Rheumatoid arthritis synovial fluid macrophages express decreased tumor necrosis factor–related apoptosis‐inducing ligand R2 and increased decoy receptor tumor necrosis factor–related apoptosis‐inducing ligand R3

Objective

To characterize the expression pattern of tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) and its cognate receptors (TRAIL R1, R2, R3, and R4) on rheumatoid arthritis (RA) synovial fluid (SF) lymphocytes and monocyte/macrophages and on cultured RA synovial fibroblasts.

Methods

The expression of TRAIL and TRAIL receptors on RA SF lymphocytes and monocyte/macrophages, normal macrophages, and RA synovial fibroblasts was examined by flow cytometry with previously characterized monoclonal antibodies. The ability of adenoviral‐mediated delivery of TRAIL to induce macrophage or RA synovial fibroblast apoptosis was examined by flow cytometry.

Results

By flow cytometry, neither TRAIL nor its cognate receptors was detectable on RA SF lymphocytes or RA synovial fibroblasts. In contrast, RA SF macrophages expressed TRAIL R3, a decoy receptor (P < 0.01 versus isotype control), but not TRAIL, or TRAIL R1, R2, or R4. Normal peripheral blood–derived monocyte‐differentiated macrophages expressed TRAIL R2 (P < 0.01), but not TRAIL or the other TRAIL receptors. Adenoviral‐mediated delivery of TRAIL had no effect on the survival of normal macrophages or RA synovial fibroblasts but readily induced apoptosis in the prostate cancer cell line (PC‐3) that expressed TRAIL R1 and R2.

Conclusion

TRAIL R1 and R2, which are required for signal transmission by TRAIL, were not detected on RA SF lymphocytes, macrophages, or synovial fibroblasts. These observations do not support a potential therapeutic role for TRAIL in RA.

     

Rheumatoid arthritis synovial fluid fibroblasts express TRAIL‐R2 (DR5) that is functionally active

Objective

To examine fibroblasts grown from the synovial fluid of rheumatoid arthritis (RA) patients for TRAIL‐R2 expression, and for susceptibility to apoptosis induced by an agonistic anti–TRAIL‐R2 monoclonal antibody (mAb).

Methods

The expression of TRAIL‐R2 (DR5) was determined by flow cytometry on fibroblasts grown from the synovial fluid of patients with RA, osteoarthritis (OA), seronegative arthritis, and unclassified monarthritis or oligoarthritis, and on fibroblasts from the synovial membrane of RA and OA patients. Susceptibility to apoptosis mediated by an agonistic anti–TRAIL‐R2 mAb was determined by alamar blue bioassay, fluorescence microscopy (annexin V/propidium iodide staining), and caspase activation.

Results

Fibroblasts grew from 35 of 50 RA synovial fluid samples, of which 26 were DR5+ (mean [±SD] fluorescence intensity [MFI] 18.74 ± 2.5). Fibroblasts also grew from 15 of 30 seronegative arthritis synovial fluid samples, 28 of 40 OA synovial fluid samples, and 8 of 20 unclassified monarthritis or oligoarthritis synovial fluid samples; all of these were DR5− (MFI 0.32 ± 0.02). All 10 of the fibroblast lines from joint replacement surgery or synovectomy specimens of RA patients were DR5+ (MFI 20.3 ± 3.2). All fibroblast lines from the synovium of 10 OA patients were DR5−, as were fibroblasts from the skin of 5 healthy subjects. DR5+ fibroblast cultures underwent apoptosis when treated in vitro with an agonistic anti‐DR5 antibody.

Conclusion

Fibroblasts grown from the synovial fluid and synovial membrane of RA patients express TRAIL‐R2 that is functionally active. An agonistic anti–TRAIL‐R2 antibody that does not induce hepatocyte toxicity may be an alternative strategy for treatment of RA.

     

Exacerbation of antigen‐induced arthritis in inducible nitric oxide synthase–deficient mice

Objective

Inhibition of nitric oxide (NO) produced by inducible NO synthase (iNOS) is suggested to be beneficial in experimental arthritis. Although NO is important for the integrity of the microcirculation, the effects of inhibition of iNOS on the synovial microcirculation are not currently known. This study investigated the synovial microcirculation and leukocyte–endothelial cell interactions in iNOS‐deficient mice with antigen‐induced arthritis (AIA) and compared these findings with disease severity.

