Shinichi Kawai

Membrane-associated prostaglandin E synthase-1 is upregulated by proinflammatory cytokines in chondrocytes from patients with osteoarthritis

Prostaglandin E synthase (PGES) including isoenzymes of membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES (cPGES) is the recently identified terminal enzyme of the arachidonic acid cascade. PGES converts prostaglandin (PG)H2 to PGE2 downstream of cyclooxygenase (COX). We investigated the expression of PGES isoenzyme in articular chondrocytes from patients with osteoarthritis (OA). Chondrocytes were treated with various cytokines and the expression of PGES isoenzyme mRNA was analyzed by the reverse transcription–polymerase chain reaction and Northern blotting, whereas Western blotting was performed for protein expression. The subcellular localization of mPGES-1 was determined by immunofluorescent microscopy. Conversion of arachidonic acid or PGH2 to PGE2 was measured by enzyme-linked immunosorbent assay. Finally, the expression of mPGES-1 protein in OA articular cartilage was assessed by immunohistochemistry. Expression of mPGES-1 mRNA in chondrocytes was significantly induced by interleukin (IL)-1β or tumor necrosis factor (TNF)-α, whereas other cytokines, such as IL-4, IL-6, IL-8, IL-10, and interferon-γ, had no effect. COX-2 was also induced under the same conditions, although its pattern of expression was different. Expression of cPGES, mPGES-2, and COX-1 mRNA was not affected by IL-1β or TNF-α. The subcellular localization of mPGES-1 and COX-2 almost overlapped in the perinuclear region. In comparison with 6-keto-PGF1α and thromboxane B2, the production of PGE2 was greater after chondrocytes were stimulated by IL-1β or TNF-α. Conversion of PGH2 to PGE2 (PGES activity) was significantly increased in the lysate from IL-1β-stimulated chondrocytes and it was inhibited by MK-886, which has an inhibitory effect on mPGES-1 activity. Chondrocytes in articular cartilage from patients with OA showed positive immunostaining for mPGES-1. These results suggest that mPGES-1 might be important in the pathogenesis of OA. It might also be a potential new target for therapeutic strategies that specifically modulate PGE2 synthesis in patients with OA.

Cyclooxygenase-1 and cyclooxygenase-2 selectivity of non-steroidal anti-inflammatory drugs: investigation using human peripheral monocytes.

Since the pharmacological profiles of various non‐steroidal anti‐inflammatory drugs (NSAIDs) might depend on their differing selectivity for cyclooxygenase 1 (COX‐1) and 2 (COX‐2), we developed a new screening method using human peripheral monocytes. Monocytes from healthy volunteers were separated, and the cells were incubated with or without lipopoly‐saccharide (LPS). Monocytes without LPS stimulation exclusively expressed COX‐1 on Western blotting analysis, whereas LPS stimulation induced COX‐2 expression. Unstimulated monocytes (COX‐1) and LPS‐stimulated monocytes (COX‐2) were then used to determine the COX selectivity of various NSAIDs. The respective mean IC50 values for COX‐1 and COX‐2 IC50 (μm), and the COX‐1/COX‐2 ratio of each NSAID were as follows: celecoxib, 82, 6.8, 12; diclofenac, 0.076, 0.026, 2.9; etodolac, > 100, 53, > 1.9; ibuprofen, 12, 80, 0.15; indometacin, 0.0090, 0.31, 0.029; meloxicam, 37, 6.1, 6.1; 6‐MNA (the active metabolite of nabumetone), 149, 230, 0.65; NS‐398, 125, 5.6, 22; piroxicam, 47,25, 1.9; rofecoxib, > 100,25, > 4.0; S‐2474, > 100,8.9, > 11; SC‐560, 0.0048, 1.4, 0.0034. The percentage inhibition of COX‐1 activity at the IC50 of COX‐2 also showed a wide variation among these NSAIDs. The bioassay system using human monocytes to assess the inhibitory effects of various NSAIDs on COX‐1 and COX‐2 may become a clinically useful screening method.

