Hiroyuki Tamemoto

Increased Levels of Interleukin 33 in Sera and Synovial Fluid from Patients with Active Rheumatoid Arthritis

Objective. To determine levels of interleukin 33 (IL-33) in serum and synovial fluid (SF) and their clinical associations in patients with rheumatoid arthritis (RA). To evaluate the ability of activated peripheral blood mononuclear cells (PBMC) and fibroblast-like synoviocytes (FLS) from RA patients to release IL-33. Methods. Sera were obtained from 59 patients with RA, 10 patients with infectious diseases, and 42 healthy volunteers. SF samples were obtained from 15 patients with RA and 13 with osteoarthritis. IL-33 levels were measured using a sandwich ELISA after removal of rheumatoid factor with protein A-Sepharose beads. FLS were stimulated with IL-1ß and tumor necrosis factor, and treated with or without chemical damage. PBMC were stimulated with anti-CD3/CD28 antibodies. The levels of IL-33 were measured in the culture supernatants and cell lysates by ELISA or immunoblotting. Results. Serum IL-33 levels were significantly higher in RA patients, especially in the high disease activity group compared to the moderate or low activity group. IL-33 levels in SF were elevated in all 15 RA patients measured. IL-33 levels were higher in SF samples than in sera in 7 RA patients measured simultaneously. The 30-kDa IL-33 precursor was detected in the culture supernatants of damaged FLS but was not detected in those of activated PBMC and non-damaged FLS. Conclusion. IL-33 levels were elevated in sera and SF samples from patients with RA, and correlated with disease activity. IL-33 was produced mainly in inflamed joints; IL-33/ST2L signaling might play an important role in joint inflammation of human RA.

Mature interleukin-33 is produced by calpain-mediated cleavage in vivo

Interleukin (IL)-33 is a novel member of the IL-1 family. IL-33 is primarily synthesized as a 30-kDa precursor (pro-IL-33). Pro-IL-33 is cleaved by caspase-1 into an 18-kDa mature form (mature IL-33) in vitro. Recombinant mature IL-33 has been known to induce T-helper type-2 (Th2)-associated cytokines and inflammatory cytokines via its receptor, ST2L. However, processing of pro-IL-33 in vivo has not been clarified yet. Here, we report that calpain mediates pro-IL-33 processing in vivo. Pro-IL-33 was expressed by stimulating human epithelial cells with phorbol 12-myristate 13-acetate. Calcium ionophore induced pro-IL-33 cleavage and mature IL-33 production. This cleavage was inhibited by treatment with a calcium chelator and calpain inhibitors. Moreover, short interfering RNA-mediated knockdown of calpains suppressed pro-IL-33 cleavage. These results indicate that calpains play a critical role in pro-IL-33 processing in vivo.

Characteristics of diabetic retinopathy in SDT rats.

We previously reported a new diabetic strain of the Sprague‐Dawley rat, named the Spontaneously Diabetic Torii (SDT) rat. The purpose of the present study was to report the histologic and ultrastructural characteristics of diabetic retinopathy (DR) in a new animal model, the SDT rat.

Sustained elevation of interleukin-33 in sera and synovial fluids from patients with rheumatoid arthritis non-responsive to anti-tumor necrosis factor: possible association with persistent IL-1β signaling and a poor clinical response

Although TNF inhibitors have dramatically improved the outcome of patients with rheumatoid arthritis, 30–40% of patients do not respond well to them and treatment needs to be changed. In an effort to discriminate good and poor responders, we focused on the change in serum and synovial fluid levels of interleukin (IL-) 33 before and after treatment with TNF inhibitors. They were also measured in synovial fluids from 17 TNF inhibitor-naïve patients, and fibroblast-like synoviocytes (FLS) in-culture from 6 patients and correlated with various pro-inflammatory cytokines. Serum levels of IL-33 at 6xa0months after treatment decreased significantly in responders, while they did not change in non-responders. Synovial fluid levels of IL-33 in 6 patients under treatment with TNF inhibitors stayed high in 3 who were refractory and slightly elevated in 2 moderate responders, while they were undetectable in one patient under remission. Among inflammatory cytokines measured in 17 synovial fluids from TNF inhibitor-naïve patients, levels of IL-33 showed a significant positive correlation only to those of IL-1β. IL-1β increased IL-33 expression markedly in FLS in vitro, compared to TNF-α. IL-1β might be inducing RA inflammation through producing pro-inflammatory IL-33 in TNF inhibitor-hypo-responders. Sustained elevation of serum and/or synovial levels of IL-33 may account for a poor response to TNF inhibitors, although how TNF inhibitors affect the level of IL-33 remains to be elucidated.

