PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (994199)

Clipboard (0)
None

Related Articles

1.  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.
Introduction:
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.
Aims:
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.
Methods:
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.
Results:
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.
Discussion:
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
2.  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
3.  Potential roles of microsomal prostaglandin E synthase-1 in rheumatoid arthritis 
Inflammation and regeneration  2011;31(2):157-166.
Rheumatoid arthritis (RA) is a chronic autoimmune disease which primarily affects the synovial joints leading to inflammation, pain and joint deformities. Nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, both of which inhibit cyclooxygenase (COX), have been extensively used for treating RA patients. Prostaglandin E synthase (PGES) is a specific biosynthetic enzyme that acts downstream of COX and converts prostaglandin (PG) H2 to PGE2. Among PGES isozymes, microsomal PGES-1 (mPGES-1) has been shown to be induced in a variety of cells and tissues under inflammatory conditions. The induction of mPGES-1 in the synovial tissue of RA patients is closely associated with the activation of the tissue by proinflammatory cytokines. Although selective mPGES-1 inhibitors have not yet been widely available, mice lacking mPGES-1 (mPGES-1–/– mice) have been generated to evaluate the physiological and pathological roles of mPGES-1 in vivo. Recent studies utilizing mPGES-1–/– mice have demonstrated the significance of mPGES-1 in the process of chronic inflammation and evocation of humoral immune response in autoimmune arthritis models. These recent findings highlight mPGES-1 as a novel therapeutic target for the treatment of autoimmune inflammatory diseases, including RA. Currently, both natural and synthetic chemicals are being tested for inhibition of mPGES-1 activity to produce PGE2. The present review focuses on the recent advances in understanding the role of mPGES-1 in the pathophysiology of RA.
PMCID: PMC3269760  PMID: 22308189
inflammation; microsomal prostaglandin E synthase-1; prostaglandin E2; rheumatoid arthritis; T-cell-dependent humoral immunity
4.  IL-1β/HMGB1 Complexes Promote The PGE2 Biosynthesis Pathway in Synovial Fibroblasts 
PGE2 is a potent lipid mediator of pain and oedema found elevated in RA. Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal enzyme of the PGE2 pathway inducible by proinflammatory cytokines. mPGES-1 is markedly upregulated in RA synovial tissue despite antirheumatic treatments, suggesting that multiple inflammatory stimuli contribute to its induction. High-mobility group box chromosomal protein 1 (HMGB1) is known to induce inflammation both by direct interaction with TLR4 and by enhancement of other proinflammatory molecules signalling, through complex formation. The high expression of extracellular HMGB1 within the inflamed synovium, implies its pro-arthritogenic role in RA. We aimed to investigate the effects of IL-1β/HMGB1 complexes on mPGES-1 and other enzymes of the PGE2 pathway in synovial fibroblasts (SFs) from patients with arthritis. Furthermore, we studied the effect of COX-2 inhibition and IL-1RI antagonism on prostanoid and cytokine production by SFs. Stimulation of SFs with HMGB1 in complex with suboptimal amounts of IL-1β significantly increased mPGES-1 and COX-2 expressions as well as PGE2 production, as compared to treatment with HMGB1 or IL-1β alone. Furthermore, NS-398 reduced the production of IL-6 and IL-8, thus indicating that IL-1β/HMGB1 complexes modulate cytokine production in part through prostanoid synthesis. Treatment with IL-1RA completely abolished the induced PGE2 and cytokine production, suggesting an effect mediated through IL-1RI. IL-1β/HMGB1 complexes promote the induction of mPGES-1, COX-2 and PGE2 in SF. The amplification of the PGE2 biosynthesis pathway by HMGB1 might constitute an important pathogenic mechanism perpetuating inflammatory and destructive activities in rheumatoid arthritis.
doi:10.1111/sji.12041
PMCID: PMC3670302  PMID: 23488692
5.  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
6.  Prostaglandin E2 Induction during Mouse Adenovirus Type 1 Respiratory Infection Regulates Inflammatory Mediator Generation but Does Not Affect Viral Pathogenesis 
PLoS ONE  2013;8(10):e77628.
Respiratory viruses cause substantial disease and are a significant healthcare burden. Virus-induced inflammation can be detrimental to the host, causing symptoms during acute infection and leading to damage that contributes to long-term residual lung disease. Prostaglandin E2 (PGE2) is a lipid mediator that is increased in response to many viral infections, and inhibition of PGE2 production during respiratory viral infection often leads to a decreased inflammatory response. We tested the hypothesis that PGE2 promotes inflammatory responses to mouse adenovirus type 1 (MAV-1) respiratory infection. Acute MAV-1 infection increased COX-2 expression and PGE2 production in wild type mice. Deficiency of the E prostanoid 2 receptor had no apparent effect on MAV-1 pathogenesis. Virus-induced induction of PGE2, IFN-γ, CXCL1, and CCL5 was reduced in mice deficient in microsomal PGE synthase-1 (mPGES-1-/- mice). However, there were no differences between mPGES-1+/+ and mPGES-1-/- mice in viral replication, recruitment of leukocytes to airways or lung inflammation. Infection of both mPGES‑1+/+ and mPGES-1-/- mice led to protection against reinfection. Thus, while PGE2 promotes the expression of a variety of cytokines in response to acute MAV-1 infection, PGE2 synthesis does not appear to be essential for generating pulmonary immunity.
doi:10.1371/journal.pone.0077628
PMCID: PMC3797793  PMID: 24147040
7.  Cytosolic Phospholipase A2 Regulates TNF-Induced Production of Joint Destructive Effectors in Synoviocytes 
PLoS ONE  2013;8(12):e83555.
Introduction
Rheumatoid arthritis (RA) is an inflammatory disease of the joint characterized by chronic synovitis causing pain, swelling and loss of function due to destruction of cartilage and bone. The complex series of pathological events occurring in RA is largely regulated via excessive production of pro-inflammatory cytokines, the most prominent being tumor necrosis factor (TNF). The objective of this work was to elucidate possible involvement of group IVA cytosolic phospholipase A2 (cPLA2α) in TNF-induced regulation of synovitis and joint destructive effectors in RA, to evaluate the potential of cPLA2α as a future therapeutic target.
