RESUMEN
Amphotericin B, an antifungal drug used to treat candidiasis, has been reported to induce pro-inflammatory cytokine production in cultured cells. This study investigated the effects of amphotericin B on pro-inflammatory cytokine production in response to lipid A, the bioactive component of lipopolysaccharide (LPS) in the cell walls of Gram-negative bacteria. Amphotericin B alone elicited a slight increase in interleukin (IL)-6 and IL-8 production by human gingival fibroblasts. However, amphotericin B synergistically up-regulated lipid A-induced production of IL-6 and IL-8. While amphotericin B minimally activated nuclear factor (NF)-κB, it synergistically increased lipid A-induced NF-κB activation. Pre-treatment with methyl-ß-cyclodextrin (MßCD), a cholesterol-binding agent, reduced IL-6 and IL-8 production in human gingival fibroblasts. Cholesterol-saturated MßCD also reversed cytokine production, suggesting that the synergistic production of cytokines by amphotericin B and lipid A is dependent on cholesterol-rich microdomains. Amphotericin B activated caspase-8. In addition, a caspase-8 inhibitor inhibited IL-6 production by amphotericin B and lipid A. This suggests that caspase-8 is required for the synergistic production of IL-6 by amphotericin B and lipid A. Collectively, our results suggest that periodontal treatment carried out before amphotericin B treatment may protect against lipid A-induced pro-inflammatory cytokine production.
Asunto(s)
Anfotericina B/farmacología , Antifúngicos/farmacología , Caspasa 8/metabolismo , Activación Enzimática/efectos de los fármacos , Encía/efectos de los fármacos , Interleucina-6/biosíntesis , Lípido A/farmacología , Células Cultivadas , Colesterol/metabolismo , Periodontitis Crónica/metabolismo , Sinergismo Farmacológico , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Encía/citología , Encía/metabolismo , Humanos , Interleucina-8/biosíntesis , Microdominios de Membrana/metabolismo , FN-kappa B/metabolismo , Activación Transcripcional , Regulación hacia Arriba , beta-Ciclodextrinas/farmacologíaRESUMEN
BACKGROUND AND OBJECTIVE: Nitrogen-containing bisphosphonates (NBPs) are widely used as anti-bone-resorptive drugs. However, use of NBPs results in inflammatory side-effects, including jaw osteomyelitis. In the present study, we examined the effects of alendronate, a typical NBP, on cytokine production by human peripheral blood mononuclear cells (PBMCs) and gingival fibroblasts incubated with lipid A. METHODS: The PBMCs and gingival fibroblasts were pretreated with or without alendronate for 24 h. Cells were then incubated in the presence or absence of lipid A for a further 24 h. Levels of secreted human interleukin (IL)-1ß, IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) in culture supernatants were measured by ELISA. We also examined nuclear factor-κB (NF-κB) activation in both types of cells by ELISA. Activation of Smad3 in the cells was assessed by flow cytometry. In addition, we performed an inhibition assay using SIS3, a specific inhibitor for Smad3. RESULTS: Pretreatment of PBMCs with alendronate promoted lipid A-induced production of IL-1ß and IL-6, but decreased lipid A-induced IL-8 and MCP-1 production. In human gingival fibroblasts, alendronate pretreatment increased lipid A-induced production of IL-6 and IL-8, and increased NF-κB activation in gingival fibroblasts but not PBMCs stimulated with lipid A. In contrast, alendronate activated Smad3 in both types of cells. Finally, SIS3 inhibited alendronate-augmented IL-6 and IL-8 production by human gingival fibroblasts but up-regulated alendronate-decreased IL-8 production by PBMCs. CONCLUSION: These results suggest that alendronate-mediated changes in cytokine production by gingival fibroblasts occur via regulation of NF-κB and Smad3 activity.
Asunto(s)
Alendronato/farmacología , Conservadores de la Densidad Ósea/farmacología , Citocinas/biosíntesis , Encía/efectos de los fármacos , FN-kappa B/metabolismo , Proteína smad3/metabolismo , Activación Transcripcional/efectos de los fármacos , Análisis de Varianza , Células Cultivadas , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Citocinas/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Encía/citología , Encía/metabolismo , Humanos , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Interleucina-6/biosíntesis , Interleucina-6/genética , Interleucina-8/biosíntesis , Interleucina-8/genética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lípido A/inmunología , Proteína smad3/antagonistas & inhibidoresRESUMEN
BACKGROUND: Chronic periodontitis is caused by mixed bacterial infection. Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are frequently detected in deep periodontal pockets. We demonstrate that these bacteria induce proinflammatory cytokine production by the mouse macrophage-like cell line J774.1. MATERIALS AND METHODS: J774.1 cells were incubated with and without bacteria for 24h in 96-well flat-bottomed plates. The culture supernatants were analyzed by enzyme-linked immunosorbent assay for secreted mouse interleukin (IL)-6, monocyte chemoattractant protein-1, IL-23, IL-1 beta and tumor necrosis factor-alpha. The cytokine concentrations were determined using a standard curve prepared for each assay. RESULTS: Mixed infection with P. gingivalis and either T. forsythia or T. denticola at 10(5)CFU/ml acted synergistically to increase IL-6 production, but not monocyte chemoattractant protein-1, IL-23, IL-1 beta or tumor necrosis factor-alpha production. Gingipain inhibitors KYT-1 and KYT-36 inhibited IL-6 production by J774.1 cells incubated with 10(5)CFU/ml of mixed bacteria. CONCLUSION: These results suggest that P. gingivalis with either T. forsythia or T. denticola directly induces synergistic IL-6 protein production and that gingipains play a role in this synergistic effect.
