RESUMO
Bisphosphonates (BPs) are major anti-bone-resorptive drugs. Among them, the nitrogen-containing BPs (NBPs) exhibit much stronger anti-bone-resorptive activities than non-nitrogen-containing BPs (non-NBPs). However, BP-related osteonecrosis of the jaw (BRONJ) has been increasing without effective strategies for its prevention or treatment. The release of NBPs (but not non-NBPs) from NBP-accumulated jawbones has been supposed to cause BRONJ, even though non-NBPs (such as etidronate (Eti) and clodronate (Clo)) are given at very high doses because of their low anti-bone-resorptive activities. Our murine experiments have demonstrated that NBPs cause inflammation/necrosis at the injection site, and that Eti and Clo can reduce or prevent the inflammatory/necrotic effects of NBPs by inhibiting their entry into soft-tissue cells. In addition, our preliminary clinical studies suggest that Eti may be useful for treating BRONJ. Notably, Eti, when administered together with an NBP, reduces the latter's anti-bone-resorptive effect. Here, on the basis of the above background, we examined and compared in vitro interactions of NBPs, non-NBPs, and related substances with hydroxyapatite (HA), and obtained the following results. (i) NBPs bind rapidly to HA under pH-neutral conditions. (ii) At high concentrations, Eti and Clo inhibit NBP-binding to HA and rapidly expel HA-bound NBPs (potency Eti>>Clo). (iii) Pyrophosphate also inhibits NBP-binding to HA and expels HA-bound NBPs. Based on these results and those reported previously, we discuss (i) possible anti-BRONJ strategies involving the use of Eti and/or Clo to reduce jawbone-accumulated NBPs, and (ii) a possible involvement of pyrophosphate-mediated release of NBPs as a cause of BRONJ.
Assuntos
Difosfatos/farmacologia , Difosfonatos/metabolismo , Durapatita/metabolismo , Cálcio/farmacologia , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , NitrogênioRESUMO
Bisphosphonates (BPs), with a non-hydrolysable P-C-P structure, are cytotoxic analogues of pyrophosphate, bind strongly to bone, are taken into osteoclasts during bone-resorption and exhibit long-acting anti-bone-resorptive effects. Among the BPs, nitrogen-containing BPs (N-BPs) have far stronger anti-bone-resorptive effects than non-N-BPs. In addition to their pyrogenic and digestive-organ-injuring side effects, BP-related osteonecrosis of jaws (BRONJ), mostly caused by N-BPs, has been a serious concern since 2003. The mechanism underlying BRONJ has proved difficult to unravel, and there are no solid strategies for treating and/or preventing BRONJ. Our mouse experiments have yielded the following results. (a) N-BPs, but not non-N-BPs, exhibit direct inflammatory and/or necrotic effects on soft tissues. (b) These effects are augmented by lipopolysaccharide, a bacterial-cell-wall component. (c) N-BPs are transported into cells via phosphate transporters. (d) The non-N-BPs etidronate (Eti) and clodronate (Clo) competitively inhibit this transportation (potencies, Clo>Eti) and reduce and/or prevent the N-BP-induced inflammation and/or necrosis. (e) Eti, but not Clo, can expel N-BPs that have accumulated within bones. (f) Eti and Clo each have an analgesic effect (potencies, Clo>Eti) via inhibition of phosphate transporters involved in pain transmission. From these findings, we propose that phosphate-transporter-mediated and inflammation/infection-promoted mechanisms underlie BRONJ. To treat and/or prevent BRONJ, we propose (i) Eti as a substitution drug for N-BPs and (ii) Clo as a combination drug with N-BPs while retaining their anti-bone-resorptive effects. Our clinical trials support this role for Eti (we cannot perform such trials using Clo because Clo is not clinically approved in Japan).