Methods
The involvement of cPLA2α in tumor necrosis factor (TNF)-induced intracellular signaling cascades in synoviocytes (synovial fibroblast-like cells) was analyzed by arachidonic acid (AA) release assay, synoviocyte enzyme activity assay, gene expression analysis by real-time PCR and ELISA immunoassay for the detection of prostaglandin E2 (PGE2), interleukin 8 (IL8) and stromelysin-1 (MMP3), respectively.
Results
Inhibitors of cPLA2α enzyme activity (AVX002, ATK) significantly reduced TNF-induced cellular release of AA, PGE2, IL8 and MMP3. This reduction was evident both at transcriptional, protein or metabolite levels. Interestingly, cPLA2α inhibition affected several key points of the arachidonyl cascade; AA-release, cyclooxygenase-2 (COX2) expression and PGE2 production. Furthermore, the results suggest that cPLA2α is subject to transcriptional auto-regulation as inhibition of cPLA2α resulted in reduced PLA2G4A gene expression in TNF-stimulated synoviocytes.
Conclusions
cPLA2α appears to be an important regulator of central effectors of inflammation and joint destruction, namely MMP3, IL8, COX2, and PGE2. Decreased transcription of the PLA2G4A and COX2 genes in response to cPLA2α enzyme inhibition further suggest a self-reinforcing effect of cPLA2α inhibition in response to TNF. Collectively, these results support that cPLA2α is an attractive therapeutic target candidate as its inhibition reduces the production of multiple key pro-inflammatory factors involved in RA pathogenesis.
doi:10.1371/journal.pone.0083555
PMCID: PMC3861525  PMID: 24349530
8.  ANTI-INFLAMMATORY PROPERTIES OF PROSTAGLANDIN E2: DELETION OF MICROSOMAL PROSTAGLANDIN E SYNTHASE-1 EXACERBATES NON-IMMUNE INFLAMMATORY ARTHRITIS IN MICE 
SUMMARY
Prostanoids and PGE2 in particular have been long viewed as one of the major mediators of inflammation in arthritis. However, experimental data indicate that PGE2 can serve both pro- and anti-inflammatory functions. We have previously shown (Kojima, F. et al. 2008 J. Immunol. 180, 8361-8368) that microsomal prostaglandin E synthase-1 (mPGES-1) deletion, which regulates PGE2 production, resulted in the suppression of collagen-induced arthritis (CIA) in mice. This suppression was attributable, at least in part, to the impaired generation of type II collagen autoantibodies. In order to examine the function of mPGES-1 and PGE2 in a non-autoimmune form of arthritis, we used the collagen antibody-induced arthritis (CAIA) model in mice deficient in mPGES-1, thereby bypassing the engagement of the adaptive immune response in arthritis development. Here we report that mPGES-1 deletion significantly increased CAIA disease severity. The latter was associated with a significant (~3.6) upregulation of neutrophil, but not macrophage, recruitment to the inflamed joints. The lipidomic analysis of the arthritic mouse paws by quantitative liquid chromatography / tandem mass-spectrometry (LC/MS/MS) revealed a dramatic (~59-fold) reduction of PGE2 at the peak of arthritis. Altogether, this study highlights mPGES-1 and its product PGE2 as important negative regulators of neutrophil-mediated inflammation and suggests that specific mPGES-1 inhibitors may have differential effects on different types of inflammation. Furthermore, neutrophil-mediated diseases could be exacerbated by inhibition of mPGES-1.
doi:10.1016/j.plefa.2013.08.003
PMCID: PMC3897272  PMID: 24055573
Nanohybrids; Room-temperature phosphorescence(RTP); Sensor; Rutin
9.  Monoarticular antigen-induced arthritis leads to pronounced bilateral upregulation of the expression of neurokinin 1 and bradykinin 2 receptors in dorsal root ganglion neurons of rats 
Arthritis Research  2000;2(5):424-427.
This study describes the upregulation of neurokinin 1 and bradykinin 2 receptors in dorsal root ganglion (DRG) neurons in the course of antigen-induced arthritis (AIA) in the rat knee. In the acute phase of AIA, which was characterized by pronounced hyperalgesia, there was a substantial bilateral increase in the proportion of lumbar DRG neurons that express neurokinin 1 receptors (activated by substance P) and bradykinin 2 receptors. In the chronic phase the upregulation of bradykinin 2 receptors persisted on the side of inflammation. The increase in the receptor expression is relevant for the generation of acute and chronic inflammatory pain.
Introduction:
Ongoing pain and hyperalgesia (enhanced pain response to stimulation of the tissue) are major symptoms of arthritis. Arthritic pain results from the activation and sensitization of primary afferent nociceptive nerve fibres ('pain fibres') supplying the tissue (peripheral sensitization) and from the activation and sensitization of nociceptive neurons in the central nervous system (central sensitization). After sensitization, nociceptive neurons respond more strongly to mechanical and thermal stimulation of the tissue, and their activation threshold is lowered. The activation and sensitization of primary afferent fibres results from the action of inflammatory mediators such as bradykinin (BK), prostaglandins and others on membrane receptors located on these neurons. BK is a potent pain-producing substance that is contained in inflammatory exudates. Up to 50% of the primary afferent nerve fibres have receptors for BK. When primary afferent nerve fibres are activated they can release neuropeptides such as substance P (SP) and calcitonin gene-related peptide from their sensory endings in the tissue. SP contributes to the inflammatory changes in the innervated tissue (neurogenic inflammation), and it might also support the sensitization of nociceptive nerve fibres by binding to neurokinin 1 (NK1) receptors. NK1 receptors are normally expressed on a small proportion of the primary afferent nerve fibres.