Asunto(s)
Citocinas/metabolismo , Forsythia/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Porphyromonas gingivalis/inmunología , Treponema denticola/inmunología , Animales , Línea Celular , RatonesRESUMEN
We recently separated a PG1828-encoded triacylated lipoprotein (Pg-LP), composed of two palmitoyl and one pentadecanoyl groups at the N-terminal of glycerocysteine from Porphyromonas gingivalis, a periodontopathic bacteria, and found that Pg-LP exhibited definite biological activities through Toll-like receptor (TLR) 2. In the present study, we synthesized 12 different Pg-LP N-terminal peptide moieties (PGTP) using four combinations of glyceryl (R and S) and cysteinyl (l and d) stereoisomers, and three different acyl group regioisomers, N-pentadecanoyl derivative (PGTP1), S-glycero 2-pentadecanoyl derivative (PGTP2) and S-glycero 3-pentadecanoyl derivative (PGTP3). All the PGTP compounds (RL, SL, SD, RD) tested showed TLR2-dependent cell activation. The activating capacities of the PGTP-R compounds were more potent than those of the PGTP-S compounds, whereas there were no differences between the PGTP-L and -D compounds. Furthermore, the production of interleukin (IL)-6 following stimulation with the PGTP1-RL, PGTP2-RL and PGTP3-RL compounds was impaired in peritoneal macrophages from TLR2 knock-out (KO), but not those from TLR1 KO or TLR6 KO mice. These results suggest that P. gingivalis triacylated lipopeptides are capable of activating host cells in a TLR2-dependent and TLR1-/TLR6-independent manner, and the fatty acid residue at the glycerol position in the PGTP molecule plays an important role in recognition by TLR2.
Asunto(s)
Proteínas Bacterianas/química , Lipoproteínas/química , Porphyromonas gingivalis/química , Adulto , Animales , Proteínas Bacterianas/inmunología , Células Cultivadas , Femenino , Humanos , Interleucina-6/biosíntesis , Lipopolisacáridos/inmunología , Lipoproteínas/inmunología , Activación de Macrófagos , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Porphyromonas gingivalis/inmunología , Transducción de Señal/fisiología , Estereoisomerismo , Relación Estructura-Actividad , Receptor Toll-Like 1/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 6/inmunologíaRESUMEN
We have reported previously that Lactobacillus casei ssp. casei, together with specific substrate dextran, exhibited an adjuvant effect of stimulating humoral immune responses against bovine serum albumin (BSA) as a model antigen in BALB/c mice. In the present study, among the Lactobacillus species tested, L. casei ssp. casei with dextran significantly elevated the natural killer (NK) cell activities in spleen mononuclear cells from BALB/c mice in comparison to L. casei ssp. casei alone or other Lactobacillus species with or without dextran. Oral administration of L. casei ssp. casei together with dextran also resulted in a significant increase of NK cell activities in healthy human volunteers. Further, L. casei ssp. casei induced significant production of interleukin (IL)-12 in human peripheral blood mononuclear cells and IL-15 mRNA expression in the human intestinal epithelial cell line Caco-2. L. casei ssp. casei with dextran in food also significantly elevated the survival rate of BALB/c mice bearing Meth-A cells. Taken together, these results demonstrate that dietary synbiotic supplementation which is a combination of the L. casei ssp. casei used as a probiotic together with the dextran, a specific substrate as a prebiotic, efficiently elicits murine and human NK cell activities.