Assuntos
Analgésicos/efeitos adversos , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/etiologia , Conservadores da Densidade Óssea/efeitos adversos , Denosumab/efeitos adversos , Difosfonatos/efeitos adversos , Analgésicos/química , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/tratamento farmacológico , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/imunologia , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/prevenção & controle , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/farmacologia , Conservadores da Densidade Óssea/uso terapêutico , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/imunologia , Denosumab/farmacologia , Difosfonatos/química , Difosfonatos/farmacologia , Difosfonatos/uso terapêutico , Humanos , Interleucina-1/imunologia , Nitrogênio/químicaRESUMO
Bisphosphonate (BP)-related osteonecrosis of the jaw (BRONJ) can occur when enhanced bone-resorptive diseases are treated with nitrogen-containing BPs (N-BPs). Having previously found, in mice, that the non-N-BP etidronate can (i) reduce the inflammatory/necrotic effects of N-BPs by inhibiting their intracellular entry and (ii) antagonize the binding of N-BPs to bone hydroxyapatite, we hypothesized that etidronate-replacement therapy (Eti-RT) might be useful for patients with, or at risk of, BRONJ. In the present study we examined this hypothesis. In each of 25 patients receiving N-BP treatment, the N-BP was discontinued when BRONJ was suspected and/or diagnosed. After consultation with the physician-in-charge and with the patient's informed consent, Eti-RT was instituted in one group according to its standard oral prescription. We retrospectively compared this Eti-RT group (11 patients) with a non-Eti-RT group (14 patients). The Eti-RT group (6 oral N-BP patients and 5 intravenous N-BP patients) and the non-Eti-RT group (5 oral N-BP patients and 9 intravenous N-BP patients) were all stage 2-3 BRONJ. Both in oral and intravenous N-BP patients (particularly in the former patients), Eti-RT promoted or tended to promote the separation and removal of sequestra and thereby promoted the recovery of soft-tissues, allowing them to cover the exposed jawbone. These results suggest that Eti-RT may be an effective choice for BRONJ caused by either oral or intravenous N-BPs and for BRONJ prevention, while retaining a level of anti-bone-resorption. Eti-RT may also be effective at preventing BRONJ in N-BP-treated patients at risk of BRONJ. However, prospective trials are still required.
Assuntos
Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/tratamento farmacológico , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/etiologia , Conservadores da Densidade Óssea/efeitos adversos , Conservadores da Densidade Óssea/uso terapêutico , Difosfonatos/efeitos adversos , Difosfonatos/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Neoplasias/tratamento farmacológico , NitrogênioRESUMO
Periodontal disease is a chronic inflammatory disease that results in the breakdown of the tooth-supporting tissues, and can ultimately lead to resorption of the alveolar bone. Recently, several studies have shown a close relationship between increased interleukin-18 (IL-18) levels and the pathogenesis of chronic periodontitis, a major cause of tooth loss. However, it has yet to be shown whether chronic periodontitis results from or causes an increase in IL-18 after bacterial infection. In the present study, we investigated how IL-18 overexpression relates to periodontal disease using IL-18 transgenic (Tg) mice. IL-18Tg and wild-type mice were inoculated intraorally with Porphyromonas (P.) gingivalis, which has been implicated in the etiology of chronic periodontitis. Seventy days after P. gingivalis infection, alveolar bone loss and gingival cytokine levels were assessed using histo-morphological analysis and enzyme-linked immuno-absorbent assay, respectively. Periodontal bone loss was evoked in IL-18Tg mice, but not in wild-type mice. Interestingly, levels of bone-resorptive cytokines, including IL-1α, IL-1ß, tumor necrosis factor-α, and IL-6, were unchanged in the gingival tissues of IL-18Tg mice infected with P. gingivalis, although levels of interferon γ (a proinflammatory T-helper 1 cytokine) decreased. RT-PCR analysis showed elevated expression of mRNAs for receptor activator of nuclear factor kappa-B ligand (a key stimulator of osteoclast development and activation) and CD40 ligand (a marker of T cell activation) in the gingiva of IL-18Tg mice infected with P. gingivalis. We conclude that increased IL-18 in the gingival tissues evokes chronic periodontitis after bacterial infection, presumably via a T cell-mediated pathway.
