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1.  Collagenase-3 (MMP-13) deficiency protects C57BL/6 mice from antibody-induced arthritis 
Arthritis Research & Therapy  2013;15(6):R222.
Matrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis.
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.
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.
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.
PMCID: PMC3979078  PMID: 24369907
2.  Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis 
Arthritis Research  2000;2(2):145-153.
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
PMCID: PMC17808  PMID: 11062605
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis
3.  Urokinase-type plasminogen activator and arthritis progression: contrasting roles in systemic and monoarticular arthritis models 
Arthritis Research & Therapy  2010;12(5):R199.
Urokinase-type plasminogen activator (u-PA) has been implicated in tissue destruction/remodeling. The absence of u-PA results in resistance of mice to systemic immune complex-driven arthritis models; monoarticular arthritis models involving an intra-articular (i.a.) antigen injection, on the other hand, develop more severe arthritis in its absence. The aims of the current study are to investigate further these contrasting roles that u-PA can play in the pathogenesis of inflammatory arthritis and to determine whether u-PA is required for the cartilage and bone destruction associated with disease progression.
To determine how the different pathogenic mechanisms leading to arthritis development in the different models may explain the contrasting requirement for u-PA, the systemic, polyarticular, immune complex-driven K/BxN arthritis model was modified to include an i.a. injection of saline as a local trauma in u-PA-/- mice. This modified model and the antigen-induced arthritis (AIA) model were also used in u-PA-/- mice to determine the requirement for u-PA in joint destruction. Disease severity was determined by clinical and histologic scoring. Fibrin(ogen) staining and the matrix metalloproteinase (MMP)-generated neoepitope DIPEN staining were performed by immunohistochemistry. Gene expression of inflammatory and destructive mediators was measured in joint tissue by quantitative PCR.
In our modified arthritis model, u-PA-/- mice went from being resistant to arthritis development following K/BxN serum transfer to being susceptible following the addition of an i.a. injection of saline. u-PA-/- mice also developed more sustained AIA compared with C57BL/6 mice, including reduced proteoglycan levels and increased bone erosions, fibrin(ogen) deposition and DIPEN expression. Synovial gene expression of the proinflammatory mediators (TNF and IL-1β), aggrecanases (ADAMTS-4 and -5) and MMPs (MMP3 and MMP13) were all sustained over time following AIA induction in u-PA-/- mice compared with C57BL/6 mice.
We propose that u-PA has a protective role in arthritis models with 'wound healing-like' processes following local trauma, possibly through u-PA/plasmin-mediated fibrinolysis, but a deleterious role in systemic models that are critically dependent on immune complex formation and complement activation. Given that cartilage proteoglycan loss and bone erosions were present and sustained in u-PA-/- mice with monoarticular arthritis, it is unlikely that u-PA/plasmin-mediated proteolysis is contributing directly to this tissue destruction/remodeling.
PMCID: PMC2991036  PMID: 20973954
4.  Regulation of inflammatory arthritis by the upstream kinase mitogen activated protein kinase kinase 7 in the c-Jun N-Terminal kinase pathway 
The c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) and cytokine production in rheumatoid arthritis (RA) and JNK deficiency markedly protects mice in animal models of arthritis. Cytokine-induced JNK activation is strictly dependent on the mitogen-activated protein kinase kinase 7 (MKK7) in fibroblast-like synoviocytes (FLS). Therefore, we evaluated whether targeting MKK7 using anti-sense oligonucleotides (ASO) would decrease JNK activation and severity in K/BxN serum transfer arthritis.
Three 2'-O-methoxyethyl chimeric ASOs for MKK7 and control ASO were injected intravenously in normal C57BL/6 mice. PBS, control ASO or MKK7 ASO was injected from Day -8 to Day 10 in the passive K/BxN model. Ankle histology was evaluated using a semi-quantitative scoring system. Expression of MKK7 and JNK pathways was evaluated by quantitative PCR and Western blot analysis.
MKK7 ASO decreased MKK7 mRNA and protein levels in ankles by about 40% in normal mice within three days. There was no effect of control ASO on MKK7 expression and MKK7 ASO did not affect MKK3, MKK4 or MKK6. Mice injected with MKK7 ASO had significantly less severe arthritis compared with control ASO (P < 0.01). Histologic evidence of synovial inflammation, bone erosion and cartilage damage was reduced in MKK7 ASO-treated mice (P < 0.01). MKK7 deficiency decreased phospho-JNK and phospho-c-Jun in ankle extracts (P < 0.05), but not phospho-MKK4. Interleukin-1beta (IL-1β), MMP3 and MMP13 gene expression in ankle joints were decreased by MKK7 ASO (P < 0.01).
MKK7 plays a critical regulatory role in the JNK pathway in a murine model of arthritis. Targeting MKK7 rather than JNK could provide site and event specificity when treating synovitis.
PMCID: PMC3392838  PMID: 22353730
C-Jun N-terminal kinase; Mitogen-activated protein kinase kinase 7; Rheumatoid arthritis; Anti-sense oligonucleotide
5.  Deficient Gadd45β in rheumatoid arthritis: Enhanced synovitis through JNK signaling 
Arthritis and rheumatism  2009;60(11):3229-3240.
c-Jun-N-terminal kinase (JNK)-mediated cell signaling plays a critical role in metalloproteinase (MMP) expression and joint destruction in rheumatoid arthritis (RA). Gadd45β (growth arrest and DNA damage inducible gene), which is an NF-κB regulated gene, was recently identified as an endogenous negative regulator of the JNK pathway by blocking the upstream kinase MKK7. We evaluated whether low Gadd45β expression in RA enhances JNK activation and overproduction of MMPs in RA and whether Gadd45β deficiency increases arthritis severity in passive K/BxN arthritis.
