Imaging and Methotrexate Response Monitoring of Systemic Inflammation in Arthritic Rats Employing the Macrophage PET Tracer [18F]Fluoro-PEG-Folate.

Background: In rheumatoid arthritis, articular inflammation is a hallmark of disease, while the involvement of extra-articular tissues is less well defined. Here, we examined the feasibility of PET imaging with the macrophage tracer [18F]fluoro-PEG-folate, targeting folate receptor ß (FRß), to monitor systemic inflammatory disease in liver and spleen of arthritic rats before and after methotrexate (MTX) treatment. Methods: [18F]Fluoro-PEG-folate PET scans (60 min) were acquired in saline- and MTX-treated (1 mg/kg, 4x) arthritic rats, followed by tissue resection and radiotracer distribution analysis. Liver and spleen tissues were stained for ED1/ED2-macrophage markers and FRß expression. Results: [18F]Fluoro-PEG-folate PET and ex vivo tissue distribution studies revealed a significant (p < 0.01) 2-fold lower tracer uptake in both liver and spleen of MTX-treated arthritic rats. Consistently, ED1- and ED2-positive macrophages were significantly (p < 0.01) decreased in liver (4-fold) and spleen (3-fold) of MTX-treated compared with saline-treated rats. Additionally, FRß-positive macrophages were also significantly reduced in liver (5-fold, p < 0.005) and spleen (3-fold, p < 0.01) of MTX- versus saline-treated rats. Conclusions: MTX treatment reduced activated macrophages in liver and spleen, as markers for systemic inflammation in these organs. Macrophage PET imaging with [18F]fluoro-PEG-folate holds promise for detection of systemic inflammation in RA as well as therapy (MTX) response monitoring.

Efficacy of an immunotoxin to folate receptor beta in the intra-articular treatment of antigen-induced arthritis.

INTRODUCTION: We previously demonstrated that synovial sublining macrophages express folate receptor beta (FRß). The aim of this study was to evaluate the efficacy of intra-articular administration of a recombinant immunotoxin to FRß for treating rat antigen-induced arthritis. METHODS: A monoclonal antibody (mAb) to rat FRß was produced by immunizing mice with B300-19 cells (murine pre-B cells) transfected with the rat FRß gene. Recombinant immunotoxin was prepared by conjugating the Fv portion of the anti-rat FRß mAb heavy chain with a truncated Pseudomonas exotoxin A and the Fv portion of the anti-rat FRß mAb light chain. Antigen-induced arthritis was induced through intra-articular injection of methylated bovine serum albumin (mBSA) after two subcutaneous injections of mBSA and complete Freund's adjuvant. Immunotoxin was intra-articularly injected into the arthritis joint every other day for seven days after arthritis onset. Joint swelling was measured and histological scores of inflammation, synovial thickness, cartilage, and bone destruction were determined. Immunohistochemistry was performed to detect osteoclast and osteoclast precursor FRß-expressing macrophages and cathepsin K-positive cells on day 21. RESULTS: Intra-articular administration of the immunotoxin attenuated joint swelling (61% suppression; P < 0.01 compared to the control on day 21) and improved histological findings, particularly cartilage and bone destruction (scores of rats treated with control versus the immunotoxin: 2.2 versus 0.5; P < 0.01), by reducing the number of FRß-expressing macrophages and cathepsin K-positive cells. CONCLUSIONS: Intra-articular administration of an immunotoxin to FRß is effective for improving rat antigen-induced arthritis.

Differential expression patterns of secreted frizzled related protein genes in synovial cells from patients with arthritis.

OBJECTIVE: To detect expression of the secreted frizzled related protein (sFRP) gene in synovial cells from patients with arthritis. METHODS: Expression of sFRP-1, 2, 3, 4, and 5 genes was detected in synovial cells from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. To identify synovial cell populations expressing sFRP-1, 3, and 4 genes, expression was compared in macrophage-rich populations and fibroblast-like cell-rich populations by RT-PCR. Levels of expression of these genes were also studied in activated peripheral blood mononuclear cells (PBMC) and activated skin fibroblasts. RESULTS: Expression of the sFRP-1, 3, and 4 genes was observed in both RA and OA synovial cells. sFRP-1 and 4 genes were expressed predominantly in fibroblast-like cell-rich populations, and the sFRP-3 gene was expressed predominantly in macrophage-rich populations. Levels of expression of sFRP-3 and 4 genes were elevated in activated PBMC and activated skin fibroblasts. CONCLUSION: Our findings suggest that sFRP-1, 3, and 4 may play different roles in the pathogenesis of synovitis.

Nitration and chlorination of folic acid by peroxynitrite and hypochlorous acid, and the selective binding of 10-nitro-folate to folate receptor beta.

The aim of this work was to characterize folates modified by ONOO(-) and HOCl and to evaluate the binding capacity of folates modified by ONOO(-) to folate receptor alpha and beta. For the modification of folate by ONOO(-), folic acid was reacted with the combination of PMA activated PMN and PAPA NONOate or chemically synthesized ONOO(-). For the modification of folate by HOCl, folic acid was reacted with the combination of MPO and H(2)O(2) or NaOCl. The structures of products were determined by 1H-NMR and MALDI-TOF mass. Nitrated folate species were identified as 10-nitro-folate and 12-nitro-folate, and chlorinated folate was identified as 12-chloro-folate. The 10-nitro-folate showed the selective binding to FR-beta, compared to folic acid.