Assuntos
Infecções Bacterianas/complicações , Infecções Bacterianas/metabolismo , Periodontite Crônica/etiologia , Periodontite Crônica/metabolismo , Gengiva/metabolismo , Interleucina-18/metabolismo , Perda do Osso Alveolar/sangue , Perda do Osso Alveolar/complicações , Perda do Osso Alveolar/microbiologia , Perda do Osso Alveolar/patologia , Animais , Infecções Bacterianas/sangue , Infecções Bacterianas/microbiologia , Biomarcadores/metabolismo , Periodontite Crônica/sangue , Periodontite Crônica/microbiologia , Gengiva/microbiologia , Gengiva/patologia , Interleucina-18/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Porphyromonas gingivalis/fisiologia , Células Th1/metabolismo , Células Th2/metabolismo , TransgenesRESUMO
Bruxism and/or clenching, resulting in fatigue or dysfunction of masseter muscles (MM), may cause temporomandibular disorders. Functional support of the microcirculation is critical for prolonged muscle activity. Histamine is a regulator of the microcirculation and is supplied by release from its stores and/or by de novo production via the induction of histidine decarboxylase (HDC). Interleukin (IL)-1, a cytokine involved in temporomandibular disorders, is an inducer of HDC. In the present study, we examined the roles of histamine, HDC and IL-1 in MM activity. Experiments were conducted using our R+G+ model. A mouse restrained (R+) inside a narrow cylinder (front end blocked with a thin plastic strip) gnaws away (G+) the strip to escape, with the weight reduction in the strip serving as an index of MM activity. Fexofenadine (a peripherally acting histamine H1 receptor antagonist) reduced MM activity in normal mice. Both H1 receptor-deficient and HDC-deficient mice exhibited low MM activity. Prolonged R+G+ induced HDC activity in MM. Mast cell-deficient mice exhibited strikingly low HDC induction in MM (and also in the quadriceps femoris muscle) in response to muscle activity or IL-1ß. Mast cells were present around blood vessels and nerves in the epimysium and perimysium of MM. These results, together with others reported previously, suggest that: (i) peripheral histamine supports strenuous MM activity; (ii) strenuous MM activity stimulates mast cells to release histamine and to induce HDC (which replenishes the histamine pool in mast cells, possibly mediated by IL-1); and (iii) peripheral histamine H1 receptor antagonists may be effective in treating temporomandibular disorders or preventing prolonged clenching and/or bruxism.
Assuntos
Antagonistas dos Receptores Histamínicos H1/farmacologia , Histamina/fisiologia , Músculo Masseter/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Animais , Bruxismo/metabolismo , Bruxismo/prevenção & controle , Histamina/metabolismo , Antagonistas dos Receptores Histamínicos H1/uso terapêutico , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Masculino , Músculo Masseter/irrigação sanguínea , Músculo Masseter/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Restrição FísicaRESUMO
The hydrophilic compound 2-hydroxyethyl methacrylate (HEMA) is a major component of dental bonding materials, and it enhances the binding of resin-composites to biomolecules. However, HEMA is a well-known contact sensitizer. We reported previously that intradermal injection of HEMA induces the production of IL-1 locally in the skin. Keratinocytes are the first barrier against chemical insults and constitutively express IL-1α. In this study, we analyzed whether HEMA induces the production of inflammatory cytokines from murine keratinocyte cell line Pam212 cells. We demonstrated that HEMA induced the release of 17-kDa mature IL-1α and caused cytotoxicity. The activity of calpain, an IL-1α processing enzyme, was significantly higher in HEMA-treated cells. The thiol-containing antioxidant N-acetyl cysteine (NAC) inhibited HEMA-induced IL-1α release but not cytotoxicity. NAC inhibited intracellular calpain activity and reactive oxygen species (ROS) production induced by HEMA. NAC post-treatment also inhibited IL-1α release and intracellular ROS production induced by HEMA. Furthermore, HEMA-induced in vivo inflammation also inhibited by NAC. NAC inhibited polymerization of HEMA through adduct formation via sulfide bonds between the thiol group of NAC and the reactive double bond of HEMA. HEMA-induced IL-1α release and cytotoxicity were also inhibited if HEMA and NAC were pre-incubated before adding to the cells. These results suggested that NAC inhibited IL-1α release through decreases in intracellular ROS and the adduct formation with HEMA. We concluded that HEMA induces IL-1α release from skin keratinocytes, and NAC may be a promising candidate as a therapeutic agent against inflammation induced by HEMA.
