Use of multispectral magnetic resonance imaging analysis to quantify erosive changes in the hands of patients with rheumatoid arthritis: Short‐term and long‐term longitudinal studies

Objective

Magnetic resonance imaging (MRI) has been shown to be more sensitive than radiography for detecting bone erosions in rheumatoid arthritis (RA). Semiquantitative scoring based on visual image assessment has been introduced. However, there is considerable interest in true quantitative measures, particularly in the context of clinical trials designed to show differences between treatment groups. This study was undertaken to investigate the use of a new quantitative approach, multispectral (MS) image analysis, for assessing erosive change.

Methods

T1‐weighted spin‐echo (SE) and fat‐suppressed gradient‐echo (GE) sequences of metacarpophalangeal joints of the dominant hand were acquired at various time points throughout a 2‐year period. MS analysis was applied to all images, resulting in segmentation into a generalized bone and a soft tissue class. Voxel changes from one to the other class identified apparent bone lesion volume change (ΔBLV). MR images were also visually scored for erosions (E score). All analyses were performed separately, on a per‐joint basis, for short‐term and long‐term data sets.

Results

Analysis of variance with adjustment for individual effect revealed similar results in the short‐term and the long‐term studies, using either GE or SE images for visual assessment. Patients with an increase in E score on visual assessment had a significantly higher ΔBLV than those without.

Conclusion

Temporal MS analysis of MRIs can be used to detect and quantify erosive changes in RA. This semiautomated method may be useful for demonstrating differences between treatment groups in clinical trials.

     

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.

     

Magnetic resonance imaging in spondyloarthritis--how to quantify findings and measure response

Sensitive and reliable tools for monitoring disease activity and damage, and for prognostication, are essential in the management of patients with spondyloarthritis, including ankylosing spondylitis and psoriatic arthritis. Magnetic resonance imaging (MRI) allows direct visualisation of inflammation in peripheral and axial joints, and peripheral and axial entheses, and has dramatically improved the possibilities for early diagnosis and objective monitoring of the disease process in spondyloarthritis. Truthful, discriminative and feasible scoring systems are available for the assessment of inflammatory activity in the spine and sacroiliac joints in axial spondyloarthritis and in the hands of patients with peripheral psoriatic arthritis. Various systems for assessment of damage in axial and peripheral joints are available, but further studies are needed to document their value in clinical trials and clinical practice. The present article reviews key aspects of the status and recent important advances in MRI in spondyloarthritis, focussing on available MRI tools for assessing activity and damage in peripheral and, particularly, axial joints.     

Prostaglandin D2 regulates joint inflammation and destruction in murine collagen‐induced arthritis

Objective

Prostaglandin D₂ (PGD₂) may exert proinflammatory or antiinflammatory effects in different biologic systems. Although this prostanoid and the enzymes responsible for its synthesis are up‐regulated by interleukin‐1β (IL‐1β) in human chondrocytes in vitro, the role of PGD₂ in arthritis remains unclear. This study was undertaken to investigate the role of PGD₂ in the inflammatory response and in joint destruction during the development of collagen‐induced arthritis (CIA) in mice.

Methods

PGD₂ and cytokine levels in mice with CIA were determined by enzyme‐linked immunosorbent assay. Expression of hematopoietic PGD synthase (h‐PGDS), lipocalin‐type PGD synthase (l‐PGDS), and DP1 and DP2 receptors was analyzed by immunohistochemical methods. PGE₂ levels were determined by radioimmunoassay.

Results

The arthritic process up‐regulated the expression of h‐PGDS, l‐PGDS, DP1, and DP2 in articular tissue. PGD₂ was produced in the joint during the early phase of arthritis, and serum PGD₂ levels increased progressively throughout the arthritic process, reaching a maximum during the late stages of CIA. Treatment of arthritic mice with the DP1 antagonist MK0524 soon after the onset of disease increased the incidence and severity of CIA as well as the local levels of IL‐1β, CXCL‐1, and PGE₂, whereas IL‐10 levels were reduced. The administration of the DP2 antagonist CAY10595 did not modify the severity of arthritis. The injection of PGD₂ into the paw, as well as the administration of the DP1 agonist BW245C, significantly lowered the incidence of CIA, the inflammatory response, and joint damage.

Conclusion

Our findings indicate that PGD₂ is produced in articular tissue during the development of CIA and plays an antiinflammatory role, acting through the DP1 receptor.