Aims:
Because the expression of receptors on the primary afferent neurons is essential for the pain-producing action of inflammatory mediators and neuropeptides, we investigated in the present study whether the expression of BK and NK1 receptors on primary afferent neurons is altered during the acute and chronic phases of an antigen-induced arthritis (AIA). AIA resembles in many aspects the inflammatory process of human rheumatoid arthritis. Because peptide receptors are expressed not only in the terminals of the primary afferent units but also in the cell bodies, we removed dorsal root ganglia (DRGs) of both sides from control rats and from rats with the acute or chronic phase of AIA and determined, after short-term culture of the neurons, the proportion of DRG neurons that expressed the receptors in the different phases of AIA. We also characterized the inflammatory process and the nociceptive behaviour of the rats in the course of AIA.
Materials and methods:
In 33 female Lewis rats 10 weeks old, AIA was induced in the right knee joint. First the rats were immunized in two steps with methylated bovine serum albumin (m-BSA) emulsified with Freund's complete adjuvant, and heat-inactivated Bordetella pertussis. After immunization, m-BSA was injected into the right knee joint cavity to induce arthritis. The joint swelling was measured at regular intervals. Nociceptive (pain) responses to mechanical stimulation of the injected and the contralateral knee were monitored in the course of AIA. Groups of rats were killed at different time points after the induction of AIA, and inflammation and destruction in the knee joint were graded by histological examination. The DRGs of both sides were dissected from segments L1–L5 and C1–C7 from arthritic rats, from eight immunized rats without arthritis and from ten normal control rats. Excised DRGs were dissociated into single cells which were cultured for 18 h.
The expression of the receptors was determined by assessment of the binding of SP-gold or BK-gold to the cultured neurons. For this purpose the cells were slightly fixed. Binding of SP-gold or BK-gold was detected by using enhancement with silver and subsequent densitometric analysis of the relative grey values of the neurons. Displacement controls were performed with SP, the specific NK1 receptor agonist [Sar9, Met(O2)11]-SP, BK, the specific BK 1 (B1) receptor agonist D-Arg (Hyp3-Thi5,8-D-Phe7)-BK and the specific BK 2 (B2) receptor agonist (Des-Arg10)-Lys-BK.
Results:
The inflammatory process in the injected right knee joint started on the first day after induction of AIA and persisted throughout the observation period of 84 days (Fig. 1). The initial phase of AIA was characterized by strong joint swelling and a predominantly granulocytic infiltration of the synovial membrane and the joint cavity (acute inflammatory changes). In the later phases of AIA (10–84 days after induction of AIA) the joint showed persistent swelling, and signs of chronic arthritic alterations such as infiltration of mononuclear leucocytes, hyperplasia of synovial lining layer (pannus formation) and erosions of cartilage and bone were predominant. The contralateral knee joints appeared normal at all time points. Destruction was observed only in the injected knee but some proteoglycan loss was also noted in the non-injected, contralateral knee. In the acute and initial chronic phases of AIA (1–29 days) the rats showed mechanical hyperalgesia in the inflamed knee (limping, withdrawal response to gentle pressure onto the knee). In the acute phase (up to 9 days) a pain response was also seen when gentle pressure was applied to the contralateral knee.
Figure 2 displays the changes in the receptor expression in the DRG neurons during AIA. The expression of SP–gold-binding sites in lumbar DRG neurons (Fig. 2a) was substantially increased in the acute phase of arthritis. In untreated control rats (n = 5), 7.7 ± 3.8% of the DRG neurons from the right side and 10.0 ± 1.7% of the DRG neurons from the left side showed labelling with SP–gold. The proportion of SP–gold-labelled neurons in immunized animals without knee injection (n = 3) was similar. By contrast, at days 1 (n = 2 rats) and 3 (n = 5 rats) of AIA in the right knee, approximately 50% of the DRG neurons exhibited labelling with SP–gold, and this was seen both on the side of the injected knee and on the opposite side. At day 10 of AIA (n = 3 rats), 26.3 ± 6.1% of the ipsilateral DRG neurons but only 15.7 ± 0.6% of the contralateral neurons exhibited binding of SP–gold. At days 21 (n = 5 rats), 42 (n = 3 rats) and 84 (n = 5 rats) of AIA, the proportion of SP–gold-positive neurons had returned to the control values, although the arthritis, now with signs of chronic inflammation, was still present. Compared with the DRG neurons of the untreated control rats, the increase in the proportion of labelled neurons was significant on both sides in the acute phase (days 1 and 3) and the intermediate phase (day 10) of AIA (Mann–Whitney U-test). The size distribution of the neurons was similar in the DRG neurons of all experimental groups. Under all conditions and at all time points, SP–gold binding was found mainly in small and medium-sized (less than 700 μm2) neurons. In the cervical DRGs the expression of NK1 receptors did not change in the course of AIA. The binding of SP–gold to the neurons was suppressed by the coadministration of the specific NK1 receptor agonist [Sar9, Met(O2)11]–SP in three experiments, showing that SP–gold was bound to NK1 receptors.
The expression of BK–gold-binding sites in the lumbar DRG neurons showed also changes in the course of AIA, but the pattern was different (Fig. 2b). In untreated control rats (n = 5), 42.3 ± 3.1% of the DRG neurons of the right side and 39.6 ± 2.6% of the DRG neurons of the left side showed binding of BK–gold. At days 1 (n = 2 rats) and 3 (n = 5 rats) of AIA, approximately 80% of the DRG neurons on the side of the knee injection (ipsilateral) and approximately 70% on the opposite side were labelled. In comparison with the untreated control group, the increase in the proportion of labelled neurons was significant on both sides. The proportion of labelled neurons in the ipsilateral DRGs remained significantly increased in both the intermediate phase (day 10, n = 3 rats) and chronic phase (days 21, n = 5 rats, and 42, n = 3 rats) of inflammation. At 84 days after the induction of AIA (n = 5 rats), 51.0 ± 12.7% of the neurons showed an expression of BK–gold-binding sites and this was close to the prearthritic values. However, in the contralateral DRG of the same animals the proportion of BK–gold-labelled neurons declined in the intermediate phase (day 10) and chronic phase (days 21–84) of AIA and was not significantly different from the control value. Thus the increase in BK–gold-labelled neurons was persistent on the side where the inflammation had been induced, and transient on the opposite side. The size distribution of the DRG neurons of the different experimental groups was similar. In the cervical DRGs the expression of BK receptors did not change in the course of AIA. In another series of experiments, we determined the subtype(s) of BK receptor(s) that were expressed in DRGs L1–L5 in different experimental groups. In neither untreated control animals (n = 5) nor immunized rats without knee injection (n = 5) nor in rats at 3 days (n = 5) and 42 days (n = 5) of AIA was the binding of BK–gold decreased by the coadministration of BK–gold and the B1 agonist. By contrast, in these experimental groups the binding of BK–gold was suppressed by the coadministration of the B2 agonist. These results show that B2 receptors, but not B1 receptors, were expressed in both normal animals and in animals with AIA.