Methods

Fourteen homozygous iNOS−/− and 14 iNOS+/+ mice were used. The severity of AIA was assessed by measuring knee joint swelling and by histologic scoring. The number of rolling and adherent leukocytes was quantitatively analyzed in synovial microvessels using intravital microscopy of intraarticular synovial tissue. Nitrite/nitrate concentrations were measured, and the expression of iNOS, E‐ and P‐selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 (VCAM‐1) was assessed by immunohistochemistry.

Results

In iNOS+/+ animals with AIA, the plasma concentration of nitrite/nitrate was increased 3‐fold and iNOS expression was detected in cells of the joint. Swelling of the knee joint as well as leukocyte infiltration were enhanced in the iNOS−/− arthritic animals compared with iNOS+/+ mice with AIA. AIA‐associated leukocyte–endothelial cell interaction in synovial postcapillary venules was more pronounced in iNOS−/−, compared with iNOS+/+, arthritic mice. A strong expression of P‐selectin and VCAM‐1 was observed in the iNOS−/− arthritic mice only.

Conclusion

These data suggest that NO production by iNOS in vivo has antiinflammatory effects in experimental arthritis, by mediating a reduction in leukocyte adhesion and infiltration.

     

Suppression of leukocyte infiltration and cartilage degradation by selective inhibition of pre–B cell colony‐enhancing factor/visfatin/nicotinamide phosphoribosyltransferase: Apo866‐mediated therapy in human fibroblasts and murine collagen‐induced arthritis

Objective

To assess the ability of pre–B cell colony‐enhancing factor (PBEF) to regulate inflammation and degradative processes in inflammatory arthritis, using the small molecule inhibitor APO866 in human fibroblasts in vitro and in murine collagen‐induced arthritis (CIA).

Methods

Enzyme‐linked immunosorbent assays were used to examine regulation of expression of metalloproteinases and chemokines in human fibroblasts. The role of PBEF was further examined using APO866 in mice with CIA, with effects on disease activity assessed using radiography, histology, in vivo imaging, and quantitative polymerase chain reaction (qPCR).

Results

In vitro activation of human fibroblasts with PBEF promoted expression of matrix metalloproteinase 3 (MMP‐3), CCL2, and CXCL8, an effect inhibited by APO866. In mice with CIA, early intervention with APO866 inhibited synovial inflammation, including chemokine‐directed leukocyte infiltration, and reduced a systemic marker of inflammation, serum hyaluronic acid. APO866 blockade led to reduced expression of MMP‐3 and MMP‐13 in joint extracts and to a reduction in a systemic marker of cartilage erosion, serum cartilage oligomeric matrix protein. Radiologic images revealed that APO866 protected against bone erosion, while qPCR demonstrated inhibition of RANKL expression. In mice with established disease, APO866 reduced synovial inflammation and cartilage destruction, and halted bone erosion. In addition, APO866 reduced the activity of MMP‐3, CCL2, and RANKL in vivo, and inhibited production of CCL2 and RANKL in synovial explants from arthritic mice, a result that was reversed with nicotinamide mononucleotide.

Conclusion

These findings confirm PBEF to be an important regulator of inflammation, cartilage catabolism, and bone erosion, and highlight APO866 as a promising therapeutic agent for targeting PBEF activity in inflammatory arthritis.

     

Mechanisms and clinical relevance of TRAIL‐triggered responses in the synovial fibroblasts of patients with rheumatoid arthritis

Objective

Results of studies in mice suggest a protective role for TRAIL in arthritis. The aim of this study was to investigate the role of TRAIL in patients with rheumatoid arthritis (RA).

Methods

In the present study, we compared RA fibroblast‐like synoviocytes (FLS) that were resistant or sensitive to TRAIL‐induced apoptosis and the expression of TRAIL receptors in these cells, and also investigated the clinical features of the patients from whom the FLS were derived. Furthermore, we evaluated the levels of TRAIL and its soluble decoy receptor osteoprotegerin (OPG) in patients with RA, patients with osteoarthritis (OA), and patients with spondylarthritis (SpA).

Results

Sensitivity to TRAIL‐induced apoptosis varied in FLS from different patients, and the severity of disease in patients with RA was inversely correlated with the susceptibility of their FLS to TRAIL‐induced apoptosis. TRAIL‐sensitive cells expressed significantly lower levels of TRAILR‐1, and silencing of TRAILR‐1 increased TRAIL‐induced apoptosis in RA FLS. TRAIL levels were elevated in the arthritic joints of patients with established RA, and TRAIL levels in the synovial fluid of these patients were elevated compared with levels in the synovial fluid of patients with OA or SpA. At baseline, a low OPG‐to‐TRAIL ratio in the sera of patients with early RA was associated with a better evolution of disease activity, but high serum levels of TRAIL at followup were associated with joint damage.

