different inbred strains of mice. Establishment of arthritis models with components of adaptive immunity in the C57BL/6J strain of mice has been difficult, and since most genetically modified mice are commonly bred on this background, there is a need to explore new ways of obtaining robust models of arthritis in this strain. This study was undertaken to establish and characterise a novel murine model of arthritis, the delayed-type hypersensitivity (DTH)-arthritis model, and evaluate whether disease can be treated with compounds currently used in the treatment of RA.
Methods
DTH-arthritis was induced by eliciting a classical DTH reaction in one paw with methylated bovine serum albumin (mBSA), with the modification that a cocktail of type II collagen monoclonal antibodies was administered between the immunisation and challenge steps. Involved cell subsets and inflammatory mediators were analysed, and tissue sections evaluated histopathologically. Disease was treated prophylactically and therapeutically with compounds used in the treatment of RA.
Results
We demonstrate that DTH-arthritis could be induced in C57BL/6 mice with paw swelling lasting for at least 28 days and that disease induction was dependent on CD4+ cells. We show that macrophages and neutrophils were heavily involved in the observed pathology and that a clear profile of inflammatory mediators associated with these cell subsets was induced locally. In addition, inflammatory markers were observed systemically. Furthermore, we demonstrate that disease could be both prevented and treated.
Conclusions
Our findings indicate that DTH-arthritis shares features with both collagen-induced arthritis (CIA) and human RA. DTH-arthritis is dependent on CD4+ cells for induction and can be successfully treated with TNFα-blocking biologics and dexamethasone. On the basis of our findings we believe that the DTH-arthritis model could hold potential in the preclinical screening of novel drugs targeting RA. The model is highly reproducible and has a high incidence rate with synchronised onset and progression, which strengthens its potential.
doi:10.1186/ar3867
PMCID: PMC3446517  PMID: 22676339
12.  RANKL inhibition by osteoprotegerin prevents bone loss without affecting local or systemic inflammation parameters in two rat arthritis models: comparison with anti-TNFα or anti-IL-1 therapies 
Arthritis Research & Therapy  2009;11(6):R187.
Introduction
Rat adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) feature bone loss and systemic increases in TNFα, IL-1β, and receptor activator of NF-κB ligand (RANKL). Anti-IL-1 or anti-TNFα therapies consistently reduce inflammation in these models, but systemic bone loss often persists. RANKL inhibition consistently prevents bone loss in both models without reducing joint inflammation. Effects of these therapies on systemic markers of bone turnover and inflammation have not been directly compared.
Methods
Lewis rats with established AIA or CIA were treated for 10 days (from day 4 post onset) with either PBS (Veh), TNFα inhibitor (pegsunercept), IL-1 inhibitor (anakinra), or RANKL inhibitor (osteoprotegerin (OPG)-Fc). Local inflammation was evaluated by monitoring hind paw swelling. Bone mineral density (BMD) of paws and lumbar vertebrae was assessed by dual X-ray absorptiometry. Markers and mediators of bone resorption (RANKL, tartrate-resistant acid phosphatase 5b (TRACP 5B)) and inflammation (prostaglandin E2 (PGE2), acute-phase protein alpha-1-acid glycoprotein (α1AGP), multiple cytokines) were measured in serum (day 14 post onset).
Results
Arthritis progression significantly increased paw swelling and ankle and vertebral BMD loss. Anti-TNFα reduced paw swelling in both models, and reduced ankle BMD loss in AIA rats. Anti-IL-1 decreased paw swelling in CIA rats, and reduced ankle BMD loss in both models. Anti-TNFα and anti-IL-1 failed to prevent vertebral BMD loss in either model. OPG-Fc reduced BMD loss in ankles and vertebrae in both models, but had no effect on paw swelling. Serum RANKL was elevated in AIA-Veh and CIA-Veh rats. While antiTNFα and anti-IL-1 partially normalized serum RANKL without any changes in serum TRACP 5B, OPG-Fc treatment reduced serum TRACP 5B by over 90% in both CIA and AIA rats. CIA-Veh and AIA-Veh rats had increased serum α1AGP, IL-1β, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), and AIA-Veh rats also had significantly greater serum PGE2, TNFα and IL-17. Anti-TNFα reduced systemic α1AGP, CCL2 and PGE2 in AIA rats, while anti-IL-1 decreased systemic α1AGP, IL-8 and PGE2. In contrast, RANKL inhibition by OPG-Fc did not lessen systemic cytokine levels in either model.
Conclusions
Anti-TNFα or anti-IL-1 therapy inhibited parameters of local and systemic inflammation, and partially reduced local but not systemic bone loss in AIA and CIA rats. RANKL inhibition prevented local and systemic bone loss without significantly inhibiting local or systemic inflammatory parameters.
doi:10.1186/ar2879
PMCID: PMC3003514  PMID: 20003323
13.  Investigation of the role of endosomal Toll-like receptors in murine collagen-induced arthritis reveals a potential role for TLR7 in disease maintenance 
Arthritis Research & Therapy  2012;14(3):R142.