Activation of the NF-κB and the JNK pathway and Gadd45β expression was analyzed in human synovium and fibroblast like synoviocytes (FLS) using quantitative PCR, immunoblotting, immunohistochemistry, electrophoretic mobility shift assay (EMSA) and luciferase reporter constructs. Gadd45β null and wild type (WT) mice were evaluated in the K/BxN serum transfer model of inflammatory arthritis and clinical signs of arthritis, osteoclast formation, and bone erosion assessed.
Gadd45β gene and protein expression were unexpectedly low in human RA synovium despite abundant NF-κB activity. Forced Gadd45β expression in human FLS attenuated TNF-induced signaling through the JNK pathway, AP-1 activation, and MMP expression. Gadd45β deficiency exacerbated K/BxN serum-induced arthritis in mice, dramatically increased signaling through the JNK pathway and MMP3 and MMP13 gene expression in joints, and increased the area of inflammation and number of osteoclasts.
Deficient Gadd45β expression in RA can contribute to activation of JNK, clinical arthritis and joint destruction. This process can be mitigated by enhancing Gadd45β expression or by inhibiting JNK or its upstream regulator MKK7.
PMCID: PMC2858378  PMID: 19877043
6.  IL-17RA Signaling Amplifies Antibody-Induced Arthritis 
PLoS ONE  2011;6(10):e26342.
To investigate the role of IL-17RA signaling in the effector phase of inflammatory arthritis using the K/BxN serum-transfer model.
Wild-type and Il17ra−/− mice were injected with serum isolated from arthritic K/BxN mice and their clinical score was recorded daily. Mice were also harvested on days 12 and 21 and ankles were analyzed for cytokine and chemokine mRNA expression by qPCR on day 12 and for bone and cartilage erosions by histology on day 21, respectively. The induction of cytokine and chemokine expression levels by IL-17A in synovial-like fibroblasts was also analyzed using qPCR.
Il17ra−/− mice were partially protected from clinical signs of arthritis and had markedly fewer cartilage and bone erosions. The expression of several pro-inflammatory mediators, including the chemokines KC/CXCL1, MIP-2/CXCL2, LIX/CXCL5 MIP-1γ/CCL9, MCP-3/CCL7, MIP-3α/CCL20, the cytokines IL-1β, IL-6, RANKL and the matrix metalloproteinases MMP2, MMP3, and MMP13 were decreased in the ankles of Il17ra−/− mice compared to wild-type mice. Many of these proinflammatory genes attenuated in the ankles of Il17ra−/− mice were shown to be directly induced by IL-17A in synovial fibroblasts in vitro.
IL-17RA signaling plays a role as an amplifier of the effector phase of inflammatory arthritis. This effect is likely mediated by direct activation of synovial fibroblasts by IL-17RA to produce multiple inflammatory mediators, including chemokines active on neutrophils. Therefore, interrupting IL-17RA signaling maybe a promising pharmacological target for the treatment of inflammatory arthritis.
PMCID: PMC3197623  PMID: 22028860
7.  Capsaicin-sensitive sensory nerves exert complex regulatory functions in the serum-transfer mouse model of autoimmune arthritis 
•Capsaicin-sensitive sensory nerves are protective against autoimmune arthritis.•Desensitization of these fibers increase immune cell activation and edema.•Sensory denervation enhances ROS production, MMP activity and arthritic changes.•Late mechanical hyperalgesia is decreased after destroying these sensory nerves.
The K/BxN serum-transfer arthritis is a widely-used translational mouse model of rheumatoid arthritis, in which the immunological components have thoroughly been investigated. In contrast, little is known about the role of sensory neural factors and the complexity of neuro–immune interactions. Therefore, we analyzed the involvement of capsaicin-sensitive peptidergic sensory nerves in autoantibody-induced arthritis with integrative methodology.
Arthritogenic K/BxN or control serum was injected to non-pretreated mice or resiniferatoxin (RTX)-pretreated animals where capsaicin-sensitive nerves were inactivated. Edema, touch sensitivity, noxious heat threshold, joint function, body weight and clinical arthritis severity scores were determined repeatedly throughout two weeks. Micro-CT and in vivo optical imaging to determine matrix-metalloproteinase (MMP) and neutrophil-derived myeloperoxidase (MPO) activities, semiquantitative histopathological scoring and radioimmunoassay to measure somatostatin in the joint homogenates were also performed.
In RTX-pretreated mice, the autoantibody-induced joint swelling, arthritis severity score, MMP and MPO activities, as well as histopathological alterations were significantly greater compared to non-pretreated animals. Self-control quantification of the bone mass revealed decreased values in intact female mice, but significantly greater arthritis-induced pathological bone formation after RTX-pretreatment. In contrast, mechanical hyperalgesia from day 10 was smaller after inactivating capsaicin-sensitive afferents. Although thermal hyperalgesia did not develop, noxious heat threshold was significantly higher following RTX pretreatment. Somatostatin-like immunoreactivity elevated in the tibiotarsal joints in non-pretreated, which was significantly less in RTX-pretreated mice.
Although capsaicin-sensitive sensory nerves mediate mechanical hyperalgesia in the later phase of autoantibody-induced chronic arthritis, they play important anti-inflammatory roles at least partially through somatostatin release.
PMCID: PMC4349500  PMID: 25524130
Capsaicin-sensitive sensory nerves; Pain; Inflammation; Somatostatin; Matrix-metalloproteinase
8.  The effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase and prostaglandin E2 production by cells of the rheumatoid lesion 
Arthritis Research  1999;1(1):63-70.
The biologically active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], acts through vitamin D receptors, which were found in rheumatoid tissues in the present study. IL-1β-activated rheumatoid synovial fibroblasts and human articular chondrocytes were shown to respond differently to exposure to 1α,25(OH)2D3, which has different effects on the regulatory pathways of specific matrix metalloproteinases and prostaglandin E2.
1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the biologically active metabolite of vitamin D3, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1α,25(OH)2D3; and cell cultures have suggested that 1α,25(OH)2D3 may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1α,25(OH)2D3 within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1α,25(OH)2D3 at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1α,25(OH)2D3 modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E2 (PGE2) production in two cell types known to express chondrolytic enzymes.