     

Velocity vector imaging to quantify ventricular dyssynchrony and predict response to cardiac resynchronization therapy

Velocity vector imaging is a novel quantitative echocardiographic technique that was applied to routine grayscale echocardiographic images of 23 patients with heart failure who underwent cardiac resynchronization therapy. The hypothesis was tested that velocity vector imaging can quantify left ventricular mechanical dyssynchrony and predict response to resynchronization therapy. Tissue velocities were determined by the automated tracking of periodic B-mode image patterns on digital cine loops from standard apical 4-chamber, 2-chamber, and long-axis views, with the user tracing the mid left ventricular wall from a single frame. Dyssynchrony was determined as the greatest opposing wall peak longitudinal systolic velocity delay from the 3 views. Responders, defined as those with >or=15% increases in the ejection fraction, had greater baseline dyssynchrony than nonresponders (opposing wall velocity delays of 131 +/- 83 vs 52 +/- 60 ms, p <0.05), and >or=75 ms predicted response with 85% sensitivity and 80% specificity when followed 8 +/- 5 months after resynchronization therapy. Baseline electrocardiographic QRS duration was not predictive in the same patients. In conclusion, echocardiographic velocity vector imaging has potential for clinical utility.     

Plasmid encoding interleukin‐4 in the amelioration of murine collagen‐induced arthritis

Objective

To evaluate the therapeutic effect of the administration of plasmid encoding interleukin‐4 (IL‐4) via gene‐gun delivery and via intradermal injection on collagen‐induced arthritis (CIA).

Methods

IL‐4 plasmid was administered by gene‐gun delivery and intradermal injection to DBA/1 mice immunized with type II collagen (CII). The therapeutic effect on the development of CIA was evaluated clinically with a visual scoring method for arthritis and serologically by enzyme‐linked immunosorbent assays and polymerase chain reaction.

Results

Treatment with IL‐4–expressing plasmid significantly reduced the incidence and severity of CIA, including a reduction in the anti‐CII antibody level. In particular, gene‐gun delivery had a higher immunosuppressive effect on CIA compared with intradermal injection. As shown by in vitro stimulation assay, the spleen cells from mice immunized with CII and treated with IL‐4 plasmid via gene gun exhibited higher Th2 cytokine responses compared with cells treated with control plasmid after in vitro stimulation with CII.

Conclusion

The results of this study suggest that treatment with IL‐4 plasmid may constitute a new clinical use of cytokine gene therapy for rheumatoid arthritis.

     

IKKβ inhibition protects against bone and cartilage destruction in a rat model of rheumatoid arthritis

Objective

The IKK complex regulates NF‐κB activation, an important pathway implicated in the rheumatoid arthritis (RA) disease process. This study was undertaken to assess the efficacy of N‐(6‐chloro‐7‐methoxy‐9H‐β‐carbolin‐8‐yl)‐2‐methylnicotinamide (ML120B), a potent and selective small molecule inhibitor of IKKβ.

Methods

Polyarthritis was induced in rats by injection of Freund's complete adjuvant into the hind footpad. ML120B was administered orally twice daily, either prophylactically or therapeutically. Paw volumes and body weights were measured every 2–3 days throughout the study. We assessed bone erosions by several methods: histologic evaluation, quantitative micro–computed tomography (micro‐CT) imaging analysis, and measurement of type I collagen fragments in the serum. Quantitative polymerase chain reaction was used to evaluate expression of messenger RNA for genes related to inflammation and to bone and cartilage integrity.

Results

Oral administration of ML120B inhibited paw swelling in a dose‐dependent manner (median effective dosage 12 mg/kg twice daily) and offered significant protection against arthritis‐induced weight loss as well as cartilage and bone erosion. We were able to directly demonstrate that NF‐κB activity in arthritic joints was reduced after ML120B administration. Also, we observed that down‐regulation of the NF‐κB pathway via IKKβ inhibition dampened the chronic inflammatory process associated with rat adjuvant‐induced arthritis.

Conclusion

The results of the present study suggest that IKKβ inhibition is an effective therapeutic approach to treat both the inflammation and the bone/cartilage destruction observed in RA. Methods for the determination of serum markers for bone and cartilage destruction, as well as micro‐CT analysis, may aid in predicting and evaluating the therapeutic efficacy of IKKβ inhibition therapy in humans.