Discussion:
These results show that in AIA in the rat the expression of SP-binding and BK-binding sites in the perikarya of DRGs L1–L5 is markedly upregulated in the course of knee inflammation. Although the inflammation was induced on one side only, the initial changes in the binding sites were found in the lumbar DRGs of both sides. No upregulation of SP-binding or BK-binding sites was observed in the cervical DRGs. The expression of SP-binding sites was upregulated only in the first days of AIA, that is, in the acute phase, in which the pain responses to mechanical stimulation were most pronounced. By contrast, the upregulation of BK-binding sites on the side of AIA persisted for up to 42 days, that is, in the acute and chronic phase of AIA. Only the B2 receptor, not the B1 receptor, was upregulated. The coincidence of the enhanced expression of NK1 and BK receptors on sensory neurons and the pain behaviour suggests that the upregulation of these receptors is relevant for the generation and maintenance of arthritic pain.
In the acute phase of AIA, approximately 50% of the lumbar DRG neurons showed an expression of SP-binding sites. Because peptide receptors are transported to the periphery, the marked upregulation of SP-binding receptors probably leads to an enhanced density of receptors in the sensory endings of the primary afferent units. This will permit SP to sensitize more neurons under inflammatory conditions than under normal conditions. However, the expression of NK1 receptors was upregulated only in the acute phase of inflammation, suggesting that SP and NK1 receptors are less important for the generation of hyperalgesia in the chronic phase of AIA.
Because BK is one of the most potent algesic compounds, the functional consequence of the upregulation of BK receptors is likely to be of immediate importance for the generation and maintenance of inflammatory pain. The persistence of the upregulation of BK receptors on the side of inflammation suggests that BK receptors should be an interesting target for pain treatment in the acute and chronic phases. Only B2 receptors were identified in normal animals and in rats with AIA. This is surprising because previous pharmacological studies have provided evidence that, during inflammation, B1 receptors can be newly expressed.
Receptor upregulation in the acute phase of AIA was bilateral and almost symmetrical. However, hyperalgesia was much more pronounced on the inflamed side. It is most likely that receptors on the contralateral side were not readily activated because in the absence of gross inflammation the local concentration of the ligands BK and SP was probably quite low. We hypothesize that the bilateral changes in receptor expression are generated at least in part by mechanisms involving the nervous system. Symmetrical segmental changes can be produced only by the symmetrical innervation, involving either the sympathetic nervous system or the primary afferent fibres. Under inflammatory conditions, primary afferent fibres can be antidromically activated bilaterally in the entry zone of afferent fibres in the spinal cord, and it was proposed that this antidromic activation might release neuropeptides and thus contribute to neurogenic inflammation. Because both sympathetic efferent fibres and primary afferent nerve fibres can aggravate inflammatory symptoms, it is also conceivable that they are involved in the regulation of receptor expression in primary afferent neurons. A neurogenic mechanism might also have been responsible for the bilateral degradation of articular cartilage in the present study.
PMCID: PMC17819  PMID: 11056677
antigen-induced arthritis; bradykinin receptor; dorsal root ganglion neurons; neurokinin 1 receptor; pain
10.  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
11.  Defective Generation of a Humoral Immune Response Is Associated with a Reduced Incidence and Severity of Collagen-Induced Arthritis in Microsomal Prostaglandin E Synthase-1 Null Mice1 
Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase and specifically catalyzes the conversion of PGH2 to PGE2. The present study demonstrates the effect of genetic deletion of mPGES-1 on the developing immunologic responses and its impact on the clinical model of bovine collagen-induced arthritis. mPGES-1 null and heterozygous mice exhibited decreased incidence and severity of arthritis compared with wild-type mice in a gene dose-dependent manner. Histopathological examination revealed significant reduction in lining hyperplasia and tissue destruction in mPGES-1 null mice compared with their wild-type littermates. mPGES-1 deficient mice also exhibited attenuation of mechanical nociception in a gene dose-dependent manner. In addition, mPGES-1 null and heterozygous mice showed a marked reduction of serum IgG against type II collagen (CII), including subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3, compared with wild-type mice, which correlated with the reduction in observed inflammatory features. These results demonstrate for the first time that deficiency of mPGES-1 inhibits the development of collagen-induced arthritis, at least in part, by blocking the development of a humoral immune response against type II collagen. Pharmacologic inhibition of mPGES-1 may therefore impact both the inflammation and the autoimmunity associated with human diseases such as rheumatoid arthritis.
PMCID: PMC2435291  PMID: 18523303
12.  Therapeutic effect of cortistatin on experimental arthritis by downregulating inflammatory and Th1 responses 
Annals of the Rheumatic Diseases  2006;66(5):582-588.
Background
Rheumatoid arthritis is a chronic autoimmune disease of unknown aetiology characterised by chronic inflammation in the joints and subsequent destruction of the cartilage and bone.
Aim
To propose a new strategy for the treatment of arthritis based on the administration of cortistatin, a newly discovered neuropeptide with anti‐inflammatory actions.
Methods
DBA/1J mice with collagen‐induced arthritis were treated with cortistatin after the onset of disease, and the clinical score and joint histopathology were evaluated. Inflammatory response was determined by measuring the levels of various inflammatory mediators (cytokines and chemokines) in joints and serum. T helper cell type 1 (Th1)‐mediated autoreactive response was evaluated by determining the proliferative response and cytokine profile of draining lymph node cells stimulated with collagen and by assaying the content of serum autoantibodies.
