Hiromi Doi

The Importance of IL-1β and TNF-α, and the Noninvolvement of IL-6, in the Development of Monoclonal Antibody-Induced Arthritis

Injection of anti-type II collagen Ab and LPS induces arthritis in mice. The levels of IL-1β, IL-6, and chemokines (macrophage inflammatory protein (MIP)-1α, MIP-2, and monocyte chemoattractant protein-1) in the hind paws increased with the onset of arthritis and correlated highly with arthritis scores. The level of TNF-α was also elevated, but only transiently. Quantitative real-time PCR analysis revealed increases in cytokine and chemokine mRNA. To elucidate the contribution of inflammatory cytokines and chemokines in arthritis development more directly, recombinant proteins, neutralizing Abs, and knockout mice were used. The injection of rIL-1β or TNF-α, but not IL-6 or chemokines, induced arthritis when mice were i.v. preinjected with anti-type II collagen Ab. However, a single injection of recombinant cytokines or chemokines into the hind paws did not induce swelling. Arthritis development was inhibited by neutralizing Ab against IL-1β, TNF-α, or MIP-1α. In contrast, the inhibitory effect by anti-MIP-2 Ab was partial and, surprisingly, Abs to IL-6 and monocyte chemoattractant protein-1 showed no inhibitory effect. Furthermore, arthritis development in IL-1R−/− mice and TNFR−/− mice was not observed at all, but severe arthritis was developed in IL-6−/− mice. These results suggest that IL-1β and TNF-α play more crucial roles than IL-6 or chemokines in this model. Because arthritis was also developed in SCID mice, the development of arthritis in the Ab-induced mice model is due to a mechanism that does not involve T or B cells.

Essential Role of Fcγ Receptors in Anti-Type II Collagen Antibody-Induced Arthritis

Anti-type II collagen (anti-CII) Ab is a well-known autoantibody observed in patients with rheumatoid arthritis. Injection of anti-CII Ab and LPS induces arthritis in mice in which anti-CII Ab as well as inflammatory cytokines, IL-1β and TNF-α, play critical roles. We investigated the involvement of IgG FcRs (FcγRs) in this arthritis model. BALB/c mice injected with the F(ab′)2 of anti-CII Ab showed no signs of arthritis. Arthritis development was not observed in FcRγ−/− mice and was partially suppressed in FcγRIII−/− mice despite the binding of anti-CII Ab and C3 to cartilage surface. Surprisingly, BALB/c mice lacking FcγRIIB, which is known as an inhibitory FcγR, developed arthritis with no exacerbation in arthritis score compared with wild-type (WT) mice, and only slight exacerbation was observed in the histopathological analysis. In contrast, aged FcγRIIB−/− BALB/c mice developed arthritis without LPS injection, suggesting an augmented susceptibility to arthritis in aged FcγRIIB−/− mice. No significant difference was observed among BALB/c-WT, -FcRγ−/−, and -FcγRIIB−/− mice on cytokine production induced by anti-CII Ab and LPS injection. Severe arthritis developed in BALB/c-WT and -FcγRIIB−/− mice, but not in BALB/c-FcRγ−/− mice, after the injection of anti-CII Ab and inflammatory cytokines. These results suggest that the reason behind the nondevelopment of arthritis in FcRγ−/− BALB/c mice is not due to a disorder in transient cytokine production, but to an irregularity downstream of cytokine production.