Introduction
Endosomal toll-like receptors (TLRs) have recently emerged as potential contributors to the inflammation observed in human and rodent models of rheumatoid arthritis (RA). This study aims to evaluate the role of endosomal TLRs and in particular TLR7 in the murine collagen induced arthritis (CIA) model.
Methods
CIA was induced by injection of collagen in complete Freund's adjuvant. To investigate the effect of endosomal TLRs in the CIA model, mianserin was administered daily from the day of disease onset. The specific role of TLR7 was examined by inducing CIA in TLR7-deficient mice. Disease progression was assessed by measuring clinical score, paw swelling, serum anti-collagen antibodies histological parameters, cytokine production and the percentage of T regulatory (Treg) cells.
Results
Therapeutic administration of mianserin to arthritic animals demonstrated a highly protective effect on paw swelling and joint destruction. TLR7-/- mice developed a mild arthritis, where the clinical score and paw swelling were significantly compromised in comparison to the control group. The amelioration of arthritis by mianserin and TLR7 deficiency both corresponded with a reduction in IL-17 responses, histological and clinical scores, and paw swelling.
Conclusions
These data highlight the potential role for endosomal TLRs in the maintenance of inflammation in RA and support the concept of a role for TLR7 in experimental arthritis models. This study also illustrates the potential benefit that may be afforded by therapeutically inhibiting the endosomal TLRs in RA.
doi:10.1186/ar3875
PMCID: PMC3446525  PMID: 22691272
14.  T cells that are naturally tolerant to cartilage-derived type II collagen are involved in the development of collagen-induced arthritis 
Arthritis Research  2000;2(4):315-326.
The immunodominant T-cell epitope that is involved in collagen-induced arthritis (CIA) is the glycosylated type II collagen (CII) peptide 256-270. In CII transgenic mice, which express the immunodominant CII 256-270 epitope in cartilage, the CII-specific T cells are characterized by a partially tolerant state with low proliferative activity in vitro, but with maintained effector functions, such as IFN-γ secretion and ability to provide B cell help. These mice were still susceptible to CIA. The response was mainly directed to the glycosylated form of the CII 256-270 peptide, rather than to the nonglycosylated peptide. Tolerance induction was rapid; transferred T cells encountered CII within a few days. CII immunization several weeks after thymectomy of the mice did not change their susceptibility to arthritis or the induction of partial T-cell tolerance, excluding a role for recent thymic emigrants. Thus, partially tolerant CII autoreactive T cells are maintained and are crucial for the development of CIA.
Introduction:
A discussion is ongoing regarding the possible role of cartilage-directed autoimmunity as a part of the pathogenesis of rheumatoid arthritis (RA). One possibility is that the association of RA with shared epitope-expressing DR molecules reflects a role for major histocompatibility complex (MHC) class II molecules as peptide receptors, and that the predilection of the inflammatory attack for the joint indicates a role for cartilage as a source of the antigenic peptides. A direct role for CII in the development of arthritis is apparent in the CIA model, in which a definite role for MHC class II molecules and a role for CII-derived peptides have been demonstrated [1,2,3]. Remarkably, it was found that the identified MHC class II molecule in the CIA model Aq has a structurally similar peptide binding pocket to that of the shared epitope, expressing DR4 molecules [4]. In fact, DR4 (DRB1*0401) and DR1 (DRB1*0101) transgenic mice are susceptible to CIA because of an immune response to a peptide that is almost identical to that which is involved in Aq-expressing mice [5,6]. They are both derived from position 260-273 of the CII molecule; the peptide binds to the Aqmolecule with isoleucine 260 in the P1 pocket, but with phenylalanine 263 in the P1 pocket of the DR4 and DR1 molecules.
Although these findings do not prove a role for CII in RA, they show that such recognition is possible and that there are structural similarities when comparing mouse with human. However, there are also strong arguments against such a possibility. First, arthritis can evolve without evidence for a cartilage-specific autoimmunity, as seen with various adjuvant-induced arthritis models [7,8] and in several observations using transgenic animals with aberrant immunity to ubiquitously expressed proteins [9,10,11]. Moreover, the MHC association in the adjuvant arthritis models correlates with severity of the disease rather than susceptibility [7,8], as has also been observed in RA [12]. Second, it has not been possible to identify the CII-reactive T cells from RA joints, or to achieve a strong and significant CII proliferative response from T cells derived from RA joints. Most recently these negative observations were corroborated using DR4+CII peptide tetramer reagents [13]. On the other hand, it has also been difficult to isolate autoreactive CII-specific T cells from CIA, and it can be anticipated that, even in the CIA model, T cells that are specific for CII will be hard to find in the joints [4].
