interleukin-8 and irsogladine

Periodontitis is an infectious disease caused by an interaction between the host and periodontopathogenic bacteria. Regulating the immune response in human gingival epithelial cells (HGEC) may contribute to the prevention of periodontitis. Irsogladine maleate (IM) has previously been shown to regulate inflammation and the cell-cell junctional barrier in HGEC. In addition to these functions, control of bacterial recognition is important for preventing inflammation in periodontal tissue. Innate immunity in gingival epithelium is the first line of defense and plays a crucial role against bacterial challenge. Therefore, the effect of IM on regulating toll-like receptor 2 (TLR2), which is part of the innate immunity, was determined in this study.. OBA-9, an immortalized human gingival epithelial cell line, and primary cultured HGEC were used in this study. Real-time PCR and western blotting were performed in OBA-9 or HGEC stimulated with whole cells of Porphyromonas gingivalis or with lipopolysaccharide (LPS) derived from P. gingivalis (PgLPS) in the presence or absence of IM to determine expression of TLR2 mRNA and production of TLR2 protein. Small interfering RNA (siRNA) against TLR2 was transfected into OBA-9 to clarify the association between the induction of TLR2 and interleukin-8 (IL-8) production.. The addition of IM into P. gingivalis or PgLPS-induced OBA-9 suppressed IL-8 production (p < 0.01). The addition of IM also abolished the induction of TLR2 by P. gingivalis or PgLPS in OBA-9 and primary cultured HGEC (p < 0.01). The suppressive effect of IM on the induction of TLR2 was also confirmed by immunohistostaining. Stimulation with peptidoglycan, a specific ligand for TLR2, suppressed the expression of toll-like receptor 4 (TLR4) mRNA in the presence of IM (p < 0.01). However, LPS derived from Escherichia coli, a ligand for TLR4, did not induce the expression of TLR2 mRNA. The PgLPS-induced expression of TLR4 mRNA was abolished by IM. Knockdown of TLR2 by siRNA transfection resulted in a weaker response of induction of IL8 mRNA in P. gingivalis or PgLPS-stimulated OBA-9.. These results suggest that IM suppresses the induction of IL-8 production by regulating increased levels of TLR2.

Topics: Cell Line; Cells, Cultured; Epithelial Cells; Gene Knockdown Techniques; Gingiva; Humans; Immunity, Innate; Immunosuppressive Agents; Interleukin-8; Lipopolysaccharides; Porphyromonas gingivalis; RNA, Small Interfering; Toll-Like Receptor 2; Triazines

The airway epithelium of the human nasal mucosa acts as the first physical barrier that protects against inhaled substances and pathogens. Irsogladine maleate (IM) is an enhancer of gastric mucosal protective factors via upregulation of gap junctional intercellular communication (GJIC). GJIC is thought to participate in the formation of functional tight junctions. However, the effects of IM on GJIC and the epithelial barrier in human nasal epithelial cells (HNECs) remain unknown. To investigate the effects of IM on GJIC and the tight junctional barrier in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with IM and the GJIC inhibitors oleamide and 18β-GA. Some cells were pretreated with IM before treatment with TLR3 ligand poly(I:C) to examine whether IM prevented the changes via TLR3-mediated signal pathways. In hTERT-HNECs, GJIC blockers reduced the expression of tight junction molecules claudin-1, -4, -7, occludin, tricellulin, and JAM-A. IM induced GJIC activity and enhanced the expression of claudin-1, -4, and JAM-A at the protein and mRNA levels with an increase of barrier function. GJIC blockers prevented the increase of the tight junction proteins induced by IM. Furthermore, IM prevented the reduction of JAM-A but not induction of IL-8 and TNF-α induced by poly(I:C). In conclusion, IM can maintain the GJIC-dependent tight junctional barrier via regulation of GJIC in upper airway nasal epithelium. Therefore, it is possible that IM may be useful as a nasal spray to prevent the disruption of the epithelial barrier by viral infections and exposure to allergens in human nasal mucosa.