To investigate VDR expression in rheumatoid tissues and to examine the effects of 1α,25-dihydroxyvitamin D3 on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE2 production.
Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chondrocytes were treated with either 1α,25(OH)2D3, or interleukin (IL)-1β or both. Conditioned medium was assayed for MMP and PGE2 by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values.
The rheumatoid synovial tissue specimens (n = 18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1α,25(OH)2D3 alone, but when added simultaneously with IL-1β the stimulation by IL-1β was reduced from expected levels by up to 50%. In contrast, 1α,25(OH)2D3 had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1β was not affected by the simultaneous addition of 1α,25(OH)2D3 whilst MMP-3 production was enhanced (Table 1). The production of PGE2 by RSFs was unaffected by 1α,25(OH)2D3 addition, but when added concomitantly with IL-1β the expected IL-1 β-stimulated increase was reduced to almost basal levels. In contrast, IL-1β stimulation of PGE2 in HACs was not affected by the simultaneous addition of 1α,25(OH)2D3 (Table 2). Pretreatment of RSFs with 1α,25(OH)2D3 for 1 h made no significant difference to IL-1β-induced stimulation of PGE2, but incubation for 16 h suppressed the expected increase in PGE2 to control values. This effect was also noted when 1α,25(OH)2D3 was removed after the 16h and the IL-1 added alone. Thus it appears that 1α,25(OH)2D3 does not interfere with the IL-1β receptor, but reduces the capacity of RSFs to elaborate PGE2 after IL-1β induction.
Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1α,25(OH)2D3 in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1α,25(OH)2D3 had little effect on basal MMP production by RSFs, although more pronounced differences were noted when IL-1β-stimulated cells were treated with 1α,25(OH)2D3, with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1α,25(OH)2D3 to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1α,25(OH)2D3 treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1α,25(OH)2D3 had little effect on basal PGE2 production by RSF, but the enhanced PGE2 production observed following IL-1β stimulation of these cells was markedly suppressed by the concomitant addition of 1α,25(OH)2D3. As with MMP production, there are disparate effects of 1α,25(OH)2D3 on IL-1β stimulated PGE2 production by the two cell types; 1α,25(OH)2D3 added concomitantly with IL-1β had no effect on PGE2 production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1α,25(OH)2D3 may have a functional role in the joint disease process. 1α,25(OH)2D3 does not appear to directly affect MMP or PGE2 production but does modulate cytokine-induced production.
Comparative effects of 1 α,25-dihydroxyvitamin D3 (1 α,25D3) on interleukin (IL)-1-stimulated matrix metalloproteinase (MMP)-1 and MMP-3 production by rheumatoid synovial fibroblasts and human articular chondrocytes in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
Comparative effects of 1α,25-dihydroxyvitamin D3 (1α,25D3) on Interleukin (IL)-1-stimulated prostaglandin E2 production by rheumatoid synovial fibroblasts and human articular chondrocyte in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
PMCID: PMC17774  PMID: 11056661
1α,25-dihydroxyvitamin D3; matrix metalloproteinase; prostaglandin E2; rheumatoid arthritis
9.  Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4 
Arthritis Research  2000;2(4):293-302.
To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that is characterized by joint inflammation, and progressive cartilage and bone erosion. Recent research has identified certain biologic agents that appear more able than conventional therapies to halt effectively the progression of disease, as well as ameliorate disease symptoms. One potential problem with the use of biologic agents for arthritis therapy is the need for daily or weekly repeat dosing. The transfer of genes directly to the synovial lining can theoretically circumvent the need for repeat dosing and reduce potential systemic side effects [1,2]. However, although many genes have been effective in treating murine CIA if administrated at a time before disease onset, local intra-articular or periarticular gene transfer has not been highly effective in halting the progression of established disease. IL-4, similar to tumor necrosis factor (TNF)-α and IL-1 inhibitors, has been shown be therapeutic for the treatment of murine CIA when administered intravenously as a recombinant protein, either alone or in combination with IL-10. IL-4 can downregulate the production of proinflammatory and T-helper (Th)1-type cytokines by inducing mRNA degradation and upregulating the expression of inhibitors of proinflammatory cytokines such as IL-1 receptor antagonist (IL-1Ra) [3,4]. IL-4 is able to inhibit IL-2 and IFN-γ production by Th1 cells, resulting in suppression of macrophage activation and the production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α by monocytes and macrophages [4,5,6,7,8,9].
In order to examine the therapeutic effects of local and systemic IL-4 expression in established CIA, an adenoviral vector carrying the gene for murine IL-4 (Ad-mIL-4) was generated. The ability of Ad-mIL-4 to treat established CIA was evaluated by local periarticular and systemic intravenous injection of Ad-mIL-4 into mice at various times after disease onset.
Materials and methods:
Male DBA/1 lacJ (H-2q) mice, aged 7-8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were immunized intradermally at the base of tail with 100 μ g bovine type II collagen. On day 21 after priming, mice received a boost injection (intradermally) with 100 μ g type II collagen in incomplete adjuvant. For the synchronous onset of arthritis, 40 μ g lipopolysaccharide (Sigma, St Louis, MO, USA) was injected intraperitoneally on day 28. Ad-mIL-4 was injected periarticularly into the hind ankle joints of mice on day 32 or intravenously by tail vein injection on day 29. Disease severity was monitored every other day using an established macroscopic scoring system ranging from 0 to 4: 0, normal; 1, detectable arthritis with erythma; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; and 4, maximal swelling with ankylosis. The average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse. Cytokine production by joint tissue or serum were assessed using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).