Conclusion

These findings suggest that TRAIL has a dual role in RA, and that the resistance of RA FLS to TRAIL‐induced apoptosis is associated with a disease‐promoting activity of TRAIL in RA.

     

Placental immunomodulator ferritin,a novel immunoregulator,suppresses experimental arthritis

Objective

To determine the effect of treatment with C48, the recombinant cytokine‐like domain of the novel human placental immunomodulator ferritin (PLIF) immunoregulator, on zymosan‐induced arthritis (ZIA) in mice and on adjuvant‐induced arthritis (AIA) in rats.

Methods

The in vitro effect of PLIF/C48 was tested in mixed lymphocyte cultures (MLCs) of allogeneic mouse splenocytes. Arthritis was induced by intraarticular injection of zymosan into naive mice and by subcutaneous injection of Mycobacterium tuberculosis into rats. C48 was injected intraperitoneally daily from day 3 to day 9 or from day 7 to day 13 after induction of synovitis by zymosan, and every other day from day 2 to day 14 after induction of AIA. Swelling of the joints and histologic features of the synovium were assessed. Th1 and Th2 cytokines were quantified by enzyme‐linked immunosorbent assay.

Results

Both PLIF and C48 significantly inhibited the in vitro immunoreactivity of mouse splenocytes in MLCs. Treatment of ZIA mice and AIA rats with C48 effectively reduced joint swelling. C48 treatment reduced synovial lining thickening, numbers of mononuclear cells and histiocytes, as well as cartilage destruction and bone erosions. In vitro, activated splenocytes from C48‐treated ZIA and AIA animals produced significantly higher levels of interleukin‐10 (IL‐10). In animals with ZIA, this was accompanied by lower levels of tumor necrosis factor and IL‐2.

Conclusion

Human PLIF and C48 were shown to exert cross‐species immunosuppressive activity in vitro. The in vivo suppression of articular inflammation in the experimental models of ZIA and AIA was the result of treatment with the antiinflammatory human C48. These results suggest that treatment with C48 may offer an effective immunotherapeutic means of controlling inflammatory polyarthritis.

     

Selective therapeutic control of C5a and the terminal complement complex by anti‐C5 single‐chain Fv in an experimental model of antigen‐induced arthritis in rats

Objective

To determine the role of the terminal complement complex (TCC) in the development of experimental antigen‐induced arthritis (AIA) and the therapeutic effects of human anti‐C5 single‐chain Fv (scFv).

Methods

Two different anti‐C5 scFv, one that inhibits both release of C5a and assembly of the TCC (TS‐A 12/22) and another that selectively blocks formation of the TCC (TS‐A 8), were injected at the onset of AIA. The effects of these scFv on disease severity were evaluated for up to 21 days and compared with the effects of injection of an unrelated scFv. AIA was also established in C6‐deficient and C6‐sufficient PVG rats to obtain further information on the role of the TCC in this model.

Results

TS‐A 12/22 and TS‐A 8 proved to be equally effective in reducing joint swelling, cell counts and tumor necrosis factor α levels in synovial lavage fluids, and the degree of histomorphologic changes compared with the effects of the unrelated scFv. TS‐A 12/22 and TS‐A 8 prevented the deposition of C9 but not that of C3, confirming the ability of the 2 scFv to neutralize C5. Administration of the 2 anti‐C5 scFv after AIA onset also reduced disease severity. In C6‐deficient rats with AIA, disease activity was reduced markedly compared with that in C6‐sufficient rats.

Conclusion

These 2 human anti‐C5 scFv could represent potential therapeutic reagents to be used in patients with rheumatoid arthritis. In addition, the finding that TS‐A 8 was as effective as TS‐A 12/22 in reducing disease severity suggests that the TCC is mainly responsible for the joint inflammation and damage observed in AIA.

     

Chemokine receptor expression in rat adjuvant‐induced arthritis

Objective

Chemokine receptors mediate leukocyte migration into inflamed rheumatoid arthritis (RA) synovial tissue (ST). Knowledge of their distribution is crucial for understanding the evolution of the inflammatory process. In this study, we used rat adjuvant‐induced arthritis (AIA), a model for RA, to define the temporospatial expression of chemokine receptors.