Results
Cortistatin treatment significantly reduced the severity of established collagen‐induced arthritis, completely abrogating joint swelling and destruction of cartilage and bone. The therapeutic effect of cortistatin was associated with a striking reduction in the two deleterious components of the disease—that is, the Th1‐driven autoimmune and inflammatory responses. Cortistatin downregulated the production of various inflammatory cytokines and chemokines, decreased the antigen‐specific Th1‐cell expansion, and induced the production of regulatory cytokines, such as interleukin 10 and transforming growth factor β1. Cortistatin exerted its effects on synovial cells through both somatostatin and ghrelin receptors, showing a higher effect than both peptides protecting against experimental arthritis.
Conclusion
This work provides a powerful rationale for the assessment of the efficacy of cortistatin as a novel therapeutic approach to the treatment of rheumatoid arthritis.
doi:10.1136/ard.2006.062703
PMCID: PMC1954632  PMID: 17151007
13.  The Death Receptor 3–TNF-like protein 1A pathway drives adverse bone pathology in inflammatory arthritis 
The Journal of Experimental Medicine  2008;205(11):2457-2464.
Rheumatoid arthritis (RA) is a chronic inflammatory disease of synovial joints that is associated with cartilage and bone destruction. Death Receptor 3 (DR3), a tumor necrosis factor (TNF) receptor superfamily member, has recently been associated with the pathogenesis of RA. We demonstrate that absence of DR3 confers resistance to the development of adverse bone pathology in experimental antigen-induced arthritis (AIA). DR3ko mice exhibited a reduction in all histopathological hallmarks of AIA but, in particular, failed to develop subchondral bone erosions and were completely protected from this characteristic of AIA. In contrast, TNF-like protein 1A (TL1A), the ligand for DR3, exacerbated disease in a dose- and DR3-dependent fashion. Analysis of osteoclast number within AIA joint revealed a reduction in areas susceptible to bone erosion in DR3ko mice, whereas in vitro osteoclastogenesis assays showed that TL1A could directly promote osteoclastogenesis in mouse and man. Treatment with antagonistic anti-TL1A mAb protected animals in a systemic model of RA disease collagen-induced arthritis. We therefore conclude that the DR3–TL1A pathway regulates joint destruction in two murine models of arthritis and represents a potential novel target for therapeutic intervention in inflammatory joint disease.
doi:10.1084/jem.20072378
PMCID: PMC2571920  PMID: 18824582
14.  Therapeutic potential of human umbilical cord mesenchymal stem cells in the treatment of rheumatoid arthritis 
Arthritis Research & Therapy  2010;12(6):R210.
Introduction
Rheumatoid arthritis (RA) is a T-cell-mediated systemic autoimmune disease, characterized by synovium inflammation and articular destruction. Bone marrow mesenchymal stem cells (MSCs) could be effective in the treatment of several autoimmune diseases. However, there has been thus far no report on umbilical cord (UC)-MSCs in the treatment of RA. Here, potential immunosuppressive effects of human UC-MSCs in RA were evaluated.
Methods
The effects of UC-MSCs on the responses of fibroblast-like synoviocytes (FLSs) and T cells in RA patients were explored. The possible molecular mechanism mediating this immunosuppressive effect of UC-MSCs was explored by addition of inhibitors to indoleamine 2,3-dioxygenase (IDO), Nitric oxide (NO), prostaglandin E2 (PGE2), transforming growth factor β1 (TGF-β1) and interleukin 10 (IL-10). The therapeutic effects of systemic infusion of human UC-MSCs on collagen-induced arthritis (CIA) in a mouse model were explored.
Results
In vitro, UC-MSCs were capable of inhibiting proliferation of FLSs from RA patients, via IL-10, IDO and TGF-β1. Furthermore, the invasive behavior and IL-6 secretion of FLSs were also significantly suppressed. On the other hand, UC-MSCs induced hyporesponsiveness of T cells mediated by PGE2, TGF-β1 and NO and UC-MSCs could promote the expansion of CD4+ Foxp3+ regulatory T cells from RA patients. More importantly, systemic infusion of human UC-MSCs reduced the severity of CIA in a mouse model. Consistently, there were reduced levels of proinflammatory cytokines and chemokines (TNF-α, IL-6 and monocyte chemoattractant protein-1) and increased levels of the anti-inflammatory/regulatory cytokine (IL-10) in sera of UC-MSCs treated mice. Moreover, such treatment shifted Th1/Th2 type responses and induced Tregs in CIA.
Conclusions
In conclusion, human UC-MSCs suppressed the various inflammatory effects of FLSs and T cells of RA in vitro, and attenuated the development of CIA in vivo, strongly suggesting that UC-MSCs might be a therapeutic strategy in RA. In addition, the immunosuppressive activitiy of UC-MSCs could be prolonged by the participation of Tregs.
doi:10.1186/ar3187
PMCID: PMC3046518  PMID: 21080925
15.  PGE2 produced by the lung augments the effector phase of allergic inflammation 
Elevated PGE2 is a hallmark of most inflammatory lesions. This lipid mediator can induce the cardinal signs of inflammation, and the beneficial actions of non-steroidal anti-inflammatory drugs are attributed to inhibition of cyclooxygenase COX-1 and COX-2, enzymes essential in the biosynthesis of PGE2 from arachidonic acid. However, both clinical studies and rodent models suggest that, in the asthmatic lung, PGE2 acts to restrain the immune response and limit physiological change secondary to inflammation. To directly address the role of PGE2 in the lung, we examined the development of disease in mice lacking microsomal prostaglandin E synthase 1 (mPGES1), which converts COX-1/COX-2 derived PGH2 to PGE2. We show that mPGES1 determines PGE2 levels in the naïve lung and is required for increases in PGE2 after ovalbumin (OVA) induced allergy. While loss of either COX-1 or COX-2 increases the disease severity, surprisingly mPGES1 −/− mice show reduced inflammation. However, an increase in serum IgE is still observed in the mPGES1 −/− mice, suggesting that loss of PGE2 does not impair induction of a TH2 response. Furthermore, mPGES1 −/− mice expressing a transgenic OVA-specific T cell receptor are also protected, indicating that PGE2 acts primarily after challenge with inhaled antigen. PGE2 produced by the lung plays the critical role in this response, as loss of lung mPGES1 is sufficient to protect against disease. Together this supports a model in which mPGES1-dependent PGE2 produced by populations of cells native to the lung contributes to the effector phase of some allergic responses.
doi:10.4049/jimmunol.1101873
PMCID: PMC3324636  PMID: 22412193
16.  Effect of phospholipase A2 inhibitory peptide on inflammatory arthritis in a TNF transgenic mouse model: a time-course ultrastructural study 
Arthritis Research & Therapy  2004;6(3):R282-R294.