Assuntos
Acetilcisteína , Calpaína , Camundongos , Animais , Acetilcisteína/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Metacrilatos/toxicidade , Metacrilatos/química , Queratinócitos/metabolismo , InflamaçãoRESUMO
Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is constitutively produced by endothelial cells and plays a vital role in maintaining vascular homeostasis. Chronic periodontitis is an inflammatory disease characterized by bleeding of periodontal tissues that support the tooth. In this study, we aimed to determine the role of PAI-1 produced by endothelial cells in response to infections caused by the primary periodontal pathogen Porphyromonas gingivalis. We demonstrated that P. gingivalis infection resulted in significantly reduced PAI-1 levels in human endothelial cells. This reduction in PAI-1 levels could be attributed to the proteolysis of PAI-1 by P. gingivalis proteinases, especially lysine-specific gingipain-K (Kgp). We demonstrated the roles of these degradative enzymes in the endothelial cells using a Kgp-specific inhibitor and P. gingivalis gingipain-null mutants, in which the lack of the proteinases resulted in the absence of PAI-1 degradation. The degradation of PAI-1 by P. gingivalis induced a delayed wound healing response in endothelial cell layers via the low-density lipoprotein receptor-related protein. Our results collectively suggested that the proteolysis of PAI-1 in endothelial cells by gingipains of P. gingivalis might lead to the deregulation of endothelial homeostasis, thereby contributing to the permeabilization and dysfunction of the vascular endothelial barrier.
Assuntos
Células Endoteliais , Porphyromonas gingivalis , Adesinas Bacterianas/metabolismo , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/farmacologia , Cisteína Endopeptidases Gingipaínas , Humanos , Inibidor 1 de Ativador de Plasminogênio , Porphyromonas gingivalis/fisiologia , CicatrizaçãoRESUMO
Among the bisphosphonates (BPs), nitrogen-containing BPs (N-BPs) have much stronger anti-bone-resorptive actions than non-N-BPs. However, N-BPs have various side effects such as acute influenza-like reactions after their initial administration and osteonecrosis of the jawbones after repeated administration. The mechanisms underlying such effects remain unclear. To overcome these problems, it is important to profile the inflammatory nature of N-BPs. Here, we analyzed the inflammatory reactions induced in mouse ear pinnae by the N-BPs alendronate (Ale) and zoledronate (Zol). We found the following: (i) Ale and Zol each induced two phases of inflammation (early weak and late strong ear swelling); (ii) both phases were augmented by lipopolysaccharides (LPSs; cell-surface constituent of gram-negative bacteria, including oral bacteria), but prevented by inhibitors of the phosphate transporters of solute carrier 20/34 (SLC20/SLC34); (iii) macrophages and neutrophils were involved in both phases of Ale+LPS-induced ear-swelling; (iv) Ale increased or tended to increase various cytokines, and LPS augmented these effects, especially that on interleukin 1ß (IL-1ß); (v) adenosine triphosphate (ATP) was involved in both phases, and Ale alone or Ale+LPS increased ATP in ear pinnae; (vi) the augmented late-phase swelling induced by Ale+LPS depended on both IL-1 and neutrophil extracellular traps (NETs; neutrophil-derived net-like complexes); (vii) neutrophils, together with macrophages and dendritic cells, also functioned as IL-1ß-producing cells, and upon stimulation with IL-1ß, neutrophils produced NETs; (viii) stimulation of the purinergic 2X7 (P2X7) receptors by ATP induced IL-1ß in ear pinnae; (ix) NET formation by Ale+LPS was confirmed in gingiva, too. These results suggest that (i) N-BPs induce both early-phase and late-phase inflammation via ATP-production and P2X7 receptor stimulation; (ii) N-BPs and LPS induce mutually augmenting responses both early and late phases via ATP-mediated IL-1ß production by neutrophils, macrophages, and/or dendritic cells; and (iii) NET production by IL-1ß-stimulated neutrophils may mediate the late phase, leading to prolonged inflammation. These results are discussed in relation to the side effects seen in patients treated with N-BPs. © 2021 American Society for Bone and Mineral Research (ASBMR).