Methods

ST from rats with AIA was immunostained, the percentage of cells expressing each receptor was determined, and findings were correlated with levels of inflammation. Chemokine receptor expression was evaluated on rat macrophages in vitro.

Results

CCR1, a receptor for macrophage inflammatory protein 1α (MIP‐1α)/CCL3 and RANTES/CCL5, exhibited high constitutive expression on macrophages in AIA. CCR5, binding MIP‐1α/CCL3 and RANTES/CCL5, was up‐regulated on ST macrophages during the course of AIA, correlating with macrophage expression of CCR2, a receptor for monocyte chemoattractant protein 1/CCL2. Endothelial cell (EC) CCR2 was down‐regulated as arthritis progressed, inversely correlating with inflammation. CCR3, another RANTES/CCL5 receptor, was constitutively high on macrophages in vivo and in vitro, with down‐regulation during AIA. CXCR4, a receptor for stromal cell–derived factor 1/CXCL12), was prominently up‐regulated on ECs, preceding the peak of inflammation.

Conclusion

These findings show that 1) constitutive expression of CCR1 on macrophages remains high during AIA; 2) CCR2 and CCR3 may play a role in initial recruitment of leukocytes to ST in AIA; 3) macrophage expression of CCR2 and CCR5 may be important for sustaining inflammatory changes; and 4) EC CXCR4 may be a harbinger of inflammatory changes. Our results may help guide chemokine receptor blockade–targeting treatment strategies in inflammatory arthritis.

     

Blocking ERK‐1/2 reduces tumor necrosis factor α–induced interleukin‐18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of interleukin‐18 binding protein A

Objective

To examine the mechanism of regulation of interleukin‐18 (IL‐18) bioactivity by IL‐18 binding protein (IL‐18BP) induction.

Methods

Levels of IL‐18 and IL‐18BPa in synovial fluid samples from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) were determined by enzyme‐linked immunosorbent assays (ELISAs), followed by calculation of free IL‐18. IL‐18 and IL‐18BPa synthesis in RA synovial fibroblasts that had been treated with proinflammatory and antiinflammatory cytokines were assessed by quantitative real‐time polymerase chain reaction and ELISA, respectively, followed by IL‐18 bioactivity determination using KG‐1 cells. Chemical signaling inhibitors were used for determination of the signal transduction pathways involved in IL‐18BPa/IL‐18 regulation. Tumor necrosis factor α (TNFα)–induced caspase 1 activity was determined by a colorimetric assay.

Results

IL‐18BPa was lower in RA synovial fluid than in OA synovial fluid (P < 0.05; n = 8), and free IL‐18 was higher in RA synovial fluid than in OA synovial fluid. TNFα induced RA synovial fibroblast IL‐18BPa and IL‐18 in a time‐dependent manner (P < 0.05). Evaluation of signaling pathways suggested that TNFα induced IL‐18 production through the ERK‐1/2, protein kinase Cδ (PKCδ), and Src pathways, whereas IL‐18BPa synthesis was mediated through the NFκB, PKC, Src, and JNK pathways. Furthermore, addition of exogenous IL‐18BPa‐Fc reduced the RA synovial fibroblast phosphorylation of ERK‐1/2 induced by TNFα.

Conclusion

These results suggest that IL‐18BPa reduces IL‐18 bioactivity induced by TNFα, by regulating the ERK‐1/2 pathway in RA synovial fibroblasts. Targeting IL‐18 bioactivity by induction or addition of IL‐18BPa may provide another therapeutic option in the management of RA.

     

In vivo imaging of experimental arthritis with near‐infrared fluorescence

Objective

To visualize early experimental arthritis with near‐infrared fluorescence (NIRF) imaging in a murine model of antigen‐induced arthritis (AIA).

Methods

The target of NIRF was the F4/80 antigen present on the surface of macrophages infiltrating the inflamed synovial membrane. Imaging was performed using anti‐F4/80 monoclonal antibodies (mAb) labeled with Cy5.5 fluorochrome. On day 7 of AIA, 6 mice received an intravenous (IV) injection of labeled mAb; control AIA mice (n = 6) received an IV injection of Cy5.5‐labeled isotype control antibody. NIRF imaging was performed before injection (baseline) and until 72 hours thereafter. Histologic evaluation of arthritis severity and immunohistochemical assessment of F4/80 antigen density were also performed on day 7.