We evaluated the therapeutic effect of secretory phospholipase A2 (sPLA2)-inhibitory peptide at a cellular level on joint erosion, cartilage destruction, and synovitis in the human tumor necrosis factor (TNF) transgenic mouse model of arthritis. Tg197 mice (N = 18) or wild-type (N = 10) mice at 4 weeks of age were given intraperitoneal doses (7.5 mg/kg) of a selective sPLA2 inhibitory peptide, P-NT.II, or a scrambled P-NT.II (negative control), three times a week for 4 weeks. Untreated Tg197 mice (N = 10) were included as controls. Pathogenesis was monitored weekly for 4 weeks by use of an arthritis score and histologic examinations. Histopathologic analysis revealed a significant reduction after P-NT.II treatment in synovitis, bone erosion, and cartilage destruction in particular. Conspicuous ultrastructural alterations seen in articular chondrocytes (vacuolated cytoplasm and loss of nuclei) and synoviocytes (disintegrating nuclei and vacuoles, synovial adhesions) of untreated or scrambled-P-NT.II-treated Tg197 mice were absent in the P-NT.II-treated Tg197 group. Histologic scoring and ultrastructural evidence suggest that the chondrocyte appears to be the target cell mainly protected by the peptide during arthritis progression in the TNF transgenic mouse model. This is the first time ultrastructural evaluation of this model has been presented. High levels of circulating sPLA2 detected in untreated Tg197 mice at age 8 weeks of age were reduced to basal levels by the peptide treatment. Attenuation of lipopolysaccharide- and TNF-induced release of prostaglandin E2 from cultured macrophage cells by P-NT.II suggests that the peptide may influence the prostaglandin-mediated inflammatory response in rheumatoid arthritis by limiting the bioavailability of arachidonic acid through sPLA2 inhibition.
doi:10.1186/ar1179
PMCID: PMC416452  PMID: 15142275
peptide; secretory phospholipase A2 inhibition; rheumatoid arthritis; TNF transgenic mouse model; ultrastructural alterations
17.  RANKL synthesized by articular chondrocytes contributes to juxta-articular bone loss in chronic arthritis 
Arthritis Research & Therapy  2012;14(3):R149.
Introduction
The receptor activator nuclear factor-kappaB ligand (RANKL) diffuses from articular cartilage to subchondral bone. However, the role of chondrocyte-synthesized RANKL in rheumatoid arthritis-associated juxta-articular bone loss has not yet been explored. This study aimed to determine whether RANKL produced by chondrocytes induces osteoclastogenesis and juxta-articular bone loss associated with chronic arthritis.
Methods
Chronic antigen-induced arthritis (AIA) was induced in New Zealand (NZ) rabbits. Osteoarthritis (OA) and control groups were simultaneously studied. Dual X-ray absorptiometry of subchondral knee bone was performed before sacrifice. Histological analysis and protein expression of RANKL and osteoprotegerin (OPG) were evaluated in joint tissues. Co-cultures of human OA articular chondrocytes with peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with macrophage-colony stimulating factor (M-CSF) and prostaglandin E2 (PGE2), then further stained with tartrate-resistant acid phosphatase.
Results
Subchondral bone loss was confirmed in AIA rabbits when compared with controls. The expression of RANKL, OPG and RANKL/OPG ratio in cartilage were increased in AIA compared to control animals, although this pattern was not seen in synovium. Furthermore, RANKL expression and RANKL/OPG ratio were inversely related to subchondral bone mineral density. RANKL expression was observed throughout all cartilage zones of rabbits and was specially increased in the calcified cartilage of AIA animals. Co-cultures demonstrated that PGE2-stimulated human chondrocytes, which produce RANKL, also induce osteoclasts differentiation from PBMCs.
Conclusions
Chondrocyte-synthesized RANKL may contribute to the development of juxta-articular osteoporosis associated with chronic arthritis, by enhancing osteoclastogenesis. These results point out a new mechanism of bone loss in patients with rheumatoid arthritis.
doi:10.1186/ar3884
PMCID: PMC3446534  PMID: 22709525
18.  Urokinase-type plasminogen activator and arthritis progression: contrasting roles in systemic and monoarticular arthritis models 
Arthritis Research & Therapy  2010;12(5):R199.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/ar3171
PMCID: PMC2991036  PMID: 20973954
19.  Treatment with recombinant interferon-β reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis 
Arthritis Research & Therapy  2004;6(3):R239-R249.