Characterization of ST2 transgenic mice with resistance to IL-33

IL‐33, a member of the IL‐1 family, activates MAPK and NF‐κB through its receptor ST2L and IL‐1RAcP. ST2, a member of the IL‐1R superfamily, is a secreted form of ST2 gene products, which has been shown to act as a decoy receptor for IL‐33 and to inhibit the IL‐33/ST2L/IL‐1RAcP signaling pathway. In this work, we generated ST2 transgenic mice. In control mice, intraperitoneal administration of IL‐33 caused an increased number of eosinophils in blood and in peritoneal cavity, an increased number of peritoneal MΦ, splenomegaly, accumulation of periodic acid‐Schiff‐positive material in the lung, and high concentrations of serum IL‐5 and IL‐13. However, these alterations were hardly detectable in ST2 Tg mice. In peritoneal MΦ from IL‐33‐stimulated mice, mRNA expression of M2 MΦ marker genes were increased compared with thioglycollate‐elicited peritoneal MΦ. The IL‐33‐stimulation also increased the secretion of IL‐6 from MΦ. However, when the IL‐33 was preincubated with ST2 prior to its addition to the MΦ cultures, the secretion of IL‐6 was attenuated. These data suggest that, though IL‐33 induced the Th2‐type immune responses and infiltration of M2 type MΦ into the peritoneal cavity, ST2 can downregulate these reactions both in vivo and in vitro.

Prospective Assessment of Proliferative Diabetic Retinopathy with Observations of Posterior Vitreous Detachment

Purpose: To study the relation between posterior vitreous detachment (PVD) and progression of diabetic retinopathy (DR), based on our observation that proliferative DR is rare in patients with complete PVD. Methods: The medical records of 403 patients with diabetes were reviewed for the relation between progressive DR and the status of PVD and HbA1c over 3xa0years. PVD was classified into none, complete PVD with collapse, complete PVD without collapse, partial PVD with a thickened posterior vitreous cortex, and partial PVD without a thickened posterior vitreous cortex. DR was classified into none, simple, preproliferative, or proliferative. When it became more extensive or when laser treatment or vitreous surgery was performed, the DR was considered progressive. Results: Progression of DR over 3xa0years occurred in 128/292 (43.8%) eyes with no PVD, 0/14 (0%) eyes with complete PVD with collapse, 2/8 (25%) eyes with complete PVD without collapse, 15/15 (100%) eyes with partial PVD with a thickened posterior vitreous cortex, and 19/74 (25.7%) eyes with partial PVD without a thickened posterior vitreous cortex. Progression of DR occurred significantly more frequently in eyes with partial PVD with a thickened posterior vitreous cortex compared to eyes with complete PVD with collapse (p<0.0001). HbA1c, did not differ significantly between these two groups (6.9 ± 0.9% and 7.5 ± 0.9%, respectively; p = 0.14), although HbA1c was significantly higher (p = 0.04) in patients with progressive DR (78 ± 1.8%) than in patients without progressive DR (7.5 ± 1.5%). Conclusion: Complete PVD is a strong negative risk factor for DR. The PVD status in patients with diabetes should be evaluated.

Association of the Glu298Asp polymorphism of the eNOS Gene with ischemic heart disease in Japanese diabetic subjects

We analysed the association of the Glu298Asp polymorphism of the endothelial nitric oxide synthase (eNOS) gene with ischemic heart disease (IHD) and albuminuria in 337 Japanese diabetes patients. Restriction fragment length polymorphism analysis of Glu298Asp of the eNOS gene was performed by amplification of genomic DNA isolated from whole blood followed by digestion with the restriction enzyme BanII. Individuals with IHD were identified by review of medical records and were identified based on apparent ischemic change of electrocardiography, significant stenosis of coronary artery, or a history of myocardial infarction, coronary artery bypass surgery, or catheter intervention. Albuminuria was confirmed by a positive test on at least two separate examinations. Of the 337 subjects analysed for polymorphisms of the eNOS gene, 45 with the GluAsp and five with the AspAsp genotype were combined into 1 group (GluAsp or AspAsp group). Among these 50 subjects, 16 (32%) had IHD, and among 287 subjects with the GluGlu genotype, 38 (13.2%) had IHD. The number of subjects with IHD was significantly greater in the GluAsp or AspAsp group than in the GluGlu group (P=0.0006). There was no difference in the frequency of albuminuria among the genotypes. The GluAsp or AspAsp genotype of the Glu298Asp polymorphism was significantly associated with IHD, but not albuminuria in these Japanese diabetic subjects.

Hypoadiponectinemia in patients with cerebral infarction: comparison with other atherosclerotic disorders.

