Shuji Mori

MAP kinase-mediated proliferation of DLD-1 carcinoma by the stimulation of protease-activated receptor 2.

Protease-activated receptor-2 (PAR-2) has been demonstrated to be highly expressed in the gastrointestinal tract. In the present study, we investigated the effects of PAR-2 stimulation on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line, in comparison with the PAR-1 stimulation. PAR-2 stimulation by agonist peptide SLIGKV concentration-dependently induced the increase in [Ca2+]i and the proliferation of DLD-1 whereas the inverse peptide LSIGKV did not. Trypin (10(-9) M), an agonist protease for PAR-2, also enhanced the proliferation of DLD-1. The proliferative response of DLD-1 to PAR-2 stimulation was associated with the transient phosphorylation of MEK and MAP kinase, but not p38 MAP kinase and JNK. Inhibition of MEK by PD98059 (50 microM) completely inhibited the proliferation-stimulating effects as well as the phosphorylation of MAP kinase induced by PAR-2 agonist peptide (100 microM) and trypsin (10(-9) M). The prolonged treatment with PAR-2 agonist peptide for more than one hour was required for the enhanced proliferative response, suggesting the existence of unknown long-lasting cooperative signaling with MAP kinase cascade. PAR-1 stimulation by the agonist peptide SFLLRN (100 microM) or thrombin (10(-8) M) produced Ca2+ signaling, however, the stimulation neither produced the cell proliferative response nor the activation of MEK-MAP kinase cascade. These results indicated that Ca2+ signaling induced by PARs activation was not enough for inducing the cell proliferation in DLD-1 cells and that stimulation of PAR-2 can induce the activation of MEK-MAP kinase cascade, leading to the growth promoting response.

Differential effect of LFA703, pravastatin, and fluvastatin on production of IL-18 and expression of ICAM-1 and CD40 in human monocytes.

A novel, proinflammatory cytokine, interleukin (IL)‐18 production was detected in the medium of human monocytes treated with 3‐hydroxy‐3‐methylglutaryl coenzyme‐A (HMG‐CoA) reductase inhibitors, pravastatin, and fluvastatin (0.1 and 1 μM) but not with the statin‐derived lymphocyte function‐associated antigen‐1 (LFA‐1) inhibitor LFA703, which did not inhibit HMG‐CoA reductase. Pravastatin and fluvastatin also induced the production of IL‐18, tumor necrosis factor α (TNF‐α) and interferon‐γ (IFN‐γ) in human peripheral blood mononuclear cells (PBMC) in contrast to LFA703. IL‐18 production by PBMC is located upstream of the cytokine cascade activated by these statins. The IL‐18‐induced cytokine production was demonstrated to be dependent on adhesion molecule expression on monocytes. In the absence and presence of lower concentrations (0.1 and 1 ng/ml) of IL‐18, pravastatin and fluvastatin inhibited the expression of intercellular adhesion molecule (ICAM)‐1 and induced the expression of CD40, whereas LFA703 had no effect. In the presence of higher concentrations (5, 10, and 100 ng/ml) of IL‐18, pravastatin, fluvastatin, and LFA703 similarly inhibited the expression of ICAM‐1 and CD40 as well as the production of IL‐12, TNF‐α, and IFN‐γ in PBMC. The effects of pravastatin and fluvastatin but not LFA703 were abolished by the addition of mevalonate, indicating the involvement of HMG‐CoA reductase in the action of pravastatin and fluvastatin. Thus, the effects of LFA703 were distinct from those of pravastatin and fluvastatin in the presence of lower concentrations of IL‐18. It was concluded that LFA703 has the inhibitory effect on an IL‐18‐initiated immune response without any activation on monocytes.

Histamine downregulates CD14 expression via H2 receptorson human monocytes

Lipopolysaccharide (LPS) binds to LPS-binding protein (LBP) in plasma and is delivered to the cell surface receptor CD14 on human monocyte. LPS is transferred to the transmembrane signaling receptor toll-like receptor (TLR) 4. In the present study, the effect of histamine on the expression of CD14 on human monocytes was investigated. Histamine concentration- and time-dependently decreased the expression of cell surface CD14, whereas histamine did not decrease mRNA for CD14 nor increase soluble CD14 (sCD14). The inhibitory effects of histamine on CD14 expression were antagonized by H2-receptor antagonist, but not by H1 and H3/H4 antagonist. The effects of selective H2-receptor agonists, 4-methylhistamine and dimaprit, on CD14 expression mimicked that of histamine indicating that histamine regulated CD14 expression through the stimulation of H2-receptors. The pretreatment with histamine partially inhibited the LPS-induced TNF-alpha production in human peripheral blood mononuclear cells (PBMC). Such inhibition might be due to the down-regulation of CD14 expression on monocytes by histamine.