     

Immunosenescence in rheumatoid arthritis: Use of CD28 negative T cells to predict treatment response

Not many years ago achieving remission in rheumatoid arthritis (RA) was difficult due to lack of effective treatment. With the advent of biologics, remission is very much within reach. But biologics are expensive. And not all patients respond adequately to biologics. Hence it will be useful if we have a marker which predicts response to any disease modifying anti-rheumatic drug (DMARD), whether conventional or biologic. Expansion of CD28-ve T cells is characteristically seen in RA. Both CD28-ve T Cells and RA are believed to be linked to immunosenescence. The available evidence is suggestive of an intimate relationship between RA and clonal expansion of CD28-ve T cells. Newer biomarkers are constantly being looked at and CD28-ve T cells is one of them. In this review the relationship between immune disorders like RA and immunosenescence and significance of CD28-ve T cells in RA is discussed.     

Interleukin‐17 as a molecular target in immune‐mediated arthritis: Immunoregulatory properties of genetically modified murine dendritic cells that secrete interleukin‐4

Objective

Our previous studies have shown that murine dendritic cells (DCs) genetically modified to express interleukin‐4 (IL‐4) reduce the incidence and severity of murine collagen‐induced arthritis. The present studies were performed to assess the immunoregulatory mechanisms underlying this response, by assessing the effects of IL‐4 DCs on cytokine production by subsets of T helper cells.

Methods

Male DBA mice ages 6–8 weeks old were immunized with type II collagen. Splenic T cells obtained during the initiation phase and the end stage of arthritis were cultured with IL‐4 DCs or untransduced DCs in the presence of collagen rechallenge. Interferon‐γ (IFNγ) and IL‐17 responses were measured. Antibodies to IL‐4, IL‐12, and IL‐23, and recombinant IL‐4, IL‐12, and IL‐23 were used to further study the regulation of T cell cytokine production by IL‐4 DCs.

Results

Splenic T cells obtained during the initiation phase of arthritis produced less IL‐17 when cultured in the presence of IL‐4 DCs, despite their production of increased quantities of other proinflammatory cytokines (IFNγ and tumor necrosis factor). T cell IL‐17 production after collagen rechallenge was not inhibited by a lack of IL‐23, since IL‐4–mediated suppression of IL‐17 was not reconstituted by IL‐23, an otherwise potent inducer of IL‐17 production by T cells. Although IL‐4 DCs can produce increased quantities of IL‐12 and IFNγ, suppression of IL‐17 production by IL‐4 DCs was independent of both. While IL‐17 production by T cells obtained during the initiation phase of arthritis was regulated by IL‐4 DCs, IL‐17 production by T cells obtained during end‐stage arthritis was not altered.

Conclusion

Our data suggest that IL‐4 DCs exert a therapeutic effect on collagen‐induced arthritis by targeting IL‐17. IL‐17 suppression by IL‐4 DCs is robust and is not reversed by IL‐23. Timing might be important in IL‐17–targeted therapy, since IL‐17 production by T cells obtained during end‐stage arthritis did not respond to suppression by IL‐4 DCs.

     

Use of multispectral magnetic resonance imaging analysis to quantify erosive changes in the hands of patients with rheumatoid arthritis: short-term and long-term longitudinal studies

OBJECTIVE: Magnetic resonance imaging (MRI) has been shown to be more sensitive than radiography for detecting bone erosions in rheumatoid arthritis (RA). Semiquantitative scoring based on visual image assessment has been introduced. However, there is considerable interest in true quantitative measures, particularly in the context of clinical trials designed to show differences between treatment groups. This study was undertaken to investigate the use of a new quantitative approach, multispectral (MS) image analysis, for assessing erosive change. METHODS: T1-weighted spin-echo (SE) and fat-suppressed gradient-echo (GE) sequences of metacarpophalangeal joints of the dominant hand were acquired at various time points throughout a 2-year period. MS analysis was applied to all images, resulting in segmentation into a generalized bone and a soft tissue class. Voxel changes from one to the other class identified apparent bone lesion volume change (Delta BLV). MR images were also visually scored for erosions (E score). All analyses were performed separately, on a per-joint basis, for short-term and long-term data sets. RESULTS: Analysis of variance with adjustment for individual effect revealed similar results in the short-term and the long-term studies, using either GE or SE images for visual assessment. Patients with an increase in E score on visual assessment had a significantly higher Delta BLV than those without. CONCLUSION: Temporal MS analysis of MRIs can be used to detect and quantify erosive changes in RA. This semiautomated method may be useful for demonstrating differences between treatment groups in clinical trials.     

Overexpression of T‐bet gene regulates murine autoimmune arthritis

Objective

To clarify the role of T‐bet in the pathogenesis of collagen‐induced arthritis (CIA).