Results

NIRF imaging showed an accumulation of fluorochrome probes in the inflamed knee joints and, to a lesser extent, in the contralateral (nonarthritic) knee joints. The signal induced by mAb F4/80 was clearly higher than that generated by the isotype control. Accumulation of fluorochrome probes in the joints was confirmed histologically by confocal laser scanning microscopy.

Conclusion

The use of fluorochromes allows imaging of arthritis in the near‐infrared range. Accumulation in the contralateral, nonarthritic knee joints can be explained by the presence of sentinel macrophages in normal synovium or by a mild contralateral response due to systemic activation or neurogenic mechanisms.

     

Evaluation of protease‐activated receptor 2 in murine models of arthritis

Objective

Protease‐activated receptor 2 (PAR‐2) activation has been linked to pro‐ and antiinflammatory cellular responses. We undertook this study to explore the importance of PAR‐2 activation in 4 murine models of arthritis and to analyze the expression of PAR‐2 in human arthritic synovium.

Methods

Zymosan‐induced arthritis (ZIA), K/BxN serum–induced arthritis, and Freund's complete adjuvant (CFA)–induced arthritis were generated in naive PAR‐2^−/− mice and PAR‐2^+/+ littermates. Antigen‐induced arthritis (AIA) was generated in immunized mice using methylated bovine serum albumin (mBSA). The severity of arthritis was assessed by clinical scoring, technetium uptake measurement, and histologic analysis. Immune responses to mBSA were also evaluated from AIA. The expression of PAR‐2 in synovial tissues from rheumatoid arthritis (RA) and osteoarthritis (OA) patients was compared.

Results

In AIA, arthritis was significantly decreased in PAR‐2–deficient mice and was associated with decreased levels of anti‐mBSA IgG antibodies and lymph node cell proliferation. No difference in arthritis severity was seen in mice with ZIA, K/BxN serum–induced arthritis, and CFA‐induced arthritis. Synovial biopsy specimens from RA patients demonstrated significantly increased expression of PAR‐2 compared with those from OA patients.

Conclusion

PAR‐2 deficiency was found to modulate articular inflammation in murine models of arthritis that require prior immunization and was associated with reduced levels of anti‐mBSA IgG and lymph node cell proliferation in AIA. Expression of PAR‐2 in RA synovium was significantly higher than that in OA synovium, and this suggests that PAR‐2 is implicated in the pathogenesis of immune‐mediated forms of arthritis.

     

Tumor necrosis factor α and RANKL blockade cannot halt bony spur formation in experimental inflammatory arthritis

Objective

To investigate the kinetics of bony spur formation and the relationship of bony spur formation to synovial inflammation and bone erosion in 2 rat arthritis models, and to address whether bony spur formation depends on the expression of tumor necrosis factor α (TNFα) or RANKL.

Methods

Analysis of the kinetics of synovial inflammation, bone erosion, osteoclast formation, and growth of bony spurs was performed in rat collagen‐induced arthritis (CIA) and adjuvant‐induced arthritis (AIA). In addition, inhibition experiments were performed to assess whether inhibition of TNFα and RANKL by pegylated soluble TNF receptor type I (pegTNFRI) and osteoprotegerin (OPG), respectively, affected bony spur formation.

Results

Bony spurs emerged from the periosteal surface close to joints, and initial proliferation of mesenchymal cells was noted as early as 3 days and 5 days after onset of CIA and AIA, respectively. Initiation of bony spur formation occurred shortly after the onset of inflammation and bone erosion. Neither pegTNFRI nor OPG could significantly halt the osteophytic responses in CIA and AIA.

Conclusion

These results suggest that bony spur formation is triggered by inflammation and initial structural damage in these rat models of inflammatory arthritis. Moreover, emergence of bony spurs depends on periosteal proliferation and is not affected by inhibition of either TNFα or RANKL. Bony spur formation can thus be considered a process that occurs independent of TNFα and RANKL and is triggered by destructive arthritis.

     

Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen‐induced arthritis

Objective

Annexin 1 (Anx‐1) is a putative mediator of the antiinflammatory actions of glucocorticoids (GCs). This study investigated the role of Anx‐1 in experimental arthritis and in GC‐mediated inhibition of inflammation, using antigen‐induced arthritis (AIA) in Anx‐1 knockout (Anx‐1^−/−) mice.