Essential role of neutrophils in anti‐type II collagen antibody and lipopolysaccharide‐induced arthritis

In mice arthritis model induced by anti‐type II collagen (CII) antibodies and lipopolysaccharide (LPS), most of cells that infiltrated into the joint space were neutrophils. To investigate the role of neutrophils in the pathogenesis of arthritis, we depleted the neutrophils in vivo by injection of the antibody against Gr‐1 expressed mainly on neutrophils. The neutrophil depletion completely inhibited the arthritis development. Furthermore, neutrophil depletion in mice that had already developed arthritis ameliorated the disease. These results showed that neutrophils are indispensable not only for the development, but also for the maintenance of arthritis. Next, we tried to develop arthritis in C5‐deficient mice to investigate the involvement of C5a, one of chemotactic factors for neutrophils. C5‐deficient mice showed significant reduction in arthritis development in comparison with wild type mice. Injection of pertussis toxin (Ptx) into the mice, which inhibits the signals from the inhibitory G‐protein coupled‐receptors including the C5a receptor, suppressed the development of arthritis. Furthermore, Ptx also ameliorated the arthritis when injected into mice that had already developed the disease. These results suggest the important role of chemotactic factors involving C5a and inhibitory G‐protein (Gi)‐coupled receptors not only in the development, but also in the maintenance of arthritis.

Discovery of CS-0777: A Potent, Selective, and Orally Active S1P1 Agonist.

CS-0777 (3) is phosphorylated in vivo, and the phosphate of CS-0777 (CS-0777-P) (4) acts as a selective S1P receptor-1 (S1P1) modulator. We report herein the synthesis of CS-0777 and CS-0777-P, pharmacological effects such as S1P1 and S1P3 agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effect on experimental autoimmune encephalomyelitis (EAE), and also the pharmacokinetics in rats. CS-0777-P had ∼320-fold greater agonist activity for human S1P1 (EC50; 1.1 nM) relative to S1P3 (EC50; 350 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-0777 in rats, lymphocyte counts decreased significantly, with a nadir at 12 h postdose and recovery to vehicle control levels by 5 days postdose. In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for the 0.1 and 1 mg/kg CS-0777 groups in rats. CS-0777 is currently in clinical trials for the treatment of multiple sclerosis (MS).

Discovery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist

S1P(3)-sparing S1P(1) agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC(50) value of 4.0 nM for human S1P(1) and over 5000-fold selectivity against S1P(3). The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID(50) value was determined at 0.407mg/kg. The docking studies of CS-2100 with the homology model of S1P(1) and S1P(3) showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P(3), not in the case of Leu276 in S1P(1). This observation gives an explanation for the excellent S1P(3)-sparing characteristic of CS-2100.

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 1

We investigated proinflammatory cytokine TNFalpha production inhibitors in order to develop novel anti-inflammatory agents. According to the results, we found that 17, a pyrrole derivative possessing a tetrahydropyridine group at the beta-position, showed potent inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS) induced TNFalpha production in human whole blood, IC(50)=1.86 microM) and in vivo (inhibition of LPS induced TNFalpha production in mice, ID(50)=5.98 mg/kg).

Anti‐type II collagen antibody accelerates arthritis via CXCR2‐expressing cells in IL‐1 receptor antagonist‐deficient mice

Arthritis can be induced in mice by the injection of anti‐type II collagen (anti‐CII) Ab and LPS. To elucidate the role of IL‐1 receptor antagonist (IL‐1ra) in Ab‐induced arthritis, WT and IL‐1ra–/– mice were administered anti‐CII Ab and LPS. These IL‐1ra–/– mice developed severe arthritis even at low doses of anti‐CII Ab and LPS, while WT mice did not. The cells that invaded the arthritic joints were mainly Gr‐1+ neutrophils, and the number of the cells in the joints remained high over 4 weeks in the IL‐1ra–/– mice. KC, a ligand for CXCR2, is found in higher levels in the arthritic paws of IL‐1ra–/– mice compared with the WT, and most of the cells that infiltrated into the joints of the IL‐1ra–/– mice were CXCR2‐expressing neutrophils. Administration of anti‐CXCR2 Ab completely inhibited arthritis development. The anti‐CXCR2 Ab decreased the number of neutrophils in the blood and also inhibited the migration of neutrophils to KC. These results suggested that the high susceptibility of IL‐1ra–/– mice to anti‐CII Ab‐induced arthritis was due to the higher expression of chemotactic factors like KC and the sustained infiltration of CXCR2‐expressing neutrophils into the joints.