Leflunomide affect on osteoclast differentiation
GPI-induced arthritis

Leflunomide affect on osteoclast differentiation
OP0107 LEFLUNOMIDE INHIBITS BONE DESTRUCTION BY INTERFERING WITH RANKL SIGNALING AND OSTEOCLASTOGENESIS
Hiroshi Takayanagi, Makoto Urushibara, Hisashi Kurosawa, Tadatsugu Taniguchi; Tokyo

In rheumatoid arthritis (RA), bone destruction occurs due to to increased osteoclastogenesis through induction of RANKL expression. Leflunomide therapy in patients with RA has been shown in clinical trials to inhibit periarticular bone destruction. Leflunomide is also known to inhibit activated T cell proliferation by inhibiting a key enzyme of the de novo pyrimidine synthesis, DHODH. The purpose of this study was to investigate whether leflunomide's suppressive effect on bone destruction is mediated through its effects on T cells.

Methods: The effect of leflunomide on osteoclast differentiation was investigated using in vitro culture system of mouse bone marrow monocytes/macrophages stimulated with RANKL and macrophage-colony stimulating factor. Expression of DHODH in osteoclasts was examined by immunostaining. T cell-independent effect of leflunomide was examined using an endotoxin-induced inflammatory bone destruction model in Rag-2-deficient mice.

Results: Leflunomide inhibited RANK-L induced osteoclast differentiation through its inhibiting effect on pyrimidine synthesis. Leflunomide inhibited RANK-L induced Ca²⁺ signalling in osteoclast precursors which, in turn, inhibited activation of a critical transcription factor of osteoclastogenesis, NFATc1. This inhibition could be overcome by overexpressing NFATc1 in these precursor cells. Leflunomide suppressed endotoxin-induced inflammatory bone destruction and osteoclastogenesis even in Rag-2 deficient mice (these mice lack T cells), indicating that the protective effect of leflunomide on bone is not mediated through T cells.

Conclusion: Leflunomide can inhibit bone destruction independent of T cells by inhibiting the induction of NFATc1, a vital molecule in osteoclast differentiation. RANK-L dependent induction of NFATc1 may be an attractive target for therapeutic intervention in RA.

Editorial Comment: These novel findings demonstrate a direct inhibitory effect of leflunomide on osteoclasts. Thus, at least one of the main therapeutic effects of leflunomide in RA, suppression of bone destruction, is not related to its known suppressive effect on T cell proliferation.

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GPI-induced arthritis
OP0013 IMMUNIZATION WITH THE GLYCOLYTIC ENZYME GLUCOSE-6-PHOSPHATE ISOMERASE (GPI) INDUCES PERIPHERAL POLYARTHRITIS IN GENETICALLY UNALTERED MICE
David Schubert, Bert Maier, Lars Morawietz, Veit Krenn, Thomas Kamradt; Germany

K/BxN TCR transgenic mice spontaneously develop an autoimmune arthritis that resembles human RA. Arthritis development depends on the recognition of the ubiquitously expressed glycolytic enzyme GPI by both the transgenic T cells and B lymphocytes. While the presence of pathogenic anti-GPI antibodies in RA was claimed and refuted, we examined whether arthritis could be induced in normal mice by immunization with GPI.

Results: Immunization with heterologous GPI in adjuvant induced a symmetric polyarthritis of the small distal joints in genetically susceptible mice. Histologically, the disease is characterized by early synovitis followed by massive cartilage destruction and erosions of the bones and later resolution of the inflammation, fibrosis, and repair processes. Although antibody titers in susceptible and non susceptible mouse strains are high, transfer of purified total IgG of sick mice alone does not transfer disease. Nonetheless, antibodies seem to play a major role since Fc-receptor gamma-chain deficient mice develop disease with a much lower frequency and reduced severity than the wild type mice. Treatment with a depleting monoclonal anti-CD4 antibody completely prevents disease. Depletion of CD4+ T cells during disease leads to an rapid resolution of arthritis. Therefore CD4+ T cells are important for both the induction of the disease and the effector phase.

Conclusion: GPI-induced arthritis in normal mice bridges the gap between the transgenic K/BxN model and the highly complex human situation, thereby providing a model in which both the induction and effector phase of antigen specific arthritis can be dissected and preventive and therapeutic strategies evaluated.

Editorial Comments: In the K/xN TCR transgenic mouse model of spontaneous arthritis, the transfer of serum (and thus anti-GPI antibodies) is necessary and sufficient to induce arthritis in recipient mice. However, anti-GPI antibodies are not found consistently in human RA and, therefore, the relevance of GPI to the development of RA has been questioned.

These studies indicate that anti-GPI antibodies occur in mice that do not develop arthritis, as well as those that do. On the surface this may seem to imply that these antibodies are not pathophysiologically important to the development of arthritis. This is not true, however, since Fc receptors and complement activation were also necessary for the development of arthritis, processes that are involved in antibody mediated effects.

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