We believe that the explanations for these observations in both experimental animals and humans are related to tolerance. The CIA model in the mouse is usually induced with heterologous CII, and is critically dependent on an immune response to the glycosylated CII peptide 256-270, which is bound to the MHC class II Aq molecule. In CII transgenic mice, expressing the heterologous (rat) form of the immunodominant CII 256-270 epitope in cartilage, we observed partial T-cell tolerance. This tolerance is characterized by a low proliferative activity, but with maintained effector functions such as production of IFN-γ and the ability to give help to B cells to produce anti-CII IgG antibodies [14]. Interestingly, these mice were susceptible to arthritis. However, a possibility was that T cells that had newly emerged from the thymus and that were not yet tolerized when the mice were immunized with CII led to the induction of arthritis. We have now addressed this possibility and found that induction of tolerance occurs within a few days, and that mice lacking recent thymic emigrants (ie thymectomized mice) display partially tolerant T cells and susceptibility to arthritis to the same extent as nonthymectomized mice. In addition we found that T cells that are reactive with the nonmodified peptides are relatively more affected by tolerance than T cells that are reactive with the more immunodominant glycosylated variants.
Objectives:
To investigate the possibility that T cells that are naturally tolerant to the cartilage protein CII are involved in the development of arthritis, and to exclude a role for nontolerized recent thymic T-cell emigrants in the development of arthritis.
Materials and methods:
A mutated mouse CII, expressing glutamic acid instead of aspartic acid at position 266, was expressed in a transgenic mouse called MMC (mutated mouse collagen) that has been described earlier [14]. The mice were thymectomized, or sham-operated, at 7 weeks of age and allowed to recover for 4 weeks before being immunized with rat CII in complete Freund's adjuvant. Arthritis development was recorded and sera analyzed for anti-CII IgG, IgG1 and IgG2a levels. To assay T-cell effector functions, other MMC and control mice were immunized in the hind footpads with rat CII in complete Freund's adjuvant, and the draining popliteal lymph nodes were taken 10 days later. The lymph node cells (LNCs) were used for proliferation assay, IFN-γ enzyme-linked immunosorbent assay (ELISA) and B-cell enzyme-linked immunospot (ELISPOT). For the proliferation assay, 106 cells were put in triplicate cultures in microtitre wells together with antigen and incubated for 72h before thymidine-labelling and harvesting 15-18h later. For IFN-γ ELISA analysis, supernatant from the proliferation plates was removed before harvesting and used in an ELISA to quantify the amount of IFN-γ produced [15]. B-cell ELISPOT was performed to enumerate the number of cells producing anti-CII IgG [16].
T-cell lines that were reactive towards rat CII were established by immunization with rat CII. An established T-cell line that was reactive with CII and specific for the CII 256-270 peptide was restimulated with freshly collected, irradiated, syngenic spleen cells and rat CII for 3 days followed by 2 weeks of IL-2 containing medium. Immediately before transfer, the cells were labelled with the cytoplasmic dye 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) [17]. Labelled cells (107) were injected intravenously into transgenic MMC mice and nontransgenic littermates. The mice were killed 4 days after cell transfer, and the concentration of CFSE-labelled cells was determined by flow cytometry.
Results and discussion:
To investigate whether and how quickly CII-reactive T cells will encounter CII in vivo, an established T-cell line that is reactive towards rat CII was labelled with the cytoplasmic dye CFSE and transferred into MMC-QD and control mice. Four days later the mice were killed, and it was found that MMC-transgenic mice had dramatically fewer CFSE-labelled cells in the spleen than did nontransgenic littermates (0.11% compared with 0.57%). Similarly, reduced numbers of CFSE-positive cells were observed in blood. This indicates that the T cells encountered the mutated CII that was present in the cartilage of MMC mice, but not in the nontransgenic littermates. Presumably, CII from cartilage is spread by antigen-presenting cells (APCs) to peripheral lymphoid organs. This observation also suggests that newly exported T cells from the thymus will be tolerized to CII in the periphery within less than 4 days.