Topics: Antineoplastic Agents; Cell Communication; Epithelial Cells; Gap Junctions; Gene Expression; Glycyrrhetinic Acid; Humans; Interleukin-8; Nasal Mucosa; Oleic Acids; Tight Junction Proteins; Triazines; Tumor Necrosis Factor-alpha

Irsogladine maleate (IM) counters Aggregatibacter actinomycetemcomitans-induced reduction of the gap junction intercellular communication and the expression of zonula occludens-1, which is a major tight junction structured protein, in cultured human gingival epithelial cells (HGEC). In addition, IM obviates the A. actinomycetemcomitans-induced increase in interleukin (IL)-8 levels in HGEC. Thus, by regulating the intercellular junctional complex and chemokine secretion in HGEC, IM may be useful to prevent periodontal disease. To clarify the effects and regulatory mechanism of IM in vivo and in vitro, we examined the expression of E-cadherin and neutrophil chemotaxis induced by A. actinomycetemcomitans under IM pretreatment. Immunohistochemical studies revealed that A. actinomycetemcomitans application to the gingival sulcus decreased the number of cells positive for E-cadherin and increased those positive for cytokine-induced neutrophil chemoattractant-2alpha (CINC-2alpha) in rat gingival epithelium. However, in IM-pretreated rats, A. actinomycetemcomitans application had little effect on CINC-2alpha and E-cadherin in gingival epithelium. In cultured HGEC, real-time PCR and Western blotting showed that IM and the ERK inhibitor PD98059 abolished the A. actinomycetemcomitans-induced increase in CXCL-1 and IL-8 in HGEC. On the other hand, IM, PD98059, and the p38 MAP kinase inhibitor SB203580 recovered the decrease in E-cadherin expression. In addition, conditioned medium from A. actinomycetemcomitans-stimulated HGEC enhanced human neutrophil chemotaxis, compared to that from un-stimulated HGEC or that from A. actinomycetemcomitans-stimulated HGEC under IM pretreatment. Furthermore, IM down-regulated the p38 MAP kinase and ERK phosphorylations induced by A. actinomycetemcomitans. In conclusion, IM may control A. actinomycetemcomitans-induced gingival inflammation by regulating neutrophil migration and E-cadherin expression in gingival epithelium.

Topics: Actins; Aggregatibacter actinomycetemcomitans; Animals; Cadherins; Chemokine CXCL1; Enzyme-Linked Immunosorbent Assay; Epithelium; Gingiva; Humans; Immunoblotting; Interleukin-8; Male; Neutrophil Infiltration; Neutrophils; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Inbred F344; Triazines

Irsogladine maleate (IM) suppresses the increase in interleukin (IL)-8 production induced by outer membrane protein (OMP) 29 from Aggregatibacter (Actinobacillus) actinomycetemcomitans in cultures of human gingival epithelial cells (HGEC). However, how IM suppresses the OMP29-induced increase in IL-8 expression remains unknown. In this study, we focused on intracellular signaling pathways to elucidate the mechanism behind the suppression.. HGEC, which had been pretreated with inhibitors of intracellular signaling molecules, were exposed to OMP29 (1 microg/mL) with or without IM (1 microM). IL-8 expression at the mRNA and protein levels was examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Extracellular signal-regulated kinase (ERK) activity was measured with a p44/42 mitogen-activated protein kinase assay kit.. An ERK inhibitor, PD98059, as well as IM, obviated the OMP29-induced increase in IL-8 levels in HGEC. A Jun kinase inhibitor, SP600125, and a nuclear factor kappaB inhibitor, PDTC, did not influence the OMP29-induced increase in IL-8 mRNA expression. The OMP29 stimulated phosphorylation of ERK in HGEC. Irsogladine maleate inhibited the phosphorylation.. The suppression of the phosphorylation of ERK by IM in HGEC culminates in inhibition of the OMP29-induced increase in IL-8.

Topics: Aggregatibacter actinomycetemcomitans; Bacterial Outer Membrane Proteins; Cells, Cultured; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Gingiva; Humans; Interleukin-8; MAP Kinase Signaling System; Phosphorylation; Protein Kinase Inhibitors; Triazines

To investigate the mucosal protective effect and the mechanisms of action of the anti-ulcer drug irsogladine maleate in gastric injury induced by indomethacin in rats.. Gastric mucosal injury was induced in male Hos:Donryu rats by oral administration of indomethacin at a dose of 48 mg/kg. One hour before indomethacin treatment, animals were orally pretreated with irsogladine maleate at doses of 1 mg/kg, 3 mg/kg or 10 mg/kg. Four hours after indomethacin administration, the animals were sacrificed and their stomachs were rapidly removed and processed for the evaluation of gastric mucosal damage and the determination of the concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-8 and myeloperoxidase (MPO) in mucosal tissues.. Linear hemorrhagic mucosal lesions were observed primarily in the glandular stomach 4 h after oral administration of indomethacin. Pretreatment with irsogladine maleate markedly reduced the number and severity of these lesions in a dose-dependent manner. The mucosal concentrations of proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-8) and MPO, which indicates the degree of mucosal infiltration by neutrophils, increased concomitantly with the occurrence of gastric injury in the indomethacin-treated rats. Pretreatment with irsogladine maleate significantly decreased the levels of these inflammatory factors in gastric tissue elicited by indomethacin.. The mucosal protective effects afforded by irsogladine maleate on gastric injury induced by indomethacin are mediated by inhibition of mucosal proinflammatory cytokine production and neutrophil infiltration, leading to suppression of mucosal inflammation and subsequent tissue destruction.