To examine the therapeutic effects of IL-4 gene transfer in a murine model of arthritis, 5×108 particles of Ad-mIL-4 and enhanced green fluorescent protein (Ad-eGFP) were administered by periarticular injection into the ankle joints of mice with established disease 4 days after lipopolysaccharide injection. All mice had established disease at time of injection. As shown in Figure 1, the severity of arthritis (Fig. 1a), paw thickness (Fig. 1b), and the number of arthritic paws (Fig. 1c) were all significantly reduced in the Ad-mIL-4 group, compared with the saline- and Ad-eGFP-treated groups. Analysis of the bones in the ankle joints of control arthritic mice showed evidence of erosion with an associated monocytic infiltrate around the joint space compared with the Ad-mIL-4-treated and nonarthritic control joints. In addition, injection of the ankle joints in the hind legs resulted in a therapeutic effect in the front paws. A similar contralateral effect has been observed with adenoviral-mediated delivery of viral (v)-IL-10. Interestingly, a high level of murine IL-10 also was detected from the joint lysates of Ad-mIL-4-treated naïve and arthritic mice, with the production of endogenous IL-10 correlating with the dose of Ad-mIL-4. The administration of recombinant IL-4 protein systemically has been shown to be therapeutic in murine CIA models if given before disease onset. To examine the effect of systemic IL-4 delivered by gene transfer, 1×109 particles of Ad-mIL-4 were injected via the tail vein of collagen-immunized mice the day after lipopolysaccharide injection. Whereas the immunized control mice, injected with Ad-eGFP, showed disease onset on day 3 after lipopolysaccharide injection, Ad-mIL-4-treated mice showed a delay in disease onset and as a reduction in the total number of arthritic paws. Also, systemic injection of Ad-mIL-4 suppressed the severity of arthritis in CIA mice according to arthritis index.
Gene therapy represents a novel approach for delivery of therapeutic agents to joints in order to treat the pathologies associated with RA and osteoarthritis, as well as other disorders of the joints. In the present study we examined the ability of local periarticular and systemic gene transfer of IL-4 to treat established and early-stage murine CIA, respectively. We have demonstrated that both local and systemic administration of Ad-mIL-4 resulted in a reduction in the severity of arthritis, as well as in the number of arthritic paws. In addition, the local gene transfer of IL-4 reduced histologic signs of inflammation and of bone erosion. Interestingly, local delivery of Ad-mIL-4 was able to confer a therapeutic effect to the untreated, front paws through a currently unknown mechanism. In addition, both local and systemic expression of IL-4 resulted in an increase in the level of endogenous IL-10, as well as of IL-1Ra (data not shown). Previous experiments have shown that gene transfer of IL-10 and IL-1 and TNF inhibitors at the time of disease initiation (day 28) is therapeutic. However, delivery of these agents after disease onset appeared to have only limited therapeutic effect. In contrast, the present results demonstrate that IL-4, resulting from local periarticular and systemic injection of Ad-mIL-4, was able partially to reverse progression of established and early-stage disease, respectively. These results, as well as those of others, support the potential application of IL-4 gene therapy for the clinical treatment of RA.
PMCID: PMC17812  PMID: 11056670
adenoviral vectors; collagen-induced arthritis; gene therapy; IL-4; IL-10; rheumatoid arthritis
10.  Inhibition of Inflammatory Arthritis Using Fullerene Nanomaterials 
PLoS ONE  2015;10(4):e0126290.
Inflammatory arthritis (e.g. rheumatoid arthritis; RA) is a complex disease driven by the interplay of multiple cellular lineages. Fullerene derivatives have previously been shown to have anti-inflammatory capabilities mediated, in part, by their ability to prevent inflammatory mediator release by mast cells (MC). Recognizing that MC can serve as a cellular link between autoantibodies, soluble mediators, and other effector populations in inflammatory arthritis, it was hypothesized that fullerene derivatives might be used to target this inflammatory disease. A panel of fullerene derivatives was tested for their ability to affect the function of human skin-derived MC as well as other lineages implicated in arthritis, synovial fibroblasts and osteoclasts. It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts. MC inhibition by fullerene derivatives was mediated through the reduction of mitochondrial membrane potential and FcγR-mediated increases in cellular reactive oxygen species and NF-κB activation. Based on these in vitro data, two fullerene derivatives (ALM and TGA) were selected for in vivo studies using K/BxN serum transfer arthritis in C57BL/6 mice and collagen-induced arthritis (CIA) in DBA/1 mice. Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls. In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α. Fullerenes remained capable of attenuating K/BxN arthritis in mast cell-deficient mice Cre-Master mice, suggesting that lineages beyond the MC represent relevant targets in this system. These studies suggest that fullerene derivatives may hold promise both as an assessment tool and as anti-inflammatory therapy of arthritis.
PMCID: PMC4400016  PMID: 25879437
11.  Decreased collagen-induced arthritis severity and adaptive immunity in mitogen activated protein kinase kinase 6 -deficient mice 
Arthritis and Rheumatism  2012;64(3):678-687.
MAPK kinases MKK3 and MKK6 regulate p38 MAPK activation in inflammatory diseases such as rheumatoid arthritis. Previous studies demonstrated that MKK3- or MKK6-deficiency inhibits K/BxN serum-induced arthritis. However, the role of these kinases in adaptive immunity-dependent models of chronic arthritis is not known. The goal of this study was to evaluate MKK3- and MKK6-deficiency in the collagen induced arthritis model.
Wildtype, MKK3−/−, and MKK6−/− mice were immunized with bovine type II collagen (CII). Disease activity was evaluated by semiquantitative scoring, histology, and microcomputed tomography. Serum anti-collagen antibody levels were quantified by ELISA. In-vitro T cell cytokine response was measured by flow cytometry and multiplex analysis. Expression of joint cytokines and matrix metalloproteinase was determined by qPCR.
MKK6-deficiency markedly reduced arthritis severity compared with WT mice, while absence of MKK3 had an intermediate effect. Joint damage was minimal in arthritic MKK6−/− mice and intermediate in MKK3−/− mice compared with wild type mice. MKK6−/− mice had modestly lower levels of pathogenic anti-collagen antibodies than WT or MKK3−/− mice. In vitro T cell assays showed reduced proliferation and IL-17 production by MKK6−/− cells in response to type II collagen. Gene expression of synovial IL-6, matrix metalloproteinases MMP3, and MMP13 was significantly inhibited in MKK6-deficient mice.