Effect of prostaglandin E2 on intercellular adhesion molecule-1 and B7 expression in mixed lymphocyte reaction.

Background. The elevation of plasma interleukin (IL)-18 levels and the expression of intercellular adhesion molecule (ICAM)-1 and B7 on monocytes are involved in acute rejection. Prostaglandin (PG) E2 suppresses the rejection in animal transplantation models; however, little is known about its action mechanism. We examined the effect of PGE2 on the expression of ICAM-1 and B7 in the human mixed leukocyte reaction (MLR) in the presence or absence of IL-18. Methods. We measured the expression of ICAM-1, B7.1, and B7.2 on human monocytes by flow cytometry and determined the associated production of interferon-&ggr; and IL-12 by enzyme-linked immunosorbent assay. The modulatory effects of PGE2 and the relevant PGE2 receptor subtypes were characterized pharmacologically. Results. PGE2 inhibited the expression of ICAM-1, B7.1, and B7.2 on monocytes in MLR in a concentration-dependent manner. Whereas IL-18 significantly induced the expression of ICAM-1, B7.1, and B7.2 on monocytes in MLR and the production of interferon-&ggr; and IL-12, PGE2 inhibited these IL-18–initiated enhancements. The effects of PGE2 were mimicked by selective EP2 and EP4 agonists, but not by EP1 and EP3 agonists. Conclusion. PGE2 strongly inhibited MLR with respect to the expression of ICAM-1, B7.1, and B7.2 via the EP2 and EP4 receptors, irrespective of the presence or absence of IL-18. In the previous study, histamine inhibited ICAM-1 expression in the presence of IL-18 but had no effect in the absence of IL-18. These results indicate that the inhibitory effect of PGE2 may be more general and stronger than that of histamine and may play an important role in future immunosuppressive strategies.

Histidine-rich glycoprotein plus zinc reverses growth inhibition of vascular smooth muscle cells by heparin

Vascular smooth muscle cell (SMC) hyperplasia is known to be an important component in the pathogenesis of arteriosclerosis and restenosis. Although heparin has been well recognized as the representative molecule suppressing SMC growth in vitro, attempts to use heparin as a therapeutic anti-restenosis drug have not favorably influenced the angiographic or clinical outcome after angioplasty in some clinical trials. In this study, we have examined the effect of histidine-rich glycoprotein (HRG), a relatively abundant serum glycoprotein (~100xa0μg/ml in human serum), on the growth inhibition of cultured vascular SMC by heparin. Vascular SMC growth was significantly inhibited by heparin, giving nearly 85% inhibition with 100xa0μg/ml heparin. HRG reversed heparin-induced SMC growth inhibition in a dose dependent manner; 75% restoration of cell growth was observed when 100xa0μg/ml of HRG was co-added with 100xa0μg/ml heparin. Interestingly, micromolar concentrations of the zinc ion (0–10xa0μM), compatible with concentrations released from activated platelets, were found to enhance the restorative action of HRG. Western blot experiment demonstrated no significant amounts of the HRG moiety in fetal bovine serum, eliminating the possible contribution of contaminant HRG from culture media. These findings indicate that HRG, in combination with the zinc ion, plays a role in modulating the SMC growth response in pathophysiological states and explain the lack of success of heparin as a therapeutic anti-restenosis drug in clinical trials.

Analysis of sensitization to carboxymethylcellulose: Identification of high risk group using ELISA and histamine release experiment

AbstractObjective and Design: Carboxymethylcellulose (CMC) has been considered to be inert and is commonly used as an additive in medicines, foods and cosmetics. However, we experienced a patient who developed an anaphylactic reaction to CMC after an upper gastrointestinal examination using a barium meal containing CMC. Therefore, we examined the incidence of sensitization by CMC in healthy subjects, and categorized the high risk group prone to developing anaphylactic response to CMC.n Methods: An ELISA for detecting CMC-specific IgE antibody was developed using serum from the patient as a positive control. In the ten subjects exhibiting high anti-CMC IgE among 387 normal populations, histamine release from isolated leukocytes was performed.n Results: Five of ten subjects with a high IgE titer showed a significant CMC-induced histamine release from leukocyte preparations in vitro as observed in the patient, and were classified as high risk group. There was a correlation between sensitization by CMC and that by Japanese cedar pollen. The incidence of sensitization in females was 2.4 fold higher than that in males.n Conclusions: The combination of ELISA and histamine release experiment made it possible to identify the high risk group for developing anaphylactic response. The administration of high dose CMC as a suspending agent in barium sulfate or injectable corticosteroids to this group should be avoided to prevent anaphylactic reactions in the clinic.