To further investigate whether the MMC mice harbours naïve or tolerized T cells, the mice were immunized with CII at different time points after thymectomy that were well in excess of the times required for their encounter with CII. After 10 days, the response was analyzed in vitro towards both the nonglycosylated and the glycosylated CII 256-270 peptides as well as towards purified protein derivative. The galactosylated form of the peptide (Fig. 1) was used because this is the most immunodominant modification [18]. In contrast to control mice, LNCs from transgenic mice did not proliferate significantly towards the nonglycosylated peptide, indicating that these cells have been specifically tolerized, which is in accordance with earlier observations [14]. A reduced, but still significant proliferation was also observed toward the immunodominant glycosylated CII peptide. Most important, however, was that the proliferative response in the MMC mice did not decrease after thymectomy. Similarly, a significant IFN-γ production towards the glycosylated CII peptide was observed in the MMC mice. The response was somewhat reduced compared with that observed in nontransgenic littermates, and this was especially true for the response toward the nonglycosylated peptide. Again, no decrease in the MMC response by thymectomy was observed. Taken together, the T-cell response in transgenic mice was reduced in comparison with that in the nontransgenic littermates. Furthermore, the response in transgenic animals did not decrease by thymectomy (4 or 8 weeks before immunization), showing that autoreactive T cells are still maintained (and partially tolerized) with significant effector functions at least up to 8 weeks after thymectomy, excluding a exclusive role for recent thymic emigrants in the autoimmune response towards CII. To investigate whether thymectomized mice, lacking recent CII-specific thymic emigrants, were susceptible to CIA, mice were immunized with CII 4 weeks after thymectomy and were observed for arthritis development during the following 10 weeks. Clearly, the thymectomized MMC mice were susceptible to arthritis (five out of 18 developed arthritis; Fig. 2), and no significant differences in susceptibility between thymectomized and sham-operated mice, or between males and females, were seen. In accordance with earlier results [14], MMC transgenic mice had a significantly reduced susceptibility to arthritis as compared with the nontransgenic littermates (P < 0.0001 for arthritic scores, disease onset and incidence). All mice were bled at 35 days after immunization, and the total levels of anti-CII IgG were determined. Transgenic mice developed levels of anti-CII IgG significantly above background, but the antibody titres were lower than in nontransgenic littermates (P < 0.0001). No effect on the antibody levels by thymectomy was observed, nor did thethymectomy affect the distribution of IgG1 versus IgG2a titres,indicating that the observed tolerance is not associated with a shift from a T-helper-1- to a T-helper-2-like immune response. These findings show that T cells that are specific for a tissue-specific matrix protein, CII, are partially tolerized within a few days after thymus export and that these tolerized cells are maintained after thymectomy. Most important, mice that lack newly exported CII reactive T cells are still susceptible to CIA, suggesting that the partially tolerant T cells are involved in development of arthritis.
In the light of these data it is possible to explain some of the findings in RA. T-cell reactivity to CII has been shown in RA patients, but with a very weak proliferative activity [19,20]. This is fully compatible with observations in mouse and rat CIA when autologous CII, and not heterologous CII, are used for immunization. This is particularly true if the responses are recorded during the chronic phase of disease, in which the antigen-specific T-cell responses seem to be suppressed in both humans and experimental animals. These observations were confirmed in a recent report [21] in which it was shown that CII-reactive T-cell activity could be detected in RA patients if IFN-γ production but not proliferation was measured. In the present studies in mice the strongest response is seen towards post-translational modifications of the peptide. Because the T-cell contact points are the same whether the peptide is bound to DR4 or to Aq, it is fully possible that post-translational modifications of the peptide also plays a significant role in humans [22]. The fact that IgG antibodies specific for CII are found in many RA patients could be explained by maintained B-cell helper functions of CII-reactive T cells. In fact, it has been reported [23,24] that the occurrence of IgG antibodies to CII is associated with shared epitope DR4 molecules. These observations are thus compatible with a role for CII reactivity in RA. To avoid any confusion, it needs to be stressed that RA is a heterogeneous syndrome in which not only CII, but also other cartilage proteins and other mechanisms are of importance. Such a pathogenic heterogeneity is reflected by the multitude of experimental animal models that have demonstrated how many different pathways may lead to arthritis [25].
PMCID: PMC17814  PMID: 11056672
autoimmunity; rheumatoid arthritis; T lymphocytes; tolerance; transgenic
15.  Immunization with T cell receptor V beta chain peptides deletes pathogenic T cells and prevents the induction of collagen-induced arthritis in mice. 
Journal of Clinical Investigation  1996;97(12):2849-2858.
Collagen-induced arthritis (CIA) in susceptible strains of mice is an animal model of T cell-mediated inflammatory polyarthritis. Analysis of T cell receptor (TCR) V beta gene usage in cells isolated from arthritic joints of BUB/BnJ (BUB) mice (H-2q, TCR V beta a) showed that TCR V beta chain gene usage was limited to TCR V beta 3 and V beta 10 gene families. All of the BUB mice immunized with a mixture of TCR V beta 3 and TCR V beta 10 peptides, but not with control TCR V beta 14 peptide, were refractory to the induction of CIA. Immunization with TCR V beta 3 and V beta 10 peptides completely blocked the development of clinical and subclinical inflammation, formation of pannus and synovial hyperplasia, and the erosion of cartilage and bone. Further studies revealed that preimmunization of BUB mice with V beta 10 peptide alone was sufficient to render the mice resistant to CIA. Analysis of TCR V beta chain gene expression in lymph node cells from arthritic and arthritis-protected mice showed the expression of TCR V beta 10 subfamily in all of the arthritic mice, but not in arthritis-protected mice. Immunization with TCR V beta peptides did not diminish the humoral responses to chicken type-II collagen and also elicited significant levels of anti-V beta 3 and anti-V beta 10 peptide antibodies. Antibodies cross-reactive with mouse chicken type-II collagen were detected in both the arthritic and arthritis-protected mice. Adoptive transfer of serum from arthritis-protected BUB mice significantly delayed the onset (P < 0.005) of arthritis in recipient BUB mice. In contrast, mice injected with serum from arthritic mice had early onset of arthritis. These results demonstrate that immunization of BUB mice with TCR V beta chain peptides elicited antibodies reactive with the self-TCR and prevented the induction of collagen-induced arthritis by eliminating or downregulating pathogenic T cells and consequently blocking the development of humoral immune response. These findings may have clinical applications in treating human autoimmune diseases characterized by common TCR gene usage.