Reduced disease severity in MKK6−/− mice correlated with decreased anti-collagen responses indicating that MKK6 is a crucial regulator of inflammation joint destruction in CIA. MKK6 is a potential therapeutic target in complex diseases involving adaptive immune responses like rheumatoid arthritis.
PMCID: PMC3267851  PMID: 21953132
12.  Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis 
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of bone and cartilage, which is mediated, in part, by synovial fibroblasts. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes responsible for matrix degradation. Macrophage migration inhibitory factor (MIF) is a cytokine that induces the production of a large number of proinflammatory molecules and has an important role in the pathogenesis of RA by promoting inflammation and angiogenesis.
In the present study, we determined the role of MIF in RA synovial fibroblast MMP production and the underlying signaling mechanisms. We found that MIF induces RA synovial fibroblast MMP-2 expression in a time-dependent and concentration-dependent manner. To elucidate the role of MIF in MMP-2 production, we produced zymosan-induced arthritis (ZIA) in MIF gene-deficient and wild-type mice. We found that MMP-2 protein levels were significantly decreased in MIF gene-deficient compared with wild-type mice joint homogenates. The expression of MMP-2 in ZIA was evaluated by immunohistochemistry (IHC). IHC revealed that MMP-2 is highly expressed in wild-type compared with MIF gene-deficient mice ZIA joints. Interestingly, synovial lining cells, endothelial cells, and sublining nonlymphoid mononuclear cells expressed MMP-2 in the ZIA synovium. Consistent with these results, in methylated BSA (mBSA) antigen-induced arthritis (AIA), a model of RA, enhanced MMP-2 expression was also observed in wild-type compared with MIF gene-deficient mice joints. To elucidate the signaling mechanisms in MIF-induced MMP-2 upregulation, RA synovial fibroblasts were stimulated with MIF in the presence of signaling inhibitors. We found that MIF-induced RA synovial fibroblast MMP-2 upregulation required the protein kinase C (PKC), c-jun N-terminal kinase (JNK), and Src signaling pathways. We studied the expression of MMP-2 in the presence of PKC isoform-specific inhibitors and found that the PKCδ inhibitor rottlerin inhibits MIF-induced RA synovial fibroblast MMP-2 production. Consistent with these results, MIF induced phosphorylation of JNK, PKCδ, and c-jun. These results indicate a potential novel role for MIF in tissue destruction in RA.
PMCID: PMC1779381  PMID: 16872482
13.  Suppression of collagen-induced arthritis in growth arrest and DNA damage inducible 45β (Gadd45β)-deficient mice 
Arthritis and rheumatism  2011;63(10):2949-2955.
Growth arrest and DNA damage inducible 45β (Gadd45β) is involved in stress responses, cell cycle regulation, and oncogenesis. Previous studies demonstrated that Gadd45β deficiency exacerbates K/BxN serum-induced arthritis and experimental allergic encephalomyelitis (EAE) in mice, indicating that Gadd45β plays a suppressive role in innate and adaptive immune responses. To further understand how Gadd45β regulates autoimmunity, we evaluated collagen-induced arthritis in Gadd45β−/− mice.
Wildtype and Gadd45β−/− DBA/1 mice were immunized with bovine type II collagen (CII). Serum anti-collagen antibody levels were quantified by ELISA. Cytokines and matrix metalloproteinase expression in the joint and spleen was determined by quantitative PCR. In-vitro T cell cytokine response to CII was measured by multiplex analysis. CD4+CD25+Treg and Th17 cells were quantified using flow cytometry.
Gadd45β−/− mice showed significantly lower arthritis severity and joint destruction compared with WT mice. MMP3 and MMP13 expression was also markedly reduced in Gadd45β−/− mice. However, serum anti-CII antibody levels were similar in both groups. Foxp3 and IL-10 expression was increased 2–3-fold in arthritic Gadd45β−/− splenocytes compared with WT. Flow cytometric analysis showed greater numbers of CD4+CD25+Treg cells in Gadd45β−/− spleen than in WT. In-vitro studies showed that interferon-γ and interleukin-17 production by T cells was significantly decreased in Gadd45β−/− mice.
Unlike in passive K/BxN arthritis model and EAE, Gadd45β-deficiency in CIA was associated with lower arthritis severity, elevated IL-10 expression, decreased IL-17 production, and increased numbers of Treg cells. The data suggest that Gadd45β plays a complex role in regulating adaptive immunity and, depending on the model, either enhances or suppresses inflammation.
PMCID: PMC3183142  PMID: 21702006
14.  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.
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.
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.
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.
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.
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
15.  Suppressive effect of secretory phospholipase A2 inhibitory peptide on interleukin-1β-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts, and its antiarthritic activity in hTNFtg mice 
Arthritis Research & Therapy  2009;11(5):R138.
Secretory phospholipase A2 (sPLA2) and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA). The objective of this study was to understand the inhibitory mechanism of phospholipase inhibitor from python (PIP)-18 peptide in cultured synovial fibroblasts (SF), and to evaluate its therapeutic potential in a human tumor necrosis factor (hTNF)-driven transgenic mouse (Tg197) model of arthritis.
Gene and protein expression of sPLA2-IIA, MMP-1, MMP-2, MMP-3, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were analyzed by real time PCR and ELISA respectively, in interleukin (IL)-1β stimulated rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts cells treated with or without inhibitors of sPLA2 (PIP-18, LY315920) or MMPs (MMP Inhibitor II). Phosphorylation status of mitogen-activated protein kinase (MAPK) proteins was examined by cell-based ELISA. The effect of PIP-18 was compared with that of celecoxib, methotrexate, infliximab and antiflamin-2 in Tg197 mice after ip administration (thrice weekly for 5 weeks) at two doses (10, 30 mg/kg), and histologic analysis of ankle joints. Serum sPLA2 and cytokines (tumor necrosis factor (TNF)α, IL-6) were measured by Escherichia coli (E coli) assay and ELISA, respectively.