Assuntos
Armadilhas Extracelulares , Lipopolissacarídeos , Trifosfato de Adenosina , Animais , Difosfonatos/farmacologia , Armadilhas Extracelulares/metabolismo , Humanos , Inflamação , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Nitrogênio , Receptores Purinérgicos P2X7RESUMO
PURPOSE: Nitrogen-containing bisphosphonates (NBPs) have powerful anti-bone-resorptive effects (ABREs). However, recent clinical applications have disclosed an unexpected side effect, osteonecrosis of the jaw. We previously found in mice that etidronate (a non-NBP), when coadministered with alendronate (an NBP), inhibited the latter's inflammatory effects. However, etidronate also reduced the ABRE of alendronate. The present study examined in mice the modulating effects of etidronate on the inflammatory and necrotic actions of zoledronate (the NBP with the strongest anti-bone-resorptive activity and the highest incidence of osteonecrosis of the jaw) and on ABREs of various NBPs including zoledronate. MATERIALS AND METHODS: NBPs were subcutaneously injected into ear pinnas of mice and ensuing inflammation and necrosis at the site of the injection were evaluated. ABREs of NBPs were evaluated by analyzing sclerotic bands induced in mouse tibias. RESULTS: Coinjection of etidronate reduced inflammatory and necrotic reactions induced by zoledronate, and also reduced the amount of zoledronate retained within the ear tissue. When both agents were intraperitoneally injected, etidronate reduced the ABRE of zoledronate and those of other NBPs. Notably, etidronate reduced the ABRE of zoledronate even when this non-NBP was injected 16 hours after the injection of zoledronate. Bone scintigram indicated that etidronate reduced the amount of zoledronate that had already bound to bone. CONCLUSIONS: These results suggest that etidronate may 1) inhibit the entry of NBPs into cells related to inflammation and/or necrosis, 2) inhibit the binding of NBPs to bone hydroxyapatite, 3) at least partly eliminate (or substitute for) NBPs that have already accumulated within bones, and thus 4) if used as a substitution drug for NBPs, be effective at treating or preventing NBP-associated osteonecrosis of the jaw.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Ácido Etidrônico/farmacologia , Alendronato/administração & dosagem , Alendronato/antagonistas & inibidores , Animais , Conservadores da Densidade Óssea/administração & dosagem , Conservadores da Densidade Óssea/antagonistas & inibidores , Reabsorção Óssea/fisiopatologia , Reabsorção Óssea/prevenção & controle , Difosfonatos/administração & dosagem , Difosfonatos/antagonistas & inibidores , Difosfonatos/farmacocinética , Orelha Externa/efeitos dos fármacos , Orelha Externa/patologia , Ácido Etidrônico/administração & dosagem , Ácido Etidrônico/análogos & derivados , Ácido Etidrônico/antagonistas & inibidores , Feminino , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Imidazóis/administração & dosagem , Imidazóis/antagonistas & inibidores , Imidazóis/farmacocinética , Mediadores da Inflamação/antagonistas & inibidores , Injeções Intraperitoneais , Injeções Subcutâneas , Interleucina-1/deficiência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Necrose , Osteonecrose/induzido quimicamente , Osteonecrose/fisiopatologia , Osteosclerose/induzido quimicamente , Osteosclerose/prevenção & controle , Pamidronato , Pravastatina/administração & dosagem , Pravastatina/farmacologia , Cintilografia , Compostos Radiofarmacêuticos , Ácido Risedrônico , Tecnécio , Tíbia/diagnóstico por imagem , Tíbia/efeitos dos fármacos , Tíbia/patologia , Fatores de Tempo , Ácido ZoledrônicoRESUMO