PMCID: PMC507379  PMID: 8675697
16.  Palmitoylethanolamide and luteolin ameliorate development of arthritis caused by injection of collagen type II in mice 
Arthritis Research & Therapy  2013;15(6):R192.
Introduction
N-palmitoylethanolamine (PEA) is an endogenous fatty acid amide belonging to the family of the N-acylethanolamines (NAEs). Recently, several studies demonstrated that PEA is an important analgesic, antiinflammatory, and neuroprotective mediator. The aim of this study was to investigate the effect of co-ultramicronized PEA + luteolin formulation on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA).
Methods
CIA was induced by an intradermally injection of 100 μl of the emulsion (containing 100 μg of bovine type II collagen (CII)) and complete Freund adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. Mice subjected to CIA were administered PEA (10 mg/kg 10% ethanol, intraperitoneally (i.p.)) or co-ultramicronized PEA + luteolin (1 mg/kg, i.p.) every 24 hours, starting from day 25 to 35.
Results
Mice developed erosive hind-paw arthritis when immunized with CII in CFA. Macroscopic clinical evidence of CIA first appeared as periarticular erythema and edema in the hindpaws. The incidence of CIA was 100% by day 28 in the CII-challenged mice, and the severity of CIA progressed over a 35-day period with a resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint. Treatment with PEA or PEA + luteolin ameliorated the clinical signs at days 26 to 35 and improved histologic status in the joint and paw. The degree of oxidative and nitrosative damage was significantly reduced in PEA + luteolin-treated mice, as indicated by nitrotyrosine and malondialdehyde (MDA) levels. Plasma levels of the proinflammatory cytokines and chemokines were significantly reduced by PEA + luteolin treatment.
Conclusions
We demonstrated that PEA co-ultramicronized with luteolin exerts an antiinflammatory effect during chronic inflammation and ameliorates CIA.
doi:10.1186/ar4382
PMCID: PMC3978572  PMID: 24246048
17.  Role of endogenous and exogenous female sex hormones in arthritis and osteoporosis development in B10.Q-ncf1*/* mice with collagen-induced chronic arthritis 
Background
Collagen-induced arthritis (CIA) is an often-used murine model for human rheumatoid arthritis (RA). Earlier studies have shown potent anti-arthritic effects with the female sex hormone estradiol and the selective estrogen receptor modulator (SERM) raloxifene in CIA in DBA/1-mice. B10.Q-ncf1*/*mice are B10.Q mice with a mutated Ncf1 gene. In B10.Q-ncf1*/*mice, CIA develops as a chronic relapsing disease, which more accurately mimics human RA. We investigated the role of endogenous and exogenous sex steroids and raloxifene in the course of this model of chronic arthritis. We also examined whether treatment would prevent the development of inflammation-triggered generalized osteoporosis.
Methods
Female B10.Q-ncf1*/*mice were sham-operated or ovariectomized, and CIA was induced. 22 days later, when 30% of the mice had developed arthritis, treatment with raloxifene, estradiol or vehicle was started, and the clinical disease was evaluated continuously. Treatment was continued until day 56 after immunization. At termination of the experiment (day 73), bone mineral density (BMD) was analyzed, paws were collected for histological examination, and sera were analyzed for markers of cartilage turnover and pro-inflammatory cytokines.
Results
Raloxifene and estradiol treatment, as well as endogenous estrogen, decreased the frequency of arthritis, prevented joint destruction and countered generalized osteoporosis. These effects were associated with lower serum levels of the pro-inflammatory cytokine IL-6.
Conclusions
This is the first study to show that raloxifene and estradiol can ameliorate established erosive arthritis and inflammation-triggered osteoporosis in this chronic arthritis model. We propose that treatment with raloxifene could be a beneficial addition to the treatment of postmenopausal RA.
doi:10.1186/1471-2474-11-284
PMCID: PMC3009959  PMID: 21159208
18.  IL-23 Dependent and Independent Stages of Experimental Arthritis: No Clinical Effect of Therapeutic IL-23p19 Inhibition in Collagen-induced Arthritis 
PLoS ONE  2013;8(2):e57553.
IL-23p19 deficient mice have revealed a critical role of IL-23 in the development of experimental autoimmune diseases, such as collagen-induced arthritis (CIA). Neutralizing IL-23 after onset of CIA in rats has been shown to reduce paw volume, but the effect on synovial inflammation and the immunological autoimmune response is not clear. In this study, we examined the role of IL-23 at different stages of CIA and during T cell memory mediated flare-up arthritis with focus on changes in B cell activity and Th1/Th17 modulation. Anti-IL-23p19 antibody (anti-IL23p19) treatment, starting 15 days after the type II collagen (CII)-immunization but before clinical signs of disease onset, significantly suppressed the severity of CIA. This was accompanied with significantly lower CII-specific IgG1 levels and lower IgG2a levels in the anti-IL-23p19 treated mice compared to the control group. Importantly, neutralizing IL-23 after the first signs of CIA did not ameliorate the disease. This was in contrast to arthritic mice that underwent an arthritis flare-up since a significantly lower disease score was observed in the IL-23p19 treated mice compared to the control group, accompanied by lower synovial IL-17A and IL-22 expression in the knee joints of these mice. These data show IL-23-dependent and IL-23-independent stages during autoimmune CIA. Furthermore, the memory T cell mediated flare-up arthritis is IL-23-mediated. These data suggest that specific neutralization of IL-23p19 after onset of autoimmune arthritis may not be beneficial as a therapeutic therapy for patients with rheumatoid arthritis (RA). However, T cell mediated arthritis relapses in patients with RA might be controlled by anti-IL-23p19 treatment.