PIP-18 inhibited sPLA2-IIA production and enzymatic activity, and suppressed production of MMPs in IL-1β-induced RA and OA SF cells. Treatment with PIP-18 blocked IL-1β-induced p38 MAPK phosphorylation and resulted in attenuation of sPLA2-IIA and MMP mRNA transcription in RA SF cells. The disease modifying effect of PIP-18 was evidenced by significant abrogation of synovitis, cartilage degradation and bone erosion in hTNF Tg197 mice.
Our results demonstrate the benefit that can be gained from using sPLA2 inhibitory peptide for RA treatment, and validate PIP-18 as a potential therapeutic in a clinically relevant animal model of human arthritis.
PMCID: PMC2787297  PMID: 19765281
16.  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.
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.
To examine whether FcγR expression on macrophages is related to severity of synovial inflammation and cartilage destruction during immune-complex-mediated joint inflammation.
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).
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.
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
17.  Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis 
Rheumatoid arthritis is an autoimmune disease characterized by hyperplasia of the synovial lining and destruction of cartilage and bone. Recent studies have suggested that a lack of apoptosis contributes to the hyperplasia of the synovial lining and to the failure in eliminating autoreactive cells. Mice lacking Fas or Bim, two pro-apoptotic proteins that mediate the extrinsic and intrinsic death cascades, respectively, develop enhanced K/BxN serum transfer-induced arthritis. Since the pro-apoptotic protein Bid functions as an intermediate between the extrinsic and intrinsic apoptotic pathways, we examined the role that it plays in inflammatory arthritis. Mice deficient in Bid (Bid-/-) show a delay in the resolution of K/BxN serum transfer-induced arthritis. Bid-/- mice display increased inflammation, bone destruction, and pannus formation compared to wild-type mice. Furthermore, Bid-/- mice have elevated levels of CXC chemokine and IL-1β in serum, which are associated with more inflammatory cells throughout the arthritic joint. In addition, there are fewer apoptotic cells in the synovium of Bid-/- compared to Wt mice. These data suggest that extrinsic and intrinsic apoptotic pathways cooperate through Bid to limit development of inflammatory arthritis.
PMCID: PMC2206343  PMID: 17509138
18.  Reduced locomotor activity correlates with increased severity of arthritis in a mouse model of antibody-induced arthritis 
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia and progressive cartilage and bone destruction that leads to a substantial loss of general functions and/or a decline in physical activities such as walking speed in humans. The K/BxN serum transfer arthritis in mice shares many immunological and pathological features with human RA. Very few studies are available in mice that investigate the changes in physical activity in relation to arthritis development. In this study we investigate the effect of arthritis on the locomotor activity of mice during K/BxN sera transfer arthritis.
Arthritis was induced in Balb/c mice by injecting intraperitoneally with 200ul of K/BxN sera; Balb/c mice injected with phosphate buffered saline (PBS) served as control. Progress of arthritis was estimated by daily measurements of joint thickness. Each mouse's locomotor activity (travel distance and travel time) was assessed every day for duration of 20 minute period using the SmartCage™ platform. Data were analyzed using the SmartCage™ analysis software (CageScore™).
Arthritic Balb/c mice showed a reduction in distance covered and travel speed when compared to arthritis-free, control Balb/c mice. Maximum decline in locomotor activity was observed during the peak period of the disease and correlated to the increase in joint thickness in the arthritic mice.
This report demonstrates that measuring locomotor activity of mice during progression of K/BxN sera-induced arthritis using the SmartCage™ platform offers a quantitative method to assess physical activity in mice during arthritis.
PMCID: PMC4264968  PMID: 25506517
K/BxN; Arthritis; Locomotion; Serum transfer
19.  Exacerbation of antigen-induced arthritis in urokinase-deficient mice. 
In rheumatoid arthritis, synovial expression of urokinase (uPA) activity is greatly increased (Busso, N., V. Péclat, A. So, and A. -P. Sappino. 1997. Ann. Rheum. Dis. 56:550- 557). We report the same effect in murine antigen-induced arthritis. uPA-mediated plasminogen activation in arthritic joints may have deleterious effects via degradation of cartilage and bone matrix proteins as well as beneficial effects via fibrin degradation. We evaluated these contrasting effects in vivo by analyzing the phenotype of uPA-deficient (uPA-/-) and control mice during antigen-induced arthritis. Joint inflammation was comparable in both groups up to day 3 and subsequently declined in control mice, remaining significantly elevated in uPA-/- mice on days 10 and 30 after arthritis onset. Likewise, synovial thickness was markedly increased in uPA-deficient mice persisting for up to 2 mo, whereas it subsided in control animals. Bone erosion was exacerbated in uPA-/- mice on day 30. By contrast, no difference in articular cartilage proteoglycan content was found between both groups. Significantly increased accumulation of fibrin was observed by day 30 in arthritic joints of uPA-/- mice. We hypothesized that synovial fibrin deposition plays a role in joint inflammation. Accordingly, defibrinogenation of uPA-/- mice by ancrod significantly decreased the sustained joint inflammation. All the above observations were reproducible in plasminogen-deficient (Pln-/-) mice. In conclusion, synovial fibrin deposition plays a role as a nonimmunological mechanism which sustains chronic arthritis.
PMCID: PMC509063  PMID: 9649555
20.  Role of Interferon Regulatory Factor 7 in Serum Transfer Arthritis: Regulation of Interferon β production 
Arthritis and Rheumatism  2011;64(4):1046-1056.
Innate immune responses activate synoviocytes and recruit inflammatory cells into the rheumatoid joint. Type I interferons (IFN) play a role in autoimmunity and IFN gene transcription is activated by IFN-regulatory factors (IRF) in response to innate sensor recognition. This study examined the effect of genetic deficiency of IRF7 in a passive K/BxN serum transfer model of arthritis.