Methods

T‐bet–transgenic (Tg) mice under the control of the CD2 promoter were generated. CIA was induced in T‐bet–Tg mice and wild‐type C57BL/6 (B6) mice. Levels of type II collagen (CII)–reactive T‐bet and retinoic acid receptor–related orphan nuclear receptor γt (RORγt) messenger RNA expression were analyzed by real‐time polymerase chain reaction. Criss‐cross experiments using CD4+ T cells from B6 and T‐bet–Tg mice, as well as CD11c+ splenic dendritic cells (DCs) from B6 and T‐bet–Tg mice with CII were performed, and interleukin‐17 (IL‐17) and interferon‐γ (IFNγ) in the supernatants were measured by enzyme‐linked immunosorbent assay. CD4+ T cells from B6, T‐bet–Tg, or T‐bet–Tg/IFNγ^−/− mice were cultured for Th17 cell differentiation, then the proportions of cells producing IFNγ and IL‐17 were analyzed by fluorescence‐activated cell sorting.

Results

Unlike the B6 mice, the T‐bet–Tg mice did not develop CIA. T‐bet–Tg mice showed overexpression of Tbx21 and down‐regulation of Rorc in CII‐reactive T cells. Criss‐cross experiments with CD4+ T cells and splenic DCs showed a significant reduction in IL‐17 production by CII‐reactive CD4+ T cells in T‐bet–Tg mice, even upon coculture with DCs from B6 mice, indicating dysfunction of IL‐17–producing CD4+ T cells. Inhibition of Th17 cell differentiation under an in vitro condition favoring Th17 cell differentiation was observed in both T‐bet–Tg mice and T‐bet–Tg/IFNγ^−/− mice.

Conclusion

Overexpression of T‐bet in T cells suppressed the development of autoimmune arthritis. The regulatory mechanism of arthritis might involve dysfunction of CII‐reactive Th17 cell differentiation by overexpression of T‐bet via IFNγ‐independent pathways.

     

Cyclin‐dependent kinase inhibitor p21, via its C‐terminal domain,is essential for resolution of murine inflammatory arthritis

Objective

The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin‐dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21‐mediated suppression of experimental inflammatory arthritis.

Methods

Experimental arthritis was induced in wild‐type or p21^−/− (C57BL/6) mice, using the K/BxN serum–transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide‐induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex‐based assay, flow cytometry, or enzyme‐linked immunosorbent assay.

Results

Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21^−/− mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21‐intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages.

Conclusion

These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.

     

Joint damage and inflammation in c‐Jun N‐terminal kinase 2 knockout mice with passive murine collagen‐induced arthritis

Objective

Previous studies have demonstrated that inhibition of c‐Jun N‐terminal kinase (JNK) decreases joint destruction in the rat adjuvant arthritis model. The present study was undertaken to investigate whether selective loss of JNK‐2 function decreases joint destruction in JNK‐2 knockout mice, in order to determine the role of this isoform in inflammatory arthritis.

Methods

Passive collagen‐induced arthritis (CIA) was induced in Jnk2^−/− and wild‐type mice by administering anti–type II collagen antibodies. Arthritis was assessed daily using a semiquantitative clinical scoring system. Fibroblast‐like synoviocytes (FLS) were prepared from Jnk2^−/− and wild‐type mice, and JNK protein expression was determined by Western blot analysis. Matrix metalloproteinase 13 (MMP‐13) expression was determined by Northern blot analysis, and activator protein 1 (AP‐1) binding activity by electromobility shift assay (EMSA).

Results

The JNK protein level in Jnk2^−/− mice with CIA was 22% of that in wild‐type mice with CIA (P < 0.001), and mainly the 46‐kd isoform was expressed in the former group. Surprisingly, clinical arthritis was slightly more severe in the Jnk2^−/− mice. Histologic scores for synovial inflammation were not significantly different. However, Safranin O–stained sections from the Jnk2^−/− mice exhibited significantly less joint damage. Although joint destruction was decreased in Jnk2^−/− mice with CIA, EMSA and Northern blot analysis of total joint extracts revealed similar levels of AP‐1 binding and MMP‐13 expression in Jnk2^−/− and wild‐type mice. The lack of correlation with AP‐1 activity and MMP expression was probably because non‐FLS cells in the joint may express more JNK‐1 than do FLS.

Conclusion

JNK‐2 is a determinant of matrix degradation, but it has little effect on inflammation in arthritis. Complete inhibition of MMP expression and joint destruction will likely require combined JNK‐1 and JNK‐2 inhibition.