doi:10.1371/journal.pone.0057553
PMCID: PMC3585376  PMID: 23469022
19.  AAV8-Mediated Gene Transfer of Interleukin-4 to Endogenous β-Cells Prevents the Onset of Diabetes in NOD Mice 
We previously demonstrated that intra-peritoneal delivery of adeno-associated virus serotype 8 (AAV8) stably transduces the pancreas, including the β-cells in the endogenous islets. We also demonstrated the ability to deliver and express genes specifically in β-cells for at least 6 months using a murine insulin promoter (mIP) in a double-stranded, self-complementary AAV vector (dsAAV8-mIP). Here we evaluated the effects of dsAAV8-mIP mediated delivery of interleukin 4 (mIL-4) to endogenous β-cells in NOD mice. In 4 week old NOD mice, the extent of gene transfer and expression in endogenous β-cells following i.p. delivery of dsAAV8-mIP-eGFP was comparable to normal BALB/c mice. Furthermore, following i.p. delivery of dsAAV8-mIP-IL4, expression of mIL-4 was detected in islets isolated and cultured from the treated mice. AAV8-mIP mediated gene expression of mIL-4 to endogenous β-cells of 4 and 8 week old NOD mice prevented the onset of hyperglycemia in NOD mice and reduced the severity of insulitis. Moreover, expression of mIL-4 also maintained the level of CD4+CD25+FoxP3+ cells and adoptive co-transfer of splenocytes from diabetes-free IL-4 vector recipients and splenocytes from wild type diabetic NOD mice prevented the onset diabetes. Taken together, these results demonstrate that local expression of mIL-4 in islets prevents islet destruction and blocks autoimmunity, in part, through regulation of T cell function. These results also demonstrate the utility of using dsAAV8-mIP gene transfer to endogenous NOD β-cells to examine the role of specific gene products in preventing or exacerbating the onset of type 1 diabetes.
doi:10.1038/mt.2008.116
PMCID: PMC3560428  PMID: 18560422
20.  Potentiation of electrochemotherapy by intramuscular IL-12 gene electrotransfer in murine sarcoma and carcinoma with different immunogenicity 
Radiology and Oncology  2012;46(4):302-311.
Background.
Electrochemotherapy provides good local tumor control but requires adjuvant treatment for increased local response and action on distant metastasis. In relation to this, intramuscular interleukin-12 (IL-12) gene electro-transfer, which provides systemic shedding of IL-12, was combined with local electrochemotherapy with cisplatin. Furthermore, the dependence on tumor immunogenicity and immunocompetence of the host on combined treatment response was evaluated.
Materials and methods.
Sensitivity of SA-1 sarcoma and TS/A carcinoma cells to electrochemotherapy with cisplatin was tested in vitro. In vivo, intratumoral electrochemotherapy with cisplatin (day 1) was combined with a single (day 0) or multiple (days 0, 2, 4) intramuscular murine IL-12 (mIL-12) gene electrotransfer. The antitumor effectiveness of combined treatment was evaluated on immunogenic murine SA-1 sarcoma in A/J mice and moderately immunogenic murine TS/A carcinoma, in immunocompetent BALB/c and immunodeficient SCID mice.
Results.
Electrochemotherapy in vitro resulted in a similar IC50 values for both sarcoma and carcinoma cell lines. However, in vivo electrochemotherapy was more effective in the treatment of sarcoma, the more immunogenic of the tumors, resulting in a higher log cell kill, longer specific tumor growth delay, and also 17% tumor cures compared to carcinoma where no tumor cures were observed. Adjuvant intramuscular mIL-12 gene electrotransfer increased the log cell kill in both tumor models, potentiating the specific tumor growth delay by a factor of 1.8-2 and increasing tumor cure rate by approximately 20%. In sarcoma tumors, the potentiation of the response by intramuscular mIL-12 gene electrotransfer was dose-dependent and also resulted in a faster onset of tumor cures. Comparison of the carcinoma response to the combined treatment modality in immunocompetent and immunodeficient mice demonstrated that the immune system is needed both for increased cell kill and for attaining tumor cures.
Conclusions.