Asunto(s)
Dextranos/administración & dosificación , Células Asesinas Naturales/inmunología , Lacticaseibacillus casei/inmunología , Probióticos , Adulto , Animales , Formación de Anticuerpos , Línea Celular , Suplementos Dietéticos , Femenino , Humanos , Interleucina-12/análisis , Interleucina-15/genética , Lacticaseibacillus casei/fisiología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/microbiología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Albúmina Sérica Bovina/administración & dosificaciónRESUMEN
Oral treponemes are well-known as causative agents of periodontal diseases; however, the details have not been fully clarified. Here, we examined the effects of Treponema medium glycoconjugate on the activation of human gingival fibroblasts using phenol-water extracts from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum subsp. nucleatum, and Actinobacillus actinomycetemcomitans. The phenol-water extracts activated human gingival fibroblasts to mediate IL-8 production, as well as IL-8 mRNA expression, phosphorylation of p38 mitogen-activated protein kinase, and expression of intercellular adhesion molecule-1. T. medium glycoconjugate exhibited no activation of human gingival fibroblasts, while phenol-water extract-induced activation of human gingival fibroblasts was clearly inhibited by T. medium glycoconjugate. Furthermore, binding of biotinylated phenol-water extracts to CD14 in the presence of LPS-binding protein was blocked with T. medium glycoconjugate. These results suggest that T. medium glycoconjugate has an inhibitory effect on host cell activation by periodontopathic bacteria caused by binding to CD14- and LPS-binding protein.
Asunto(s)
Extractos Celulares/farmacología , Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Glicoconjugados/farmacología , Bacterias Gramnegativas/fisiología , Enfermedades Periodontales/microbiología , Treponema/fisiología , Proteínas de Fase Aguda/inmunología , Aggregatibacter actinomycetemcomitans/fisiología , Proteínas Portadoras/inmunología , Células Cultivadas , Fibroblastos/inmunología , Fusobacterium nucleatum/fisiología , Encía/citología , Encía/inmunología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-8/biosíntesis , Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/inmunología , Glicoproteínas de Membrana/inmunología , Fenoles , Fosforilación , Porphyromonas gingivalis/fisiología , Prevotella intermedia/fisiología , Treponema/inmunología , Agua , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The atmospheric chemistry of (CF3)2CHOCH3, a possible HCFC/HFC alternative, was studied using a smog chamber/FT-IR technique. OH radicals were prepared by the photolysis of ozone in a 200-Torr H2O/O3/O2 gas mixture held in an 11.5-dm3 temperature-controlled chamber. The rate constant, k1, for the reaction of (CF3)2CHOCH3 with OH radicals was determined to be (1.40 +/- 0.28) x 10(-12) exp[(-550 +/- 60)/T] cm3 molecule(-1) s(-1) by means of a relative rate method at 253-328 K. The value of k1 at 298 K was (2.25 +/- 0.04) x 10(-13) cm3 molecule(-1) s(-1). The random errors are reported with +/-2 standard deviations, and potential systematic errors of 15% could increase k(1). In considering OH-radical reactions, we estimated the tropospheric lifetime of (CF3)2CHOCH3 to be 2.0 months using the rate constant at 288 K. The degradation mechanism of (CF3)2CHOCH3 initiated by OH radicals was also investigated using FT-IR spectroscopy at 298 K. Products (CF3)2CHOC(O)H, CF3C(OH)2CF3, CF3C(O)OCH3, and COF(2) were identified and quantified. The branching ratio, k1a/k1b, was estimated to be 2.1:1 for reactions (CF3)2CHOCH3 + OH --> (CF3)2CHOCH2*+ H2O (k1a) and (CF3)2CHOCH3 + OH --> (CF3)2C*OCH3 + H2O (k1b).
RESUMEN
We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin-8 (IL-8) responses of the cells to Salmonella lipopolysaccharide, a water-soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll-like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR-related molecules, i.e. TLR2, TLR4, MD-2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL-8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL-8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2- and TLR4-independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti-CD14 MAb.
Asunto(s)
Proteínas de Drosophila , Encía/inmunología , Interleucina-8/biosíntesis , Receptores de Lipopolisacáridos/fisiología , Glicoproteínas de Membrana/fisiología , Ligamento Periodontal/inmunología , Receptores de Superficie Celular/fisiología , Acetilmuramil-Alanil-Isoglutamina/inmunología , Análisis de Varianza , Proteínas Bacterianas/inmunología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/microbiología , Citometría de Flujo , Encía/citología , Encía/metabolismo , Encía/microbiología , Bacterias Gramnegativas/inmunología , Bacterias Grampositivas/inmunología , Humanos , Lipopolisacáridos/inmunología , Peptidoglicano/inmunología , Ligamento Periodontal/citología , Ligamento Periodontal/metabolismo , Ligamento Periodontal/microbiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no Paramétricas , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Gingival epithelial cells may form the first barriers of defense against oral bacteria in periodontal tissues. We stimulated human gingival epithelial cells (keratinocytes) in primary culture, the oral epithelial cell line KB and the colonic epithelial cell line SW620 with various bacterial cell-surface components in the presence or absence of soluble CD14 (sCD14). The SW620 produced interlukin-8 (IL-8) in an sCD14-dependent manner in response to lipopolysaccharide, lipoteichoic acid and peptidoglycan. However, the primary gingival epithelial cells and KB cells did not show enhanced production of IL-8 upon stimulation with these components even in the presence of serum. These human epithelial cells were devoid of membrane CD14, as determined by flow cytometry, and CD14 mRNA expression, as determined by reverse transcriptase-PCR. In contrast, gingival epithelial cells and KB cells expressed the mRNA expression for Toll-like receptor (TLR) 2, TLR4, MD2 and MyD88 to the similar extent to those observed in SW620 cells.