Selective cyclooxygenase‐2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells

Although the influence of selective cyclooxygenase (COX)‐2 inhibitors on the proliferation of colon adenocarcinoma cells have been the subject of much investigation, relatively little research has compared the effects of different COX‐2 inhibitors. Celecoxib strongly suppressed the proliferation of COX‐2 expressing HT‐29 cells at 10–40 μM. NS‐398 and nimesulide also inhibited cell proliferation, whereas rofecoxib, meloxicam, and etodolac did not. Only celecoxib induced apoptosis of HT‐29 cells, as detected on the basis of DNA fragmentation, TUNEL positivity, and caspase‐3/7 activation. DNA fragmentation was also increasd in COX‐2 non‐expressing cell lines (SW‐480 and HCT‐116) by exposure to celecoxib for 6–24 h. All six COX‐2 inhibitors suppressed the production of prostaglandin E2 by HT‐29 cells, suggesting that the pro‐apoptotic effect of celecoxib was unrelated to inhibition of COX‐2. Inactivation of Akt might explain the differential pro‐apoptotic effect of these selective COX‐2 inhibitors on colon adenocarcinoma cells.

Comparison of cyclooxygenase-1 and -2 inhibitory activities of various nonsteroidal anti-inflammatory drugs using human platelets and synovial cells.

Recent studies have shown that cyclooxygenase exists in two isozyme forms. Since differences in the pharmacological profiles of nonsteroidal anti-inflammatory drugs (NSAIDs) might be accounted for by varying degrees of selectivity for these isozymes, cyclooxygenase-1 and -2, the relative potency of various NSAIDs in inhibiting their activities was examined in intact human cells. We used human platelets cyclooxygenase-1 and interleukin-1beta-stimulated human synovial cell cyclooxygenase-2 for measuring cyclooxygenase selectivity. The presence of the enzymes was confirmed by immunoblotting and immunoprecipitation analysis, and by the reverse transcriptase-polymerase chain reaction. Mean IC50 values (microM) for human platelet cyclooxygenase-1 and interleukin-1beta-stimulated human synovial cell cyclooxygenase-2 and cyclooxygenase-1/-2 IC50 ratio of various NSAIDs were as follows: aspirin, 3.2, 26, 0.12; diclofenac, 0.037, 0.00097, 38; etodolac, 122, 0.68, 179; ibuprofen, 3.0, 3.5, 0.86; indomethacin, 0.013, 0.044, 0.30; loxoprofen (active metabolite), 0.38, 0.12, 3.2; NS-398, 12, 0.0095, 1263; oxaprozin, 2.2, 36, 0.061; zaltoprofen, 1.3, 0.34, 3.8; respectively. Our bioassay system employing intact human cells to assess the cyclooxygenase selectivity of NSAIDs may provide clinically useful information.

Cyclosporine and tacrolimus for the treatment of rheumatoid arthritis

Purpose of reviewThe calcineurin inhibitors cyclosporine and tacrolimus are important treatments for patients with active rheumatoid arthritis, especially in cases of resistance or intolerance to methotrexate or other disease-modifying antirheumatic drugs. Here, we discuss the mechanism, efficacy and safety of cyclosporine and tacrolimus in the treatment of rheumatoid arthritis. Recent findingsRecent clinical trials of cyclosporine have shown the advantages of its combination with methotrexate, glucocorticoids and leflunomide in the treatment of active rheumatoid arthritis. In Japan, tacrolimus monotherapy was found to be quite effective and combination therapy with methotrexate had positive results in an American study. The inhibitory effects of both drugs not only on T lymphocytes, but also on human osteoclast formation, have been demonstrated in basic studies. SummaryCyclosporine and tacrolimus are clinically available disease-modifying antirheumatic drugs. Numerous clinical studies have shown the usefulness of these calcineurin inhibitors in monotherapy and also when combined with methotrexate. Although these drugs have similar effects, there are some differences in adverse reactions.

Japan College of Rheumatology 2009 guidelines for the use of tocilizumab, a humanized anti-interleukin-6 receptor monoclonal antibody, in rheumatoid arthritis

The introduction of biological agents targeting tumor necrosis factor-alpha (TNF-α) has brought about a paradigm shift in the treatment of rheumatoid arthritis (RA). Although these anti-TNF agents have excellent efficacy against RA, a substantial number of patients still show inadequate responses. In Western countries, such patients are already being treated with new classes of antirheumatic drugs such as abatacept and rituximab. Tocilizumab (TCZ) is a humanized monoclonal antibody developed in Japan against the human interleukin-6 (IL-6) receptor. TCZ does not only alleviate the signs and symptoms of RA but also seems to prevent progressive bone and joint destruction. However, there is a concern that TCZ might increase the risk of adverse events such as infections since IL-6 plays a pivotal role in the immune system. Calculating the relative risks of specific adverse outcomes with TCZ use remains difficult, due to insufficient patient numbers enrolled in clinical trials to date. This review presents tentative guidelines for the use of TCZ for RA patients prepared by the Japan College of Rheumatology and based on results of clinical trials in Japan and Western countries. The guidelines are intended as a guide for postmarketing surveillance and clinical practice, and will be revised periodically based on the surveillance.