Methods

Arthritis was induced by intraarticular injection of methylated BSA (mBSA) in mice preimmunized with mBSA. Disease was assessed after 7 days by histologic examination of the knee joints. Serum levels of anti‐mBSA IgG were determined by enzyme‐linked immunosorbent assay. Cytokine messenger RNA (mRNA) expression was detected by real‐time polymerase chain reaction.

Results

A significant exacerbation of arthritis was observed in the Anx‐1^−/− mice compared with wild‐type (WT) mice. This was associated with increased mRNA expression of synovial interleukin‐1β, tumor necrosis factor α, interleukin‐6, and macrophage migration inhibitory factor. Dexamethasone significantly reduced the histologic severity of synovitis and bone damage in the WT mice, but exerted no inhibitory effects in the Anx‐1^−/− mice, and also significantly reduced the serum levels of anti‐mBSA IgG and the numbers of peripheral blood neutrophils and lymphocytes in WT mice, but had no such effect in Anx‐1^−/− mice.

Conclusion

Anx‐1 exerts endogenous antiinflammatory effects on AIA via the regulation of cytokine gene expression, and also mediates the antiinflammatory actions of dexamethasone in AIA.

     

Protective effect of RC‐3095, an antagonist of the gastrin‐releasing peptide receptor,in experimental arthritis

Objective

To evaluate the antiinflammatory effects of RC‐3095 in 2 experimental models of arthritis, collagen‐induced arthritis (CIA) and antigen‐induced arthritis (AIA), and to determine the mechanisms of action involved.

Methods

RC‐3095 was administered daily to mice with CIA and mice with AIA, after induction of disease with methylated bovine serum albumin. Disease incidence and severity were assessed using a clinical index and evaluation of histologic features, respectively. In mice with CIA, gastrin‐releasing peptide receptor (GRPR) was detected by immunohistochemical analysis, while in mice with AIA, migration of neutrophils, presence of glycosaminoglycans, and lymphocyte proliferation, determined using the MTT assay, were assessed. Expression of cytokines interleukin‐17 (IL‐17), IL‐1β, and tumor necrosis factor α (TNFα) was evaluated in all mouse knees using enzyme‐linked immunosorbent assay. Treg cell production was assessed by flow cytometry in the joints of mice with AIA.

Results

In mice with AIA, administration of RC‐3095 reduced neutrophil migration, mechanical hypernociception, and proteoglycan loss. These findings were associated with inhibition of the levels of all 3 proinflammatory cytokines, decreased lymphocyte proliferation, and increased Treg cell numbers. In the CIA model, treatment with RC‐3095 led to a significant reduction in arthritis clinical scores and the severity of disease determined histologically. Synovial inflammation, synovial hyperplasia, pannus formation, and extensive erosive changes were all dramatically reduced in the arthritic mice treated with RC‐3095. Furthermore, arthritic mice treated with RC‐3095 showed a significant reduction in the concentrations of IL‐17, IL‐1β, and TNFα, and showed a diminished expression of GRPR.

Conclusion

These findings suggest that the GRP pathway has a significant role in chronic arthritis, and its inhibition can be explored as a possible therapeutic strategy in rheumatoid arthritis.

     

Reduction of joint inflammation and bone erosion in rat adjuvant arthritis by treatment with interleukin‐17 receptor IgG1 Fc fusion protein

Objective

To investigate the role of interleukin‐17 (IL‐17) in inflammatory arthritis by blockade with an IL‐17 receptor/human IgG1 Fc fusion protein (muIL‐17R:Fc) in adjuvant‐induced arthritis (AIA) in the rat.

Methods

AIA was induced in 39 DA rats with the use of Freund's complete adjuvant. Rats received either 7.3 or 20 mg/kg of muIL‐17R:Fc or phosphate buffered saline intraperitoneally every other day from the time of arthritis induction for ∼17 days. Paw volume, arthritis severity, and weight were assessed every 3–4 days. Rats were killed between days 21 and 23 postinduction. Ankles were removed for quantitative radiology and histology and for immunohistochemistry for T cells.

Results

Treatment with muIL‐17R:Fc attenuated paw volume in a dose‐dependent manner. Both the 7.3 and 20 mg/kg doses of muIL‐17R:Fc significantly reduced radiographic scores in the treated rats compared with the controls. The 20 mg/kg dose of muIL‐17R:Fc significantly reduced histology scores compared with the controls. T cell numbers were unchanged in the muIL‐17R:Fc–treated rats as a function of dose.