Asunto(s)
Colon/inmunología , Proteínas de Drosophila , Células Epiteliales/inmunología , Encía/inmunología , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/inmunología , Peptidoglicano/inmunología , Adulto , Células Cultivadas , Niño , Colon/citología , Citocinas/metabolismo , Femenino , Encía/citología , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Ácidos Teicoicos/inmunología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Células Tumorales CultivadasRESUMEN
Lipopolysaccharide (LPS) preparations from gram-negative black-pigmented bacteria such as Porphyromonas gingivalis and Prevotella intermedia activate cells from non-LPS-responsive C3H/HeJ mice, but it is still unclear whether this activity is due to the unique structure of LPS or to a minor component(s) responsible for the activity in the preparation. A nonendotoxic glycoprotein with bioactivity against cells from C3H/HeJ mice was purified from a hot phenol-water extract of P. intermedia ATCC 25611 and designated Prevotella glycoprotein (PGP). Treatment of human monocytic THP-1 cells with 22-oxyacalcitriol (OCT) induced maturation and marked expression of CD14 on the cells, but the cells constitutively expressed Toll-like receptor 2 (TLR2) and TLR4 on the cells irrespective of the treatment. PGP induced a high level of interleukin-8 production at doses of 100 ng/ml and higher in OCT-treated THP-1 cells compared with Salmonella LPS, and the production was significantly inhibited by anti-CD14 and anti-TLR2 but not anti-TLR4 antibodies. Consistent with this, TLR2-deficient murine macrophages did not respond to PGP. It was also shown that PGP activity on the THP-1 cells was LPS-binding protein dependent and was inhibited by a synthetic lipid A precursor IV(A). These results indicate that PGP activates monocytic cells in a CD14- and TLR2-dependent manner.
Asunto(s)
Proteínas de Fase Aguda , Proteínas Bacterianas/metabolismo , Proteínas de Drosophila , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Prevotella intermedia/metabolismo , Receptores de Superficie Celular/metabolismo , Glicoesfingolípidos Acídicos/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Calcitriol/análogos & derivados , Calcitriol/farmacología , Proteínas Portadoras/metabolismo , Interleucina-8/biosíntesis , Receptores de Lipopolisacáridos/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/metabolismo , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Receptores de Superficie Celular/biosíntesis , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
An analog of 1alpha,25-dihydroxyvitamin D3, 22-oxyacalcitriol (OCT), differentiated human monocytic THP-1 and U937 cells to express membrane CD14 and rendered the cells responsive to bacterial cell surface components. Both THP-1 and U937 cells expressed Toll-like receptor 4 (TLR4) on the cell surface and TLR4 mRNA in the cells, irrespective of OCT treatment. In contrast, OCT-treated U937 cells scarcely expressed TLR2 mRNA, while OCT-treated THP-1 cells expressed this transcript. Muramyldipeptide (MDP) by itself exhibited only a weak ability to induce secretion of inflammatory cytokines such as interleukin-8 (IL-8) in the OCT-differentiated THP-1 cells but showed marked synergistic effects with Salmonella lipopolysaccharide (LPS) or lipoteichoic acid (LTA) from Staphylococcus aureus, both of which exhibited strong activities. Combinatory stimulation with LPS plus LTA did not show a synergistic effect on OCT-differentiated THP-1 cells. Similar results were observed in OCT-differentiated U937 cells, although combination experiments were carried out only with MDP plus LPS. Anti-CD14 monoclonal antibody (MAb) MY4, anti-TLR4 MAb HTA125, and the synthetic lipid A precursor LA-14-PP almost completely inhibited the IL-8-inducing activities of LTA as well as LPS on OCT-treated THP-1 cells, but these treatments increased MDP activity. OCT-treated THP-1 cells primed with MDP exhibited enhanced production of IL-8 upon stimulation with LPS, while the cells primed with LPS showed no change in production upon stimulation with MDP. MDP up-regulated mRNA expression of an adapter molecule to TLRs, MyD88, to an extent similar to that for LPS in OCT-treated THP-1 cells. These findings suggested that LTA as well as LPS activated human monocytic cells in a CD14- and TLR4-dependent manner, whereas MDP exhibited activity in a CD14-, TLR4-, and probably TLR2-independent manner and exhibited synergistic and priming effects on the cells for cytokine production in response to various bacterial components.