WP 9.Validation of targets and development of new therapy in murine arthritis models

This area of work will be lead by Partner 8 (Nijmegen) with input from Partner 1 (KI), Partner 2 (Leiden), Partner 4 (Vienna), Partner 9 (Montpellier), Partner 10 (Zürich), Partner 21 (EULAR), Partner 23 (Future Partner), Partner 24 (Biovitrum) and Partner 27 (Arthrogen)

•  Develop a new model of arthritis using DR4 transgenic mice immunised with human citrullinated fibrinogen.

•  Develop specific immunotherapy using autologous dendritic cells specific for the antigen.

Description of the work

Activity 1: We will evaluate and determine the procedure to induce arthritis in humanized mice bearing DR404/CD4. As a first step, 100 microgram human citrullinated fibrinogen (hCF) will be emulsioned in FCA and injected at 5 different intradermal points at the base of the tail. A boost with 100 microg hCF will be performed on day 21. Analysis will be performed using clinical score, X-ray of the paws, and paw histology at 50 days. The control groups will include collagen-induced arthritis in the DR4 transgenic animals and non-modified fibrinogen.

Activity 2 : The humoral immune response will be determined using ELISA. The cell immunity will be assessed by lymph nodes and spleen T cell proliferation after stimulation with hCP. The positive controls will be Con A stimulated cells, and the negative cells stimulated with non-modified human fibrinogen. The arthritogenic peptide responsible for the immune response will be determined by synthesis of antigenic epitopes.

Activity 3: We will purify and characterise dendritic cells (DC) in the DR4Tg mice. Bone marrow cells will be washed and cultivated for 10 days in the presence of GMCSF and IL4. The DC phenotype will be assessed by FACS analysis (expression of CD80, CD86, DR4 and CD14 negative). The functionality of the DC will be assessed through MLR using the arthritogenic peptide. The cells will then be manipulated to obtain an immunosuppressive effect specific for hCF (culture in the presence of rIL10, genetical modification of DC using lentiviral vectors to obtain long-term expression of IL10 or IL4).

Activity 4: To obtain induction of immune tolerance we will determine the number of DC, the route of injection, the time of injection both in a preventive and curative way. The specificity will be determined using human citrullinated fibrinogen in order to pulse the DC. DC will be labelled with GFP (using lentivral vector containing GFP gene) and followed by a CCD camera in vivo at different time points (days 2, 7, 14, 21, 28, 35) after injection the GFP positive cells.

Deliverables

Dl1: description of a new model of arthritis in humanised transgenic animals.

Dl2: procedure to obtain immunosuppressive DC.

Dl3: procedure for induction of tolerance to arthritis.

Use of targeted therapies in antigen-induced monoarthritis, in adjuvant-arthritis and collagen induced polyarthritis as well as in TNF-transgenic arthritis-prone mice

The involved laboratories have an extensive experience of using these models to evaluate effects of various targeted therapies in humans. This work will now be extended and coordinated between the various collaborating groups. Several targeted therapies will be possible to analyse in these systems during the first 18 months. One of these is the targeted therapy against HMGB1 (see details below). Another is against PGE-synthase (details given below). Other examples are OPG-based therapy which may inhibit joint destruction as well as neutralization of IL-17 and IL-18 in various arthritic conditions and identification of its validity as therapeutic targets. Studies will be extended at later stages with potential targets which are in the pipeline after first having been identified via use of genomics.

HMGB1

This is a novel pro-inflammatory cytokine (programme developed by Prof Ulf Andersson and Ass Prof Helena Erlandsson-Harris at Karolinska Institutet in collaboration with Prof Kevin Tracey, North Shore Hospital , New York ). HMGB1 (high mobility group box chromosomal protein 1) is an endogenous factor of great potential interest for RA research since:

•  HMGB1 is a potent inducer of TNF and IL-1 formation signalling via RAGE (the receptor for advanced glycation end products) Andersson, U. et al . J Exp Med, 2000. 192 (4): p. 565-570

•  A polymorphism of the RAGE gene of the ligand-binding domain of the receptor has been identified and the RAGE 82S allele enhancing cytokine production has an increased prevalence in RA patients (Andersson, U. et al . J Leukoc Biol, 2002. 72 (6): p. 1084-91.

•  HMGB1 induces local proteolytic tissue destruction by activating plasminogen and metalloproteinase; Andersson, U. et al . J Exp Med, 2000. 192 (4): p. 565-570.

•  HMGB1 is massively released by cells in the rheumatic synovitis; Andersson, U. et al . J Leukoc Biol, 2002. 72 (6): p. 1084-91.

•  Intra-articular HMGB1 injection causes arthritis in mice.

•  Neutralising HMGB1 therapy is successful in mice and rats affected by collagen-induced arthritis. Kokkola, R. et al . Arthritis Rheum, 2002. 46 (10): p. 2598-2603.

Further verification of the mode of action of this extremely efficient targeted therapy will be achieved within the next 18 months in several different animal models.

Generation of neutralising anti-HMGB1 mAb is in good progress and these antibodies will be used for further therapy studies initially in experimental arthritis and later in a clinical setting. Such reagents will also be required for further quantitative assessments of HMGB1 in disease. It is possible that such experiments will be initiated within the next 18 months, but an absolute time for this cannot be exactly foreseen at present.

Prostaglandin synthase

Programme developed by Ass Prof Per-Johan Jacobsson, in collaboration with Prof Bengt Samuelsson and others at Karolinska Institutet.

Microsomal, inducible prostaglandin E synthase (mPGES1) catalyses the last committed step in the formation of PGE 2 from cyclooxygenase derived PGH 2 (Jakobsson et al . Proc. Natl. Acad. Sci. U. S. A. , 1999 , 96 , 7220.1) . The role of PGE 2 in inflammation and particularly in RA has recently begun to be understood, and mice lacking pG-synthase do not develop collagen-induced arthritis. As an initial step in analyses aimed at exploring the role of PG-synthase in RA and in experimental arthritis, we have investigated the presence of mPGES and related enzymes in synovial biopsies and synovial fluid cells obtained from patients with RA at different stages of diseases and during different therapies (Westman et al Arthritis Rheum June 2004). The results will further bring clarity in the role of the biologically very active eicosanoids in rheumatoid arthritis and possibly the validation of novel targets against this inflammatory disease