Based on the comparison of the antitumor effectiveness of electrochemotherapy to intratumoral cisplatin administration, we can conclude that the fraction of cells killed and the tumor cure rate are higher in immunogenic sarcoma tumor compared to moderately immunogenic carcinoma tumor. The tumor cell kill and cure rate depend on the immune response elicited by the destroyed tumor cells, which might depend on the tumor immunogenicity. The effect of adjuvant intramuscular mIL-12 gene electrotransfer is dependent on the amount of IL-12 in the system and the immune competence of the host, as demonstrated by the dose-dependent increase in the cure rate of SA-1 tumors after multiple intramuscular mIL-12 gene electrotransfer and in the differential cure rate of TS/A tumors growing in immunocompetent and immunodeficient mice.
doi:10.2478/v10019-012-0044-9
PMCID: PMC3572893  PMID: 23412658
IL-12; gene electrotransfer; cisplatin; electrochemotherapy; sarcoma; carcinoma; mice
21.  Granulocyte-macrophage colony stimulating factor exacerbates collagen induced arthritis in mice 
Annals of the Rheumatic Diseases  1997;56(6):364-368.
OBJECTIVE—To examine the effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on disease progression in the collagen induced arthritis (CIA) model in mice.
METHODS—DBA/1 mice were primed for a suboptimal CIA response by intradermal injection of chick type II collagen without a secondary immunisation. Three weeks after immunisation the mice were given four to five consecutive daily intraperitoneal injections of recombinant murine GM-CSF (15 µg; 5 × 105 U), or vehicle, and arthritis development was monitored by clinical scoring of paws and calliper measurements of footpad swelling. At approximately six to eight weeks after immunisation mice were killed, their limbs removed and processed for histological analyses of joint pathology.
RESULTS—Control animals receiving a single immunisation with collagen exhibited a varied CIA response both in terms of incidence and severity. Mice treated with GM-CSF at 20 to 25 days after immunisation with collagen had a consistently greater incidence and more rapid onset of disease than the vehicle treated control mice, based on clinical assessment. GM-CSF treated mice showed higher average clinical scores and greater paw swelling than controls. Histological analyses of joints reflected the clinical scores with GM-CSF treated mice displaying more pronounced pathology (synovitis, pannus formation, cartilage and bone damage) than control mice.
CONCLUSION—GM-CSF is a potent accelerator of the pathological events leading to chronic inflammatory polyarthritis in murine CIA supporting the notion that GM-CSF may play a part in inflammatory polyarthritis, such as rheumatoid arthritis.


PMCID: PMC1752394  PMID: 9227165
22.  Efficacy of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats 
Arthritis Research & Therapy  2003;5(3):R122-R131.
To evaluate the antioxidant activity of the glycosaminoglycans hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S), we used a rat model of collagen-induced arthritis (CIA). Arthritis was induced in Lewis rats by multiple intradermal injections of 250 μl of emulsion containing bovine type II collagen in complete Freund's adjuvant at the base of the tail and into three to five other sites on the back. Rats were challenged again with the same antigen preparation 7 days later. Disease developed about 11 days after the second immunization. The effects of treatment in the rats were monitored by biochemical parameters and by macroscopic and histological evaluations in blood, synovial tissue and articular cartilage. Arthritis produced the following symptoms: severe periarticular erythema, edema and inflammation in the hindpaws; membrane peroxidation in the cartilage of the joints; endogenous antioxidant wasting; high tumour necrosis factor-α (TNF-α) plasma levels; and synovial neutrophil accumulation. Treatment with HYA and C4S, starting at the onset of arthritis for 10 days, limited the erosive action of the disease in the articular joints of knee and paw, reduced lipid peroxidation, restored the endogenous antioxidants reduced glutathione (GSH) and superoxide dismutase, decreased plasma TNF-α levels, and limited synovial neutrophil infiltration. These data confirm that erosive destruction of the joint cartilage in CIA is due at least in part to free radicals released by activated neutrophils and produced by other biochemical pathways. The beneficial effects obtained with the treatment suggest that HYA and C4S could be considered natural endogenous macromolecules to limit erosive damage in CIA or as a useful tool with which to study the involvement of free radicals in rheumatoid arthritis.
doi:10.1186/ar748
PMCID: PMC165044  PMID: 12723984
antioxidants; collagen-induced arthritis; free radicals; glycosaminoglycans; lipid peroxidation
23.  Transgenic mice over-expressing carbonic anhydrase I showed aggravated joint inflammation and tissue destruction 
Background
Studies have demonstrated that carbonic anhydrase I (CA1) stimulates calcium salt precipitation and cell calcification, which is an essential step in new bone formation. Our study had reported that CA1 encoding gene has a strong association with rheumatoid arthritis (RA) and ankylosing spondylitis (AS), two rheumatic diseases with abnormal new bone formation and bone resorption in joints. This study investigated the effect of CA1 on joint inflammation and tissue destruction in transgenic mice that over-express CA1 (CA1-Tg).
Methods
CA1-Tg was generated with C57BL/6J mice by conventional methods. CA1-Tg was treated with collagen-II to induce arthritis (CIA). Wild-type mice, CA1-Tg treated with bovine serum albumin (BSA) and transgenic mice over-expressing PADI4 (PADI4-Tg), a gene known to be involved in rheumatoid arthritis, were used as controls. Histochemistry and X-ray radiographic assay were used to examine joint destruction. Western blotting and real time-PCR were used to examine CA1 expression.