Nickel (Ni) is the most frequent metal allergen and induces Th1-dependent type-IV allergies. In local skin, epidermal Langerhans cells (LCs) and/or dermal dendritic cells (DCs) uptake antigens and migrate to draining lymph nodes (LNs). However, the subsets of antigen-presenting cells that contribute to Ni presentation have not yet been identified. In this study, we analyzed the Ni-binding capabilities of murine DCs using fluorescent metal indicator Newport Green. Elicitation of Ni allergy was assessed after intradermal (i.d.) injection of Ni-treated DCs into ear pinnae of Ni-sensitized mice. The Ni-binding capabilities of MHC class IIhi CD11cint migratory DCs were significantly stronger than those of MHC class IIint CD11chi resident DCs and CD11cint PDCA1+ MHC class IIint B220+ plasmacytoid DCs. Migratory DCs in skin-draining and mandibular LNs showed significantly stronger Ni-binding capabilities than those in mesenteric and medial iliac LNs. An i.d. injection of IL-1ß induced the activation of LCs and dermal DCs with strong Ni-binding capabilities. Ni-binding LCs were detected in draining LNs after i.d. challenge with IL-1ß and Ni. Moreover, an i.d. injection of Ni-treated DCs purified from skin-draining LNs elicited Ni-allergic inflammation. These results demonstrated that migratory DCs in skin-draining LNs have strong Ni-binding capabilities and elicit Ni allergy.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Derme/citologia , Níquel/imunologia , Alérgenos/imunologia , Animais , Antígenos CD11/imunologia , Células Cultivadas , Derme/imunologia , Humanos , Interleucina-1beta/imunologia , CamundongosRESUMO
The ultimate goal of periodontal disease treatment is the reorganization of functional tissue that can regenerate lost periodontal tissue. Regeneration of periodontal tissues is clinically possible by using autogenic transplantation of MSCs. However, autologous MSC transplantation is limited depending on age, systemic disease and tissue quality, thus precluding their clinical application. Therefore, we evaluated the efficacy of allogeneic transplantation of adipose-derived multi-lineage progenitor cells (ADMPC) in a micro-mini pig periodontal defect model. ADMPC were isolated from the greater omentum of micro-mini pigs, and flow cytometry analysis confirmed that the ADMPC expressed MSC markers, including CD44 and CD73. ADMPC exhibited osteogenic, adipogenic and periodontal ligament differentiation capacities in differentiation medium. ADMPC showed high expression of the immune suppressive factors GBP4 and IL1-RA upon treatment with a cytokine cocktail containing interferon-γ, tumor necrosis factor-α and interleukin-6. Allogeneic transplantation of ADMPC in a micro-mini pig periodontal defect model showed significant bone regeneration ability based on bone-morphometric analysis. Moreover, the regeneration ability of ADMPC by allogeneic transplantation was comparable to those of autologous transplantation by histological analysis. These results indicate that ADMPC have immune-modulation capability that can induce periodontal tissue regeneration by allogeneic transplantation.