We investigated the therapeutic potential and mechanism of action of IFN-β protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-β or saline for 7 days. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis. Proteoglycan depletion was determined by safranin O staining. Expression of cytokines, receptor activator of NF-κB ligand, and c-Fos was evaluated immunohistochemically. The IL-1-induced expression of IL-6, IL-8, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) was studied by ELISA in supernatant of RA and osteoarthritis fibroblast-like synoviocytes incubated with IFN-β. We also examined the effect of IFN-β on NF-κB activity. IFN-β, at 0.25 μg/injection and higher, significantly reduced disease severity in two experiments, each using 8–10 mice per treatment group. IFN-β-treated animals displayed significantly less cartilage and bone destruction than controls, paralleled by a decreased number of positive cells of two gene products required for osteoclastogenesis, receptor activator of NF-κB ligand and c-Fos. Tumor necrosis factor α and IL-6 expression were significantly reduced, while IL-10 production was increased after IFN-β treatment. IFN-β reduced expression of IL-6, IL-8, and GM-CSF in RA and osteoarthritis fibroblast-like synoviocytes, correlating with reduced NF-κB activity. The data support the view that IFN-β is a potential therapy for RA that might help to diminish both joint inflammation and destruction by cytokine modulation.
doi:10.1186/ar1165
PMCID: PMC416442  PMID: 15142270
antibodies; cytokines; inflammation; rheumatoid arthritis
20.  Feedback Control of the Arachidonate Cascade in Osteoblastic Cells by 15-deoxy-Δ12,14-Prostaglandin J2 
15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and an anti-diabetic thiazolidinedione, troglitazone (TRO) are peroxisome proliferator-activated receptor (PPAR)-γ ligands, which regulate immuno-inflammatory reactions as well as adipocyte differentiation. We previously reported that 15d-PGJ2 can suppress interleukin (IL)-1β-induced prostaglandin E2 (PGE2) synthesis in synoviocytes of rheumatoid arthritis (RA). IL-1 also stimulates PGE2 synthesis in osteoblasts by regulation of cyclooxygenase (COX)-2 and regulates osteoclastic bone resorption in various diseases such as RA and osteoporosis. In this study, we investigated the feedback mechanism of the arachidonate cascade in mouse osteoblastic cells, MC3T3-E1 cells, which differentiate into mature osteoblasts. Treatment with 15d-PGJ2 led to a significant increase in IL-1α-induced COX-2 expression and PGE2 production in a dose dependent manner. The effect of 15d-PGJ2 was stronger than that of TRO. However, it did not affect the expression of COX-1. In addition, cell viability of MC3T3-E1 cells was not changed in the condition we established. This means that 15d-PGJ2 exerts a positive feedback regulation of the arachidonate cascade of PGE2 in osteoblastic cells. These results may provide important information about the pathogenesis and treatment of bone resorption in a variety of diseases such as RA and osteoporosis.
doi:10.3164/jcbn.2008011
PMCID: PMC2212349  PMID: 18231633
15d-PGJ2; PPAR-γ; osteoblast; PGE2; COX-2
21.  Consumption of Hydrolyzable Tannins Rich Pomegranate Extract (POMx) Suppresses Inflammation and Joint Damage In Rheumatoid Arthritis 
Objective
Although consumption of dietary supplements containing pomegranate extract by arthritis patients is on the rise, efficacy of such preparations in suppressing joint inflammation and damage is not known. The present study was designed to evaluate a standardized preparation of pomegranate extract (POMx) using collagen-induced arthritis in mice (CIA)-a widely used animal model of rheumatoid arthritis (RA).
Methods
CIA susceptible DBA/1 mice were fed POMx by gavage before and after immunization with chicken type-II collagen (CII). Severity of clinical arthritis was scored using a visual scoring system. Arthritic joints were analyzed by histopathology and graded. Lysates were generated from mouse joints and the levels of anti-type-II collagen IgG and inflammatory cytokines IL-1β, IL-6 and TNF-α were quantified by ELISA. Effect of POMx on LPS-induced NO production was determined by Griess reaction and MAP kinase activation was studied by Western immunoblotting in mouse macrophages.
Results
Consumption of POMx potently delayed the onset and reduced the incidence of CIA in mice. Severity of arthritis was also significantly lower in POMx -fed animals. Histopathology of the arthritic joints from POMx-fed mice demonstrated reduced joint infiltration by the inflammatory cells and the destruction of bone and cartilage were alleviated. Levels of IL-6 were significantly decreased in the joints of POMx-fed mice with CIA. In mouse macrophages, POMx abrogated multiple signal transduction pathways and downstream mediators implicated in RA pathogenesis.
Conclusions
Our studies suggest that inhibition of a spectrum of signal transduction pathways and the downstream pathogenic cellular response by POMx or compounds derived from it may be a useful approach for the prevention of onset and severity of inflammatory arthritis.
doi:10.1016/j.nut.2008.03.013
PMCID: PMC2577876  PMID: 18490140
Pomegranate; Collagen-induced arthritis; Rheumatoid Arthritis; IL-6; TNF-α
22.  Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo 
The FASEB Journal  2013;27(6):2328-2341.
The potent inflammatory mediator prostaglandin E2 (PGE2) is implicated in the pathogenesis of several chronic inflammatory conditions, including periodontitis. The inducible enzyme microsomal prostaglandin E synthase-1 (mPGES-1), catalyzing the terminal step of PGE2 biosynthesis, is an attractive target for selective PGE2 inhibition. To identify mPGES-1 inhibitors, we investigated the effect of aminothiazoles on inflammation-induced PGE2 synthesis in vitro, using human gingival fibroblasts stimulated with the cytokine IL-1β and a cell-free mPGES-1 activity assay, as well as on inflammation-induced bone resorption in vivo, using ligature-induced experimental periodontitis in Sprague-Dawley rats. Aminothiazoles 4-([4-(2-naphthyl)-1,3-thiazol-2-yl]amino)phenol (TH-848) and 4-(3-fluoro-4-methoxyphenyl)-N-(4-phenoxyphenyl)-1,3-thiazol-2-amine (TH-644) reduced IL-1β-induced PGE2 production in fibroblasts (IC50 1.1 and 1.5 μM, respectively) as well as recombinant mPGES-1 activity, without affecting activity or expression of the upstream enzyme cyclooxygenase-2. In ligature-induced experimental periodontitis, alveolar bone loss, assessed by X-ray imaging, was reduced by 46% by local treatment with TH-848, compared to vehicle, without any systemic effects on PGE2, 6-keto PGF1α, LTB4 or cytokine levels. In summary, these results demonstrate that the aminothiazoles represent novel mPGES-1 inhibitors for inhibition of PGE2 production and reduction of bone resorption in experimental periodontitis, and may be used as potential anti-inflammatory drugs for treatment of chronic inflammatory diseases, including periodontitis.—Kats, A., Båge, T., Georgsson, P., Jönsson, J., Quezada, H. C., Gustafsson, A., Jansson, L., Lindberg, C., Näsström, K., Yucel-Lindberg, T. Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo.