Passive transfer arthritis was induced in Irf7−/− mice and additional groups were treated with IFNβ or poly (I-C). Clinical arthritis scores, histology, microcomputed tomography, and synovial tissue Q-PCR were performed. Mouse serum was analyzed by ELISA.
In the K/BxN passive model, arthritis severity was significantly increased in Irf7−/− mice compared with wild type (WT). In addition, synovial and serum IFNβ expression was decreased, potentially contributing to increased arthritis. Irf7−/− mice injected with replacement IFNβ had a decrease in arthritis. Poly (I-C) treatment diminished arthritis in Irf7−/− mice, restored synovial IFNβ gene expression, and increased serum IFNβ. In vitro studies demonstrated that poly (I-C) stimulation of Irf7−/− fibroblast-like synoviocytes (FLS) resulted in increased induction of pro-inflammatory gene expression compared with WT FLS; however, IFNβ expression was not significantly different. In contrast, Irf7−/− macrophages showed significantly less induction of IFNβ in response to poly (I-C) stimulation.
IRF7 deficiency exacerbated arthritis and replacement treatment with IFNβ or poly (I-C) decreased arthritis severity. Both macrophage and synoviocyte specific roles for IRF7 likely contribute to increased arthritis. IRF7 might play an anti-inflammatory role in passive transfer arthritis through regulation of macrophage IFNβ production.
PMCID: PMC3290705  PMID: 22076939
transcription factors; signal transduction; inflammation; rheumatoid arthritis
21.  Anti-arthritic activity of Fu-Fang-Lu-Jiao-Shuang on collagen-induced arthritis in Balb/c mice and its underlying mechanisms 
Pharmacognosy Magazine  2015;11(42):242-249.
Rheumatoid arthritis (RA) is a common, autoimmune disorder characterized by progressive multiple joint destruction, deformity, disability and premature death in most patients. Fu-Fang-Lu-Jiao-Shuang (FFLJS) is an effective traditional Chinese medicine, which has long been used clinically to treat RA patients.
The objective of this study is aimed to evaluate the anti-rheumatic effects of FFLJS on collagen induced arthritis (CIA) model, as well as the underlying mechanisms, which have not previously been explored.
Materials and Methods:
CIA was induced by immunization with type II collagen (CII) in male Balb/c mice. The mice in the onset of arthritis were treated daily with FFLJS (125 or 500 mg/kg) or 1% carboxymethyl cellulose-Na for 28 days. Paw thickness and arthritic score were evaluated to confirm the anti-arthritic effect of FFLJS on CIA in mice. Levels of anti-CII antibody, proinflammatory cytokines interleukin-1 (IL-1) β, IL-17, and tumor necrosis factor-α (TNF-α) as well as prostaglandin E-2 (PGE-2) in serum and histological changes in the ankle joint were also analyzed. In addition, expressions of matrix metalloproteinases-1 (MMP-1), MMP-3 and tissue inhibitors of matrix metalloproteases-1 (TIMP-1) in synovial tissue were also detected to further study the molecular mechanism of the anti-arthritic effects of FFLJS.
During therapeutic treatment, FFLJS significantly reduced paw thickness and arthritic score in CIA mice, decreased the amounts of TNF-α, IL-1 β, IL-17, PGE-2 and anti-CII antibody in serum. In addition, FFLJS treatment could prevent the bone destruction by reducing the expression of MMP-1 and MMP-3, increasing the expression of TIMP-1 in synovial tissue of CIA mice.
These findings offer the convincing evidence for the first time that the anti-rheumatic effects of FFLJS might be related to down-regulation of TNF-α, IL-1 β, IL-17 and PGE-2 levels for acute arthritis, and regulation of MMP-1, MMP-3 and TIMP-1 protein expression for chronic arthritis.
PMCID: PMC4378120  PMID: 25829761
Anti-arthritic activity; Fu-Fang-Lu-Jiao-Shuang; matrix metalloproteinases; traditional Chinese medicine
22.  The Arthritis Severity Locus Cia5d Is a Novel Genetic Regulator of the Invasive Properties of Synovial Fibroblasts 
Arthritis and rheumatism  2008;58(8):2296-2306.
The synovial fibroblast, or fibroblast-like synoviocyte (FLS), has a central role in pannus invasion and destruction of cartilage and bone in rheumatoid arthritis (RA). However, regulation of the FLS remains incompletely understood. The aim of this study was to determine whether the invasive properties of FLS are genetically regulated by arthritis severity loci.
DA rats (arthritis susceptible) and rat strains congenic for arthritis-protective intervals were studied. Primary FLS cell lines were generated from each strain and used in a well-established FLS invasion model through a collagen-rich barrier. Cells or culture supernatants were analyzed for gene expression, activity of different matrix metalloproteinases (MMPs), cytoskeleton integrity, and cell proliferation.
The median number of FLS from DA.F344(Cia5d) rats that invaded through the collagen-rich barrier was reduced 86.5% compared with the median number of invading FLS from DA rats. Histologic examination showed that DA.F344(Cia5d) rats preserved a normal joint without pannus, hyperplasia, or erosions. FLS from DA.F344(Cia5d) rats produced significantly lower levels of active MMP-2 compared with FLS from DA rats, but the levels of proMMP-2 and MMP-2 messenger RNA in DA.F344(Cia5d) rats were similar to those in DA rats. Treatment of FLS from DA rats with an MMP-2 inhibitor reduced cell invasion to a level similar to that in DA.F344(Cia5d) rats, demonstrating that MMP-2 activity accounted for the difference between FLS from these 2 strains. Analysis of MMP-2–activating pathways revealed increased levels of soluble membrane type 1 (MT1)–MMP in DA rats compared with DA.F344(Cia5d) rats.
These data represent the first evidence for a genetic component in the regulation of FLS invasion. A gene located within the Cia5d interval accounts for this effect and operates via the regulation of soluble MT1-MMP production and MMP-2 activation. These observations suggest novel potential pathways for prognostication and therapy.