Conclusion

In vivo blockade of IL‐17 by muIL‐17R:Fc treatment attenuated AIA and reduced joint damage, suggesting that IL‐17 plays an important role in the inflammation and joint destruction of AIA. IL‐17 may be a potential therapeutic target for inflammatory diseases in humans, such as rheumatoid arthritis.

     

Endothelial dysfunction in rat adjuvant‐induced arthritis: Up‐regulation of the vascular arginase pathway

Objective

To investigate whether arginase pathway abnormalities occur in vessels from rats with adjuvant‐induced arthritis (AIA), and to determine whether the up‐regulation of arginase, which reciprocally regulates nitric oxide synthase (NOS) by competing for the same substrate, L ‐arginine, contributes to endothelial dysfunction in AIA.

Methods

We performed vascular reactivity experiments on thoracic aortic rings from AIA rats and control rats, and we investigated the response of rings to norepinephrine (NE), sodium nitroprusside (SNP), and acetylcholine (ACh). ACh‐induced relaxation was evaluated in the presence (or not in the presence) of the NOS inhibitor N^G‐nitro‐L ‐arginine methyl ester (L‐NAME), the arginase inhibitor N^ω‐hydroxy‐nor‐L ‐arginine (nor‐NOHA), or both. Aortic arginase activity was measured using a spectrophotometric method, and the expression of arginase and endothelial NOS (eNOS) was evaluated by Western blotting.

Results

ACh‐induced vasodilation was significantly impaired in AIA rats, while the responses to NE and to SNP did not differ from those in control rats. L‐NAME reduced ACh‐induced vasodilation to a lesser extent in AIA rats than in control rats. Incubation of aortic rings with nor‐NOHA enhanced the vascular response to ACh in AIA rats and reversed the effects of L‐NAME. Compared with control rats, AIA rats exhibited increased vascular expression of arginase II (by 22%) (P < 0.05) as well as increased arginase activity (by 49%) (P < 0.05), whereas eNOS expression was unchanged. Finally, arginase activity and expression correlated positively with arthritis severity.

Conclusion

Our results are consistent with the notion that arginase up‐regulation plays a role in AIA‐associated endothelial dysfunction. They suggest that arginase might be an attractive new target for treating endothelial dysfunction in arthritis.

     

Additive bone‐protective effects of anabolic treatment when used in conjunction with RANKL and tumor necrosis factor inhibition in two rat arthritis models

Objective

To investigate whether the bone‐preserving effects of a RANKL antagonist or a tumor necrosis factor (TNF) antagonist could be further improved by the addition of a bone anabolic agent in inflammatory arthritis.

Methods

Lewis rats with either adjuvant‐induced arthritis (AIA) or collagen‐induced arthritis (CIA) were treated for 10 days with PEGylated soluble tumor necrosis factor receptor type I (PEG sTNFRI), interleukin‐1 receptor antagonist (IL‐1Ra), osteoprotegerin (OPG), parathyroid hormone (PTH), or combinations of these agents starting on day 4 after disease onset. Treatment effects were assessed clinically, radiologically, and histologically, and by morphometry for the extent of paw swelling, bone erosive changes, and synovial inflammation.

Results

Paw swelling and synovial inflammation were significantly inhibited by PEG sTNFRI in AIA and CIA, and by IL‐1Ra in CIA. OPG and PTH had no significant effect on these parameters. Analysis of bone erosion revealed a significant bone‐sparing effect of monotherapy with PEG sTNFRI or OPG in both models, whereas IL‐1Ra was only effective in CIA. PTH treatment alone did not show a bone‐protective effect in either model. With the combination of PEG sTNFRI and PTH, erosion scores (−74% in AIA and −61% in CIA versus controls) were significantly lower than those elicited by PEG sTNFRI alone (−41% and −29%, respectively, versus controls). Similar results were also obtained with the combination of OPG and PTH (−88% in AIA and −73% in CIA, compared with −70% and −55%, respectively, with OPG monotherapy). Coadministration of IL‐1Ra and PTH had no synergistic bone‐sparing effect. Morphometric analysis revealed that the addition of PTH to PEG sTNFRI or OPG resulted in higher bone volume and higher osteoblast numbers in both AIA and CIA.

Conclusion

The bone‐protective effects resulting from RANKL or TNF antagonism can be further improved by the addition of a bone anabolic agent.