Prostaglandin E synthase in the pathophysiology of arthritis

Prostaglandin E synthase (PGES) is a recently identified terminal enzyme that acts downstream of cyclooxygenase and catalyzes the conversion of prostaglandin (PG) H2 to PGE2. At least three isozymes have been cloned so far, which are called membrane‐associated PGES (mPGES)‐1, mPGES‐2, and cytosolic PGES. Among them, mPGES‐1 is induced by various inflammatory stimuli in some cells and tissues. Induction of mPGES‐1 in the component of articular tissues of patients with rheumatoid arthritis and osteoarthritis has been demonstrated in vitro. Recent studies using adjuvant induced arthritis model have shown the increase of mPGES‐1 expression resulted in the increase of PGE2 production at the sites of inflammation. In addition, reports of mPGES‐1‐deficient mice clearly suggest the role of mPGES‐1 in the process of chronic inflammation such as collagen‐induced arthritis and collagen antibody induced arthritis in vivo. Thus, recent in vitro and in vivo findings suggest that mPGES‐1 may be a novel therapeutic target for arthritis. This paper introduces recent advances in research about the role of PGES in the pathophysiology of arthritis.

Serum lipoprotein(a) and apolipoprotein(a) phenotypes in patients with rheumatoid arthritis

OBJECTIVE To determine serum lipoprotein(a) (Lp[a]) concentrations and to analyze the apolipoprotein(a) (Apo[a]) phenotype in patients with rheumatoid arthritis (RA). METHODS The subjects included 131 patients with RA and 200 healthy control subjects. Serum Lp(a) concentrations were measured by enzyme-linked immunosorbent assay, and the Apo(a) phenotype was determined by immunoblotting. HLA-DR typing was also done. RESULTS The mean serum Lp(a) level was significantly higher (P < 0.001) in the RA patients (27.5 mg/dl) than in the controls (15.0 mg/dl). The S3 allele was found in 70.0% of the patients versus 39.5% of the controls (P < 0.001). There was no significant difference in HLA-DR4 positivity between patients with and without the S3 phenotype. CONCLUSION The serum Lp(a) level was increased in patients with RA, possibly partly because of S3 phenotype predominance.

Cyclooxygenase selectivity and the risk of gastro-intestinal complications of various non-steroidal anti-inflammatory drugs: A clinical consideration

Abstract. Severe gastro-intestinal complications are a major cause of NSAID-induced deaths in cases of rheumatoid arthritis. We measured COX selectivity by using an intact cell assay system, and found that NS-398 is a highly COX-2–selective inhibitor. Meloxicam, etodolac and diclofenac also showed high COX-2 selectivity. Zaltoprofen, loxoprofen-SRS (active metabolite of loxoprofen), 6-MNA (active metabolite of nabumetone) and ibuprofen showed intermediate COX-2 selectivity. The lowest COX-2 selectivities, which means the highest COX-1 selectivities, were observed in indomethacin, aspirin, and oxaprozin. There appears to be a good relationship between our data and some clinical data of severe gastro-intestinal toxicity. The more a given NSAID is selective for COX-2, the safer it is for clinical use. In conclusion, to anticipate the safety of NSAIDs, we find that an intact cell assay system, using human cells for measurement of COX selectivity, may be more useful than using direct enzyme assay systems.

Adiponectin stimulates IL-8 production by rheumatoid synovial fibroblasts

The adipokines are linked not only to metabolic regulation, but also to immune responses. Adiponectin, but not leptin or resistin induced interleukin-8 production from rheumatoid synovial fibroblasts (RSF). The culture supernatant of RSF treated with adiponectin induced chemotaxis, although adiponectin itself had no such effect. Addition of antibody against adiponectin, and inhibition of adiponectin receptor gene decreased adiponectin-induced IL-8 production. Nuclear translocation of nuclear factor-kappa B was increased by adiponectin. The induction of interleukin-8 was inhibited by mitogen-activated protein kinase inhibitors. These findings suggest that adiponectin contributes to the pathogenesis of rheumatoid arthritis.