Assuntos
Tecido Adiposo/citologia , Regeneração Óssea , Regeneração Tecidual Guiada Periodontal , Transplante de Células-Tronco , Células-Tronco/citologia , Animais , Biomarcadores , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Citocinas/metabolismo , Imuno-Histoquímica , Imunomodulação , Mediadores da Inflamação/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Modelos Biológicos , Osteogênese , Periodonto/diagnóstico por imagem , Periodonto/patologia , Transplante de Células-Tronco/métodos , Células-Tronco/imunologia , Células-Tronco/metabolismo , Suínos , Porco Miniatura , Engenharia Tecidual , Transplante Homólogo , Microtomografia por Raio-XRESUMO
Toll-like receptor (TLR) family members are pattern-recognition receptors and very important molecules in innate immunity. Although TLRs are originally type I transmembrane receptors, soluble forms of TLRs are detected in human plasma and milk. This study showed that soluble TLR2 (sTLR2) is detected in human parotid saliva. Western blotting with anti-TLR2 antibodies (Abs) showed that three polypeptides are detected as sTLR2 with molecular weights of 55, 40 and 27kDa, respectively. Parotid saliva neutralized the binding of anti-TLR2 polyclonal Ab to cell-surface TLR2 on THP-1, a human monocytic cell line. Immunohistochemical analysis revealed that TLR2 is expressed in serous and interlobular ductal cells of human salivary gland. Human salivary gland cell lines, AZA3 and HSY, constitutively expressed TLR2. Parotid saliva augmented IL-8 production of THP-1 cells stimulated with a synthetic TLR2 ligand, Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)). Depletion of sCD14 from parotid saliva by immunoprecipitation eliminated the augmentation of IL-8 production, indicating that the augmentable effects depended on sCD14 in parotid saliva. On the other hand, preincubation of Pam(3)CSK(4) with parotid saliva abrogated the augmentation of IL-8 production, indicating that sTLR2 in saliva bound to Pam(3)CSK(4) and neutralized its function. These results suggest that parotid saliva modulates the TLR2-mediated immune responses with binary mechanisms via sTLR2 and sCD14 in the oral cavity.
Assuntos
Interleucina-8/imunologia , Monócitos/imunologia , Saliva/imunologia , Receptor 2 Toll-Like/imunologia , Linhagem Celular Tumoral , Humanos , Interleucina-8/biossíntese , Receptores de Lipopolissacarídeos/imunologia , Proteínas do Leite/imunologia , Proteínas do Leite/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Boca/citologia , Boca/imunologia , Boca/metabolismo , Glândula Parótida/citologia , Glândula Parótida/imunologia , Glândula Parótida/metabolismo , Saliva/metabolismo , Receptor 2 Toll-Like/sangueRESUMO
Lactoferrin (Lf) is a member of the transferrin family of iron-binding anti-bacterial proteins, present in most exocrine secretions, such as saliva, and plays an important role in mucosal defense. In this study, we identified small Lf peptides with Con A low-affinity in the parotid saliva of chronic periodontitis patients by Con A two-dimensional immunoelectrophoresis, Con A affinity chromatography and Western blotting using anti-human Lf polyclonal Ab. N-terminal amino acid sequencing of the four Con A low-affinity Lf peptides confirmed them to be fragments of intact Lf. The detection ratio of the proteinase 3 (PR3)-like activity was elevated in the parotid saliva of periodontitis patients and was associated with the severity of clinical symptoms. PR3 protein was also detected in the parotid saliva of periodontitis patients, and PR3, but not human leukocyte elastase and cathepsin G, degraded intact Lf. Con A low-affinity saliva Lf peptides showed no anti-bacterial activity against Escherichia coli, and had a reduced iron-chelating capacity. Con A low-affinity saliva Lf peptides, PR3-treated Lf preparation and two of four synthetic polypeptides induced the production of interleukin IL-6, monocyte chemoattractant protein-1 and IL-8, and the activation of NF-kappaB in human oral epithelial HSC-2 cells. Furthermore, concentrations of the Lf peptides in the parotid saliva of periodontitis patients were increased with a correlation to the severity of clinical symptoms. These results suggest that Lf in the parotid saliva of periodontitis patients was degraded into small peptides by the PR3-like activity with the capability to induce inflammatory mediators.