PMCID: PMC2714698  PMID: 18668563
23.  c-Fms-mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis 
Tyrosine kinases are key mediators of multiple signaling pathways implicated in rheumatoid arthritis (RA). We previously demonstrated that imatinib mesylate--a Food and Drug Administration (FDA)-approved, antineoplastic drug that potently inhibits the tyrosine kinases Abl, c-Kit, platelet-derived growth factor receptor (PDGFR), and c-Fms--ameliorates murine autoimmune arthritis. However, which of the imatinib-targeted kinases is the principal culprit in disease pathogenesis remains unknown. Here we examine the role of c-Fms in autoimmune arthritis.
We tested the therapeutic efficacy of orally administered imatinib or GW2580, a small molecule that specifically inhibits c-Fms, in three mouse models of RA: collagen-induced arthritis (CIA), anti-collagen antibody-induced arthritis (CAIA), and K/BxN serum transfer-induced arthritis (K/BxN). Efficacy was evaluated by visual scoring of arthritis severity, paw thickness measurements, and histological analysis. We assessed the in vivo effects of imatinib and GW2580 on macrophage infiltration of synovial joints in CIA, and their in vitro effects on macrophage and osteoclast differentiation, and on osteoclast-mediated bone resorption. Further, we determined the effects of imatinib and GW2580 on the ability of macrophage colony-stimulating factor (M-CSF; the ligand for c-Fms) to prime bone marrow-derived macrophages to produce tumor necrosis factor (TNF) upon subsequent Fc receptor ligation. Finally, we measured M-CSF levels in synovial fluid from patients with RA, osteoarthritis (OA), or psoriatic arthritis (PsA), and levels of total and phosphorylated c-Fms in synovial tissue from patients with RA.
GW2580 was as efficacious as imatinib in reducing arthritis severity in CIA, CAIA, and K/BxN models of RA. Specific inhibition of c-Fms abrogated (i) infiltration of macrophages into synovial joints of arthritic mice; (ii) differentiation of monocytes into macrophages and osteoclasts; (iii) osteoclast-mediated bone resorption; and (iv) priming of macrophages to produce TNF upon Fc receptor stimulation, an important trigger of synovitis in RA. Expression and activation of c-Fms in RA synovium were high, and levels of M-CSF were higher in RA synovial fluid than in OA or PsA synovial fluid.
These results suggest that c-Fms plays a central role in the pathogenesis of RA by mediating the differentiation and priming of monocyte lineage cells. Therapeutic targeting of c-Fms could provide benefit in RA.
PMCID: PMC2875666  PMID: 20181277
24.  Focal adhesion kinase is required for synovial fibroblast invasion, but not murine inflammatory arthritis 
Synovial fibroblasts invade cartilage and bone, leading to joint destruction in rheumatoid arthritis. However, the mechanisms that regulate synovial fibroblast invasion are not well understood. Focal adhesion kinase (FAK) has been implicated in cellular invasion in several cell types, and FAK inhibitors are in clinical trials for cancer treatment. Little is known about the role of FAK in inflammatory arthritis, but, given its expression in synovial tissue, its known role in invasion in other cells and the potential clinical availability of FAK inhibitors, it is important to determine if FAK contributes to synovial fibroblast invasion and inflammatory arthritis.
After treatment with FAK inhibitors, invasiveness of human rheumatoid synovial fibroblasts was determined with Matrigel invasion chambers. Migration and focal matrix degradation, two components of cellular invasion, were assessed in FAK-inhibited rheumatoid synovial fibroblasts by transwell assay and microscopic examination of fluorescent gelatin degradation, respectively. Using mice with tumor necrosis factor α (TNFα)–induced arthritis in which fak could be inducibly deleted, invasion and migration by FAK-deficient murine arthritic synovial fibroblasts were determined as described above and arthritis was clinically and pathologically scored in FAK-deficient mice.
Inhibition of FAK in human rheumatoid synovial fibroblasts impaired cellular invasion and migration. Focal matrix degradation occurred both centrally and at focal adhesions, the latter being a novel site for matrix degradation in synovial fibroblasts, but degradation was unaltered with FAK inhibitors. Loss of FAK reduced invasion in murine arthritic synovial fibroblasts, but not migration or TNFα-induced arthritis severity and joint erosions.
FAK inhibitors reduce synovial fibroblast invasion and migration, but synovial fibroblast migration and TNFα-induced arthritis do not rely on FAK itself. Thus, inhibition of FAK alone is unlikely to be sufficient to treat inflammatory arthritis, but current drugs that inhibit FAK may inhibit multiple factors, which could increase their efficacy in rheumatoid arthritis.
PMCID: PMC4203874  PMID: 25280866
25.  Critical Roles for Interleukin 1 and Tumor Necrosis Factor α in Antibody-induced Arthritis 
In spontaneous inflammatory arthritis of K/BxN T cell receptor transgenic mice, the effector phase of the disease is provoked by binding of immunoglobulins (Igs) to joint surfaces. Inflammatory cytokines are known to be involved in human inflammatory arthritis, in particular rheumatoid arthritis, although, overall, the pathogenetic mechanisms of the human affliction remain unclear. To explore the analogy between the K/BxN model and human patients, we assessed the role and relative importance of inflammatory cytokines in K/BxN joint inflammation by transferring arthritogenic serum into a panel of genetically deficient recipients. Interleukin (IL)-1 proved absolutely necessary. Tumor necrosis factor (TNF)–α was also required, although seemingly less critically than IL-1, because a proportion of TNF-α–deficient mice developed robust disease. There was no evidence for an important role for IL-6. Bone destruction and reconstruction were also examined. We found that all mice with strong inflammation exhibited the bone erosion and reconstruction phenomena typical of K/BxN arthritis, with no evidence of any particular requirement for TNFα for bone destruction. The variability in the requirement for TNF-α, reminiscent of that observed in treated rheumatoid arthritis patients, did not appear genetically programmed but related instead to subtle environmental changes.
PMCID: PMC2194010  PMID: 12093872
transgenic; cytokine; knockout; inflammatory; TNF

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