Kiyoshi Ohura

DNA microarray analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues.

In the inflammatory gingival tissues of patients with periodontitis, cytokines such as interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha have been detected. Gingival fibroblasts are the major constituents of gingival tissue. We recently demonstrated that lipopolysaccharide (LPS) from periodontopathic bacteria induces inflammatory reactions in various tissues via CD14 and/or Toll-like receptors (TLRs) in gingival tissues [Biochem. Biophys. Res. Commun. 273 (2000) 1161]. To confirm this, we examined the expression of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, CD14, TLR2, and TLR4 in human gingival fibroblasts (HGFs) obtained from patients with healthy or inflammatory gingiva using DNA microarray analysis. We also studied the expression levels of these proteins by flow cytometric analysis (FACS). The expression levels of all eight genes in the HGFs of the Inflammatory group were significantly higher than those in the Healthy group on DNA microarray analysis. FACS revealed that the expression levels of all eight proteins on the HGFs of the Inflammatory group were higher than those on the Healthy group. Our data indicated that these eight proteins in HGFs are involved in inflammatory conditions in the gingiva, including periodontal disease. Our results suggested that these eight proteins, in turn, act directly or indirectly on the immune response by activating host cells involved in inflammatory processes.

Comparison of inhibitory effects of local anesthetics on immune functions of neutrophils

Immunological effects of a variety of local anesthetics on adhesion, phagocytosis, and the production of superoxide anion and hydrogen peroxide by neutrophils were compared. Neutrophils were isolated by peritoneal lavage from rats, 4 h after injection of 1% glycogen. Lidocaine, mepivacaine, procaine, prilocaine and tetracaine at 1 mg/ml inhibited adhesion, phagocytosis, and the production of superoxide anion and hydrogen peroxide in neutrophils. Moreover, lidocaine, mepivacaine, procaine and prilocaine at 0.1 mg/ml inhibited the production of superoxide anion and hydrogen peroxide but not adhesion or phagocytosis. In contrast, tetracaine at 0.1 mg/ml inhibited phagocytosis, and the production of superoxide anion and hydrogen peroxide but not adhesion. At 0.01 mg/ml, however, tetracaine inhibited the production of superoxide anion and hydrogen peroxide; in contrast, other drugs failed to affect neutrophil function. These results suggest that the local anesthetics may affect adhesion, phagocytosis, and the production of superoxide anion and hydrogen peroxide by neutrophils.

Involvement of CD14 on human gingival fibroblasts in Porphyromonas gingivalis lipopolysaccharide-mediated interleukin-6 secretion.

The lipopolysaccharides (LPS) of Porphyromonas gingivalis are implicated in the initiation and development of periodontal diseases. However, the mechanisms underlying P. gingivalis LPS-mediated periodontal destruction are still unknown. Here, it was found that P. gingivalis LPS activates human gingival fibroblasts (HGF) to release interleukin 6 (IL-6) via CD14. Flow-cytometric analysis showed that HGFs bind to fluorescein-isothiocyanate (FITC)-labelled LPS, and express CD14 on their surfaces. The binding of FITC LPS was competitively suppressed by unlabelled synthetic lipid A as well as by LPS. LPS-induced IL-6 production was inhibited by anti-CD14 monoclonal antibody in a dose-dependent manner. The binding of FITC LPS to HGF was abrogated by anti-CD14 monoclonal antibody. Engagement of LPS initiated the protein tyrosine phosphorylation of several intracellular proteins including extracellular signal-regulated kinase (ERK) 1 and 2, and these events were suppressed by the anti-CD14 monoclonal. These results suggest that CD14 is a cell surface binding site for LPS and is involved in the LPS-mediated activation of HGF.

Histamine inhibits chemotaxis, phagocytosis, superoxide anion production, and the production of TNFα and IL-12 by macrophages via H2-receptors

Histamine is released from stimulated basophils and mast cells, and plays an important role in the pathogenesis of allergic inflammatory processes. In vitro treatment of macrophages with histamine resulted in inhibition of chemotaxis. Moreover, histamine at l0(-5) M markedly inhibited the production of superoxide anions by both opsonized zymosan-A and phorbol 12-myristate 13-acetate (PMA) stimulated macrophages and histamine at a concentration range of 10(-7) to 10(-5) M significantly inhibited phagocytosis of Escherichia coli by macrophages. In addition, H2-selective receptor agonist dimaprit resulted in inhibition of macrophage chemotaxis and markedly inhibited the production of superoxide anion by PMA-stimulated macrophages and phagocytosis of E. coli by macrophages. On the other hand, histamine and dimaprit both resulted in a concentration-dependent inhibition of lipopolysaccharide-induced production of TNFalpha and IL-12 by macrophages. These results suggest that histamine and dimaprit may inhibit chemotaxis, phagocytosis, superoxide anion production, and the production of TNFalpha and IL-12 by macrophages via H2-histamine receptors. reserved.

Identification of specific autoantigens in Sjögren's syndrome by SEREX.

We carried out SEREX (serological analysis of antigens by recombinant cDNA expression cloning) using sera from patients with Sjögrens syndrome (SjS) and investigated the frequencies of autoantibodies against autoantigens identified by SEREX in the sera of healthy individuals (HI) and patients with SjS, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). IFI16 and two kelch‐like proteins, KLHL12 and KLHL7, were found to be novel autoantigens in SjS by SEREX. A markedly high frequency of anti‐IFI16 autoantibodies was observed in the sera of SjS (SjS, 70%; RA, 13%; SLE, 33%; HI, 0%). Interestingly, all serum samples from SjS demonstrated immunoreactivity against one or both of IFI16 and SS‐B/La. The presence of autoantibodies against KLHL12 and KLHL7 in the sera was significantly specific to SjS (23% and 17%, respectively), as they were not detected in RA, SLE or HI. Furthermore, we confirmed that transcripts of these autoantigens were expressed preferentially in the salivary glands and immuno‐privileged testes. Our results suggest these autoantigens may be useful as serological markers for the clinical diagnosis of SjS and may play a crucial role as organ‐specific autoantigens in the aetiopathogenesis of SjS. This study warranted clinical evaluations of autoantibodies against IFI16, KLHL12 and KLHL7 in combination with anti‐SS‐B/La autoantibodies.