The present study was undertaken to determine serum adiponectin level in patients with cerebral infarction and to further analyze any difference in serum adiponectin levels among atherosclerotic disorders. One hundred fifty-two subjects with atherosclerotic disorders were enrolled, 110 males and 42 females, with the age of 67.0 ± 9.9 years (mean ± SD). They were divided into 62 patients with cerebral infarction, 48 patients with ischemic heart disease, and 42 patients with arteriosclerosis obliterans. Thirty-two subjects matched by age, gender, and body mass index served as controls. Serum adiponectin levels were 7.2 ± 0.6 &mgr;g/mL (mean ± SE) in the patients with cerebral infarction, 7.2 ± 0.8 &mgr;g/mL in those with ischemic heart disease, and 6.9 ± 0.9 &mgr;g/mL in those with arteriosclerosis obliterans. They were significantly less than the level of 12.6 ± 1.9 &mgr;g/mL in the control group (P < 0.01). However, there was no difference in serum adiponectin level among three groups of atherosclerotic disorders. In the patients with acute cerebral infarction, serum adiponectin level was temporarily reduced from 7.3 ± 0.9 to 6.2 ± 0.8 &mgr;g/mL 14 days after the hospitalization (P < 0.01), followed by recovery to the basal value. The present findings indicate that serum adiponectin levels are equivalently reduced in patients with atherosclerotic disorders, and that serum adiponectin is changeable under acute phase of cerebral infarction.

Hypotonicity reduces the activity of murine aquaporin-2 promoter induced by dibutyryl cAMP

The present study was undertaken to determine whether hypotonicity regulates the aquaporin‐2 (AQP‐2) gene in vitro. The 5′‐flanking region of the AQP‐2 gene contains the tonicity‐response enhancer (TonE) promoter located between −570 and −560 bp, and another distinct hypertonicity‐responsive region between −6.1 and −4.3 kb of the AQP‐2 gene. The 5′‐flanking region of murine AQP‐2 gene up to −9.5 kb was cloned into a luciferase (Luc) reporter plasmid. The constructs, which have TonE and/or the hypertonicity‐responsive region, together with the murine AQP‐2 gene, were co‐transfected into murine IMCD3 cells. When the cells were co‐transfected with the construct containing more than 1.1 kb of the 5′‐flanking region of murine AQP‐2 gene (–9.5AQP2, −6.1AQP2 and −1.1AQP2) and the AQP‐2 gene, 24 h exposure to 5 μmol l−1 dibutyryl cAMP (DBcAMP) significantly increased the Luc activity by 2.3‐fold in the isotonic medium (300 mosmol kg−1). In the hypotonic medium (225 mosmol kg−1), basal activity was not altered, and the response of Luc activity to 24 h exposure to 5 μmol l−1DBcAMP was abolished. Similar findings were obtained in isosmotic, urea‐supplemented medium (estimated tonicity, 225 mosmol kg−1). The response of Luc activity to 5 μmol l−1 DBcAMP in the hypotonic medium was not affected in cells either transfected with 0.36 kb of the 5′‐flanking region of AQP‐2 or co‐transfected with −1.1AQP2 and a dominant‐negative TonE binding protein (pDNTonEBP). Pre‐incubation of cells with 1 μmol l−1 SP600125, an inhibitor of c‐Jun N‐terminal kinase (JNK), restored the response of Luc activity to 5 μmol l−1 DBcAMP under hypotonic conditions. These findings may indicate that hypotonicity reduces the cAMP‐induced AQP‐2 promoter activity mediated via TonE by activating JNK kinase.

Soluble ST2 protein inhibits LPS stimulation on monocyte-derived dendritic cells

ST2 protein is a soluble splicing variant of ST2L protein, which is the receptor for interleukin-33 (IL-33). Previously, we reported that soluble ST2 suppressed the signal transduction of lipopolysaccharide (LPS) and cytokine production in monocytic cells. To investigate whether or not this inhibitory effect occurs in dendritic cells, which are the key players in innate and adaptive immunity, human monocyte-derived dendritic cells were pre-treated with soluble ST2 protein before LPS stimulation. Although soluble ST2 did not attenuate the LPS-induced maturation of dendritic cells, pre-treatment with soluble ST2 suppressed cytokine production and inhibited LPS signaling. Moreover, the proliferation of naive T cells was inhibited significantly by soluble ST2 pre-treatment. IL-33 had little effect on the cytokine production of immature monocyte-derived dendritic cells. Furthermore, soluble ST2 protein was internalized into dendritic cells, suggesting that soluble ST2 protein acts by a noncanonical mechanism other than the sequestration of IL-33.