doi:10.1096/fj.12-214445
PMCID: PMC3659347  PMID: 23447581
cyclooxygenase-2; gingival fibroblasts; interleukin-1β; PGE2; mPGES-1 inhibitor; anti-inflammatory
23.  Effects of antibody to receptor activator of nuclear factor κ-B ligand on inflammation and cartilage degradation in collagen antibody-induced arthritis in mice 
Background
Rheumatoid arthritis (RA) is an inflammatory disease that leads to destruction of both articular cartilage and bone tissues. In rheumatic joints, synoviocytes and T-lymphocytes as well as bone cells produce the receptor activator of nuclear factor κ-B (RANK) ligand (RANKL), which binds to RANK on the surface of osteoclasts and their precursor cells to induce differentiation and activation of osteoclasts. Hence, inhibition of RANKL may be a promising approach to suppress osteolysis in RA. On the other hand, RANKL production by lymphocytes indicates the possibility that its inhibition would be effective to suppress inflammation in RA. In addition, it has been reported that cathepsin K, a predominant cysteine protease in osteoclasts, is involved in cartilage destruction in RA model mice. Here, we evaluated the effects of an anti-RANKL antibody on inflammation in footpads and degradation of articular cartilage in RA model mice.
Results
We induced arthritis in mice by injection of anti-type II collagen antibodies and lipopolysaccharide (LPS). Inhibition of RANKL by an anti-RANKL antibody (OYC1, Oriental Yeast, Tokyo, Japan) was confirmed by increased bone volume in the metaphysis of tibias. Swelling in either limb until day 14 was seen in 5 of 6 mice injected with anti-collagen antibodies and LPS without treatment with OYC1, while that was seen in 4 of 5 mice treated with OYC1. The average arthritis scores on day 14 in those groups were 2.17 and 3.00, respectively, indicating that OYC1 did not ameliorate inflammation in the limbs. Histological analyses indicated that OYC1 does not protect articular cartilage from destruction in mice with arthritis.
Conclusions
Our present study failed to show the effectiveness of an anti-RANKL antibody to ameliorate inflammation in the limbs or protect articular cartilage from degradation in a collagen antibody-induced arthritis mouse model.
Electronic supplementary material
The online version of this article (doi:10.1186/s12952-014-0018-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12952-014-0018-0
PMCID: PMC4272563  PMID: 25495344
Rheumatoid arthritis; Collagen antibody-induced arthritis; RANKL; Antibody; Inflammation; Articular cartilage
24.  Differential regulation of c-Met signaling pathways for synovial cell function 
SpringerPlus  2014;3:554.
We previously demonstrated that blocking the hepatocyte growth factor (HGF) receptor, c-Met, using a HGF antagonist, NK4, inhibited arthritis in a rheumatoid arthritis (RA) model mice. In the present study, we investigated the role of c-Met signaling in synovial cell function. We demonstrated that synovial tissues from RA patients and MH7A cells, a human RA synovial cell line, expressed HGF and c-Met. HGF and c-Met expression in RA synovium was increased compared to osteoarthritis synovium suggesting increased c-Met signaling in RA synovial cells. The c-Met inhibitor, SU11274, inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated MH7A cells. MEK and PI3K inhibitors suppressed production of matrix metalloproteinase-3 (MMP-3), vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) by MH7A cells, suggesting that c-Met-MEK-ERK and c-Met-PI3K-AKT pathways are involved positively regulating MH7A cell function. Although SU11274 suppressed MMP-3 and VEGF production it enhanced PGE2 production by MH7A cells suggesting that negative regulation by c-Met signaling, independent of the MEK-ERK and PI3K-AKT pathways, is involved in PGE2 production. Blocking c-Met signaling may be therapeutically useful to inhibit angiogenesis and cartilage and bone destruction by inhibiting VEGF and MMP-3 production, while enhancing PGE2 production in synovial cells in RA.
doi:10.1186/2193-1801-3-554
PMCID: PMC4192143  PMID: 25332857
c-Met; Hepatocyte growth factor; Rheumatoid arthritis; Synovial cell
25.  A proinflammatory role for Fas in joints of mice with collagen-induced arthritis 
Arthritis Research & Therapy  2004;6(5):R404-R414.
Collagen-induced arthritis (CIA) is a chronic inflammatory disease bearing all the hallmarks of rheumatoid arthritis, e.g. polyarthritis, synovitis, and subsequent cartilage/bone erosions. One feature of the disease contributing to joint damage is synovial hyperplasia. The factors responsible for the hyperplasia are unknown; however, an imbalance between rates of cell proliferation and cell death (apoptosis) has been suggested. To evaluate the role of a major pathway of cell death – Fas (CD95)/FasL – in the pathogenesis of CIA, DBA/1J mice with a mutation of the Fas gene (lpr) were generated. The susceptibility of the mutant DBA-lpr/lpr mice to arthritis induced by collagen type II was evaluated. Contrary to expectations, the DBA-lpr/lpr mice developed significantly milder disease than the control littermates. The incidence of disease was also significantly lower in the lpr/lpr mice than in the controls (40% versus 81%; P < 0.05). However DBA-lpr/lpr mice mounted a robust immune response to collagen, and the expression of local proinflammatory cytokines such as, e.g., tumor necrosis factor α (TNF-α) and IL-6 were increased at the onset of disease. Since the contribution of synovial fibroblasts to inflammation and joint destruction is crucial, the potential activating effect of Fas on mouse fibroblast cell line NIH3T3 was investigated. On treatment with anti-Fas in vitro, the cell death of NIH3T3 fibroblasts was reduced and the expression of proinflammatory cytokines TNF-α and IL-6 was increased. These findings suggest that impairment of immune tolerance by increased T-cell reactivity does not lead to enhanced susceptibility to CIA and point to a role of Fas in joint destruction.
doi:10.1186/ar1205
PMCID: PMC546278  PMID: 15380040
apoptosis; Fas; rheumatoid arthritis; tolerance

Results 1-25 (994199)