Hyperglycemia in diabetic rats reduces the glutathione content in the aortic tissue

The glutathione redox cycle plays a major role in scavenging hydrogen peroxide (H2O2) under physiological conditions. Recently, we demonstrated that a high glucose concentration in the culture medium reduced the level of H2O2 scavenging activity of human vascular smooth muscle cells (hVSMCs). We also showed that a high glucose concentration reduced the intracellular glutathione (GSH) content and the rate of uptake of cystine, which itself is a rate-limiting factor that maintains the GSH level (FEBS Lett.421: 19-22,1998). In the present study, we investigated whether the hyperglycemic condition in diabetic rats impairs the glutathione content in the aortic tissue in vivo. Wistar rats were divided into the following three groups: streptozotocin-induced diabetic rats (STZ-D, n=7), insulin-treated STZ-D rats (I-STZ-D, n=8), and non-diabetic controls (C, n=7). Fourteen days after streptozotocin injection, the aortic tissue was extracted and the GSH content in the aortic tissue was measured. Furthermore, the relationship between the GSH content in the aortic tissue and blood glucose level in Otsuka Long-Evans Tokushima Fatty (OLETF) rats aged 30 weeks, which developed diabetes spontaneously, was investigated. The GSH content in the aortic tissue of the STZ-D group (0.99+/-0.14 nmol/mg protein) was significantly lower than that of the control group (1.68+/-0.15 nmol/mg protein). Insulin treatment to the diabetic rats restored the GSH content in the aortic tissue (I-STZ-D group; 1.45+/-0.11 nmol/mg protein). Among the 22 Wistar rats, the GSH content in the aortic tissue was negatively correlated with the blood glucose level (r=-0.69, p<0.01, n=22). Among the OLETF rats, a similar negative correlation between the GSH content in the aortic tissue and blood glucose level was seen (r=-0.64, p<0.05, n=10). We demonstrated in vivo that the hyperglycemic condition in STZ-induced diabetic Wistar rats and OLETF rats reduced the GSH content in aortic tissue. This suggested reduced glutathione redox cycle function of aorta.

Induction of apoptosis in human gingival fibroblasts by a Porphyromonas gingivalis protease preparation

Proteases produced by Porphyromonas gingivalis are believed to contribute to the pathogenesis of periodontal diseases. Here the cytotoxic effects of a purified preparation of a P. gingivalis protease with trypsin-like specificity were tested on human gingival fibroblasts in vitro. The active protease induced apoptotic cell death in the fibroblasts, as indicated by DNA fragmentation and the expression of 7A6 antigen. Thus, the production of proteases by periodontopathic bacteria could be an important factor in the induction of apoptosis of host cells in the aetiology of periodontal diseases.

Endomorphin-2 Modulates Productions of TNF-α, IL-1β, IL-10, and IL-12, and Alters Functions Related to Innate Immune of Macrophages

We evaluate immunological effects of opioid peptide endomorphin-2 on the production of cytokines related to inflammation and Th1/Th2 balance, and functions related to innate immune of rat peritoneal macrophages. Endomorphin-2 inhibited TNF-α, IL-10, and IL-12 productions, but potentiated IL-1β production by macrophages. Moreover, endomorphin-2 potentiated macrophage adhesion to fibronectin, and the expression of adhesion molecule Mac-1 on macrophages. In contrast, endomorphin-2 suppressed phagocytosis of opsonized E. coli by macrophages, without affecting phagocytosis of non-opsonized E. coli. In addition, endomorphin-2 inhibited macrophage chemotaxis, and the production of superoxide anion by macrophages. These results suggest that endomorphin-2 may alter macrophage functions such as cytokine productions and functions related to innate immune.

Immunomodulation of the neutrophil respiratory burst by endomorphins 1 and 2

Opioid peptides were found to be released from cells of the immune system during inflammation and stress, and were associated with altered immune responses. Production of superoxide anions by PMA-stimulated neutrophils was markedly inhibited in a concentration-dependent manner by preincubation for 15 min with 10(-18) - 10(-6) M of the endogenous opioid peptides endomorphin 1 or 2. Inhibition was prevented by prior treatment with the micro-opioid receptor-selective antagonist beta-funaltrexamine at 10(-12) - 10(-8) M, but not the delta-opioid receptor-selective antagonist naltrindole. In contrast, endomorphins 1 and 2 caused significant potentiation of superoxide anion production in unstimulated neutrophils. These results suggest that the endogenous opioid peptides endomorphins 1 and 2 may modulate the production of superoxide anions in neutrophils via mu-opioid receptors.

15-deoxy-Δ12,14-prostaglandin J2, a ligand for peroxisome proliferators-activated receptor-γ, induces apoptosis in human hepatoma cells

Background/Aims: 15‐deoxy‐Δ12,14‐prostaglandin J2 (15‐d‐PGJ2) induces apoptosis in several carcinoma cell lines and is a potent activator of peroxisome proliferators‐activated receptor‐γ (PPAR‐γ). In the present study, we examined the effect of 15‐d‐PGJ2 on human hepatoma cells.