Topics: Animals; Anti-Ulcer Agents; Cyclic Nucleotide Phosphodiesterases, Type 4; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Gastric Mucosa; Indomethacin; Interleukin-1beta; Interleukin-8; Male; Peroxidase; Phosphodiesterase 4 Inhibitors; Phosphodiesterase Inhibitors; Rats; Stomach Ulcer; Time Factors; Triazines; Tumor Necrosis Factor-alpha

Our previous report has shown that Irsogladine maleate (IM) counters and obviates the reduction in gap junction intercellular communication (GJIC) and the increase in IL-8 levels, respectively, induced by outer membrane protein 29 from Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) in cultured human gingival epithelial cells (HGEC). In addition, IM suppresses the increase in the secretion of IL-8 caused by whole live A. actinomycetemcomitans. These findings implicate the modulation of IL-8 levels by IM in abolishment of the reduction of GJIC in HGEC. Tight junctions are also responsible for cell-cell communication. Zonula occludens protein-1 (ZO-1) is a major tight junction protein. To investigate the regulatory mechanism of intercellular communication mediated by IM, in the present study, we focused on the involvement of IL-8 in A. actinomycetemcomitans-induced change in GJIC and ZO-1 expression in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in levels of Connexin (CX) 43, suggesting that it could abolish the A. actinomycetemcomitans-induced reduction in GJIC in HGEC. CXCR-1 is a receptor of IL-8. The simultaneous addition of A. actinomycetemcomitans and anti-CXCR-1 antibody also abrogated the repression of GJIC and CX43 expression by A. actinomycetemcomitans in HGEC, although the anti-CXCR-1 antibody was less effective than IM. IM inhibited the IL-8-induced reduction in CX43 levels and GJIC in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in the expression of ZO-1, although anti-CXCR-1 antibody did not influence the decrease in ZO-1 mRNA levels caused by A. actinomycetemcomitans. Furthermore, IL-8 had little effect on the mRNA levels of ZO-1. These findings suggest that IL-8 mediates the A. actinomycetemcomitans-induced reduction of GJIC and CX43 expression in HGEC. The regulation of IL-8 levels by IM in HGEC is partially involved in abrogation of the reduction of GJIC and CX43 expression by A. actinomycetemcomitans. Furthermore, the regulatory effect of IM on the expression of CX43 and ZO-1 is different.

Topics: Aggregatibacter actinomycetemcomitans; Cell Communication; Cells, Cultured; Connexin 43; Epithelial Cells; Gap Junctions; Humans; Interleukin-8; Triazines

Gingival epithelial cells first encounter periodontopathogenic bacteria and their metabolic products to produce inflammatory cytokines. Gap junctional intercellular communication (GJIC) is thought to play a critical role in cellular coordination in tissue homeostasis. Gap junctions are structured by connexins (CXs). GJIC response of gingival epithelial cells to the bacteria may be involved in the initiation of periodontal disease. Irsogladine maleate (IM) is known to enhance GJIC through cAMP. In the present study, we examined an effect of IM on GJIC response and on interleukin-8 (IL-8) levels in human gingival epithelial cells (HGEC) exposed to a periodontopathogenic bacterium, Actinobacillus actinomycetemcomitans, and its outer membrane protein (OMP) 29 in order to test the hypothesis that IM has the ability to modulate GJIC and inflammatory responses of gingival epithelial cells to periodontopathogenic bacteria. IM countered the OMP29-induced reduction of GJIC, CX43 levels and cAMP levels in HGEC. The simultaneous addition of OMP29 and dibutyryl cAMP also abrogated the repression of GJIC by OMP29. Furthermore, IM obviated the increase in IL-8 levels in HGEC stimulated by whole live A. actinomycetemcomitans and by OMP29. These findings suggest that IM counters the OMP29-induced GJIC reduction in HGEC through cAMP. IM may eliminate initial perturbation of gingival epithelial cells by regulating responses of GJIC and IL-8 to periodontopathogenic bacterial exposure.

Topics: Aggregatibacter actinomycetemcomitans; Bacterial Outer Membrane Proteins; Cell Communication; Cells, Cultured; Dose-Response Relationship, Drug; Gap Junctions; Gingiva; Humans; Interleukin-8; Triazines