Results
CIA was observed in 60% of CA1-Tg, 20% of PADI4-Tg and 20% of wild-type mice after collagen injections. No CIA was found in CA1-Tg mice that received injections of BSA. The arthritic score was 5.5 ± 0.84 in the CA1-Tgs but the score was less than 2 in the injected wild-type mice and the PADI4-Tgs. The thickness of the hind paws in the CA1-Tgs was 3.46 ± 0.11 mm, which was thicker than that of PADI4-Tgs (2.23 ± 0.08 mm), wild-type mice (2.08 ± 0.06 mm) and BSA-treated CA1-Tgs (2.04 ± 0.07 mm). Histochemistry showed obvious inflammation, synovial hyperplasia and bone destruction in the joints of CA1-Tg that was not detected in PADI4-Tgs or wild-type mice. X-ray assays showed bone fusion in the paws and spines of CA1-Tg mice.
Conclusion
Over-expression of CA1 may aggravate joint inflammation and tissue destruction in the transgenic mice.
doi:10.1186/1471-2474-13-256
PMCID: PMC3575273  PMID: 23256642
Carbonic anhydrase I (CA1); New bone formation; Transgenic mice; collagen-induced arthritis (CIA); Ankylosing spondylitis (AS); Rheumatoid arthritis (RA)
24.  Radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma 
BMC Cancer  2013;13:38.
Background
Interleukin-12 (IL-12) based radiosensitization is an effective way of tumor treatment. Local cytokine production, without systemic shedding, might provide clinical benefit in radiation treatment of sarcomas. Therefore, the aim was to stimulate intratumoral IL-12 production by gene electrotransfer of plasmid coding for mouse IL-12 (mIL-12) into the tumors, in order to explore its radiosensitizing effect after single or multiple intratumoral gene electrotransfer.
Methods
Solid SA-1 fibrosarcoma tumors, on the back of A/J mice, were treated intratumorally by mIL-12 gene electrotransfer and 24 h later irradiated with a single dose. Treatment effectiveness was measured by tumor growth delay and local tumor control assay (TCD50 assay). With respect to therapeutic index, skin reaction in the radiation field was scored. The tumor and serum concentrations of cytokines mIL-12 and mouse interferon γ (mIFNγ) were measured. Besides single, also multiple intratumoral mIL-12 gene electrotransfer before and after tumor irradiation was evaluated.
Results
Single intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral but not serum mIL-12 and mIFNγ concentrations, and had good antitumor (7.1% tumor cures) and radiosensitizing effect (21.4% tumor cures). Combined treatment resulted in the radiation dose-modifying factor of 2.16. Multiple mIL-12 gene electrotransfer had an even more pronounced antitumor (50% tumor cures) and radiosensitizing (86.7% tumor cures) effect.
Conclusions
Single or multiple intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral mIL-12 and mIFNγ cytokine level, and may provide an efficient treatment modality for soft tissue sarcoma as single or adjuvant therapy to tumor irradiation.
doi:10.1186/1471-2407-13-38
PMCID: PMC3562515  PMID: 23360213
IL-12; Gene electrotransfer; Irradiation; Sarcoma; Mice
25.  Collagenase-3 (MMP-13) deficiency protects C57BL/6 mice from antibody-induced arthritis 
Arthritis Research & Therapy  2013;15(6):R222.
Introduction
Matrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis.
Methods
For this study we employed the K/BxN serum-induced arthritis model. Arthritis was induced in C57BL/6 wild type (WT) and MMP-13-deficient (MMP-13–/–) mice by intraperitoneal injection of 200 μl of K/BxN serum. Arthritis was assessed by measuring the ankle swelling. During the course of the experiments, mice were sacrificed every second day for histological examination of the ankle joints. Ankle sections were evaluated histologically for infiltration of inflammatory cells, pannus tissue formation and bone/cartilage destruction. Semi-quantitative PCR was used to determine MMP-13 expression levels in ankle joints of untreated and K/BxN serum-injected mice.
Results
This study shows that MMP-13 is a regulator of inflammation. We observed increased expression of MMP-13 in ankle joints of WT mice during K/BxN serum-induced arthritis and both K/BxN serum-treated WT and MMP-13–/– mice developed progressive arthritis with a similar onset. However, MMP-13–/– mice showed significantly reduced disease over the whole arthritic period. Ankle joints of WT mice showed severe joint destruction with extensive inflammation and erosion of cartilage and bone. In contrast, MMP-13–/– mice displayed significantly decreased severity of arthritis (50% to 60%) as analyzed by clinical and histological scoring methods.
Conclusions
MMP-13 deficiency acts to suppress the local inflammatory responses. Therefore, MMP-13 has a role in the pathogenesis of arthritis, suggesting MMP-13 is a potential therapeutic target.
doi:10.1186/ar4423
PMCID: PMC3979078  PMID: 24369907

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