Expression of 5-lipoxygenase and 15-lipoxygenase in rheumatoid arthritis synovium and effects of intraarticular glucocorticoids

The past years have witnessed tremendous progress in the treatment of rheumatoid arthritis, a chronic debilitating autoimmune disease mainly characterized by joint inflammation with progressive tissue destruction and loss of function. This condition affects 0.5-1% of the population, is associated with important co-morbidities and represents a heavy economical burden. New strategies, employing early and aggressive therapies with classical drugs or new agents, have resulted in impressive improvements in controlling disease activity. In some cases they even lead to clinical remission. Despite potent and efficient biological agents that specifically modulate distinct pathological pathways a large proportion of patients remain unresponsive to these therapies; drug-free remission is also difficult to achieve since attempting discontinuation of treatment usually results in disease flare. In rheumatoid arthritis joints there is a constant activation of complex networks of cytokines and factors mediating immune interactions and inflammation, in which prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) are important players and contributors to pathogenesis. Our research aimed to investigate the synovial expression of enzymes controlling prostaglandin E2 synthesis and degradation – cyclooxygenase (COX) 1 and 2, microsomal prostaglandin E2 synthase 1 (MPGES1) and 15-prostaglandin dihydrogenase (15-PGDH) as well as enzymes involved in leukotriene synthesis, such as 5-lipoxygenase (LO) and 15-LO. In addition, we evaluated how traditional and new therapies influence these pathways, by analyzing enzyme expression before and after systemic treatment with tumor necrosis factor (TNF) antagonists, rituximab or methotrexate, as well as before and after intra-articular treatment with glucocorticoids. We also evaluated the in vitro effects of TNF antagonists and glucocorticoids on synovial fluid cells and that of methotrexate on synovial fibroblasts. We demonstrated that synovial tissue from RA patients displayed an important expression of enzymes involved in the metabolism of PGE2, as well as 5-LO and 15-LO. MPGES1 and COX-2, the inflammation-inducible enzymes co-localized mainly in fibroblasts and macrophage-like cells and accounted for the local PGE2 production. Intra-articular glucocorticoids significantly reduced all enzymes involved in the PGE2 cascade – COX-1 and COX-2, MPGES1 and 15-PGDH, but also 5-LO, responsible for leukotriene formation. However, they did not influence the expression of 15-LO, an enzyme involved in the formation of both pro-and anti-inflammatory lipid mediators. Regarding the effects of TNF blockers, rituximab or methotrexate, they did not alter the expression profile of enzymes involved in PGE2 metabolism despite showing clinical efficiency in improving disease activity. Although anti-TNF agents reduced the in vitro expression of MPGES1 and COX-2 in synovial fluid cells, the lack of effect ex vivo in biopsies emphasized once again the differences between synovial compartments and possibly the difficulty in mimicking the micro-environment at the site of inflammation in vitro. In conclusion, this thesis demonstrates that potent anti-rheumatic drugs currently used in the clinic with good efficiency also leave inflammatory pathways un-affected, which may account for subclinical ongoing disease activity. Blocking the PGE2 pathway by using MPGES1 inhibitors as combination therapy may show benefit in dampening ongoing local inflammation

Prostaglandin E2 Synthesizing Enzymes in Rheumatoid Arthritis B Cells and the Effects of B Cell Depleting Therapy on Enzyme Expression

Introduction: B cells may play an important role in promoting immune activation in the rheumatoid synovium and can produce prostaglandin E-2 (PGE(2)) when activated. In its turn, PGE(2) formed by cyclooxygenase (COX) and microsomal prostaglandin E-2 synthase 1 (MPGES1) contributes to the rheumatoid arthritis (RA) pathological process. Therapeutic depletion of B cells results in important improvement in controlling disease activity in rheumatoid patients. Therefore we investigated the expression of PGE(2) pathway enzymes in RA B cells and evaluated the effects of B cell depleting therapy on their expression in RA tissue. Methods: B cells expressing MPGES1 and COX-2 were identified by flow cytometry in in vitro stimulated and control mononuclear cells isolated from synovial fluid and peripheral blood of RA patients. Synovial biopsies were obtained from 24 RA patients before and at two consecutive time points after rituximab therapy. Expression of MPGES1, COX-1 and COX-2, as well as interleukin (IL)-1 beta and IL-6, known inducers of MPGES1, was quantified in immunostained biopsy sections using computerized image analysis. Results: Expression of MPGES1 or COX-2 was significantly upregulated upon stimulation of B cells from blood and synovial fluid while control cells displayed no detectable enzymes. In synovial biopsy sections, the expression of MPGES1, COX-1 or COX-2 was resistant to rituximab therapy at 8 or 16 weeks after start of treatment. Furthermore expression of IL-1 beta in the synovial tissue remained unchanged, while IL-6 tended to decrease after therapy. Conclusions: Therapy with B cell depleting agents, although efficient in achieving good clinical and radiographic response in RA patients, leaves important inflammatory pathways in the rheumatoid synovium essentially unaffecte

MPGES1 is not expressed in B cell-rich areas in RA synovium.

<p>Frozen sections of synovial tissue showing diaminobenzidine (brown) staining of MPGES1 (A), COX-2 (B), COX-1 (C) and CD20 B cells (D), CD138 plasma cells (E) (hematoxiline counterstained). Insets represent higher power of image A and D. Original magnification 80x and 200x (insets). (F) and (G) Immunofluorescence pictures of double stained MPGES1 (red) and CD20+ B cells (F) and CD138+ plasma cells (G) (green). Magnification 400x.</p

Minimal influence of rituximab treatment on expression of MPGES1, COX-1 and COX-2 in synovial tissue.

<p>Immunohistochemical staining of frozen biopsy sections from rheumatoid arthritis patients shows diaminobenzidine staining (brown) of MPGES1 (A–C), COX-2 (E-G) and COX-1 (I–K) before treatment, 4 weeks and 16 weeks after treatment (hematoxiline counterstained). Graphs depict image analysis of MPGES1 (D), COX-2 (H) and COX-1 (L) expression in stained sections from patients' biopsies at the different time points. Original magnification 150x.</p

MPGES1 expression in synovial lining fibroblasts before and 16 weeks after initiation of rituximab therapy.

<p>Double immunofluorescence pictures show the presence of MPGES1 (red) expression in CD55 positive fibroblasts (green) in the rheumatoid tissue before rituximab initiation (A) and 16 weeks later (B). Original magnification 500x. Arrows point to double stained cells.</p

Rituximab treatment exerts limited effects on the synovial tissue expression of IL-1β and IL-6.

<p>Graphs show image analysis of positive stained sections for (A) IL-1β and (B) IL-6 before and at consecutive time points after initiation of rituximab therapy. At week 16 a trend towards decrease occurred in IL-6 but not in IL-1β.</p