Assuntos
Lactoferrina/metabolismo , Mieloblastina/metabolismo , Glândula Parótida/metabolismo , Fragmentos de Peptídeos/análise , Periodontite/metabolismo , Saliva/química , Sequência de Aminoácidos , Quimiocinas/metabolismo , Concanavalina A/química , Citocinas/metabolismo , Eletroforese em Gel Bidimensional , Humanos , Lactoferrina/química , Dados de Sequência Molecular , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/imunologia , NF-kappa B/metabolismo , Fragmentos de Peptídeos/farmacologia , Periodontite/diagnósticoRESUMO
Nitrogen-containing bisphosphonates (NBPs) are powerful anti-bone-resorptive drugs, but they frequently induce various inflammatory side effects. Recent clinical applications have disclosed an unexpected new side effect, jaw-bone necrosis and exposure. In vitro studies suggest that the inflammatory effects of NBPs are due to Vgamma2Vdelta2 T-cells, stimulated directly and/or indirectly [the latter via isopentenylpyrophosphate (IPP) in the mevalonate pathway]. Rats and mice, however, lack Vgamma2Vdelta2 T-cells, yet NBPs still induce necrotic and inflammatory reactions. In mice, NBPs induce IL-1-dependent inflammatory reactions, such as inductions of histidine decarboxylase (HDC, the histamine-forming enzyme) in the liver, lung, spleen, and bone marrow, an increase in granulocytic cells in the peritoneal cavity, pleural exudation, and splenomegaly. Here, we examined the involvement of IPP, TNF, macrophages, and T-cells in the inflammatory actions of alendronate (a typical NBP) in mice. Various statins (mevalonate-synthesis inhibitors) suppressed the alendronate-induced HDC inductions, while mevalonate itself augmented such inductions. IPP injection also induced HDC. Like IL-1-deficient mice, TNF-deficient mice were resistant to alendronate-stimulated HDC induction. Alendronate-stimulated HDC inductions were significantly weaker in macrophage-depleted mice and in nude mice than in control mice. Similar, though generally less clear-cut, results were obtained when other alendronate-induced inflammatory reactions were examined. These results suggest that (i) inhibition of the mevalonate pathway causes and/or modifies at least some inflammatory actions of alendronate in mice, (ii) in addition to IL-1, TNF is also involved in the inflammatory actions of alendronate, and (iii) alendronate may act on a variety of cells, including macrophages and T-cells.
Assuntos
Alendronato/farmacologia , Conservadores da Densidade Óssea/farmacologia , Histidina Descarboxilase/metabolismo , Inflamação/induzido quimicamente , Animais , Ácido Clodrônico/farmacologia , Feminino , Histidina Descarboxilase/imunologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Interleucina-1alfa/deficiência , Interleucina-1alfa/genética , Interleucina-1alfa/imunologia , Interleucina-1beta/deficiência , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Lipossomos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/imunologia , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Pulmão/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Ácido Mevalônico/farmacologia , Camundongos , Camundongos Knockout , Cavidade Peritoneal/citologia , Baço/efeitos dos fármacos , Baço/enzimologia , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
To investigate the role of human gingival fibroblasts (HGF), the major constituents of gingival tissue in periodontal inflammatory disease, the expression of interleukin-2 receptor (IL-2R) alpha, beta, and gamma chains was examined. Immunohistochemistry showed a pronounced accumulation of CD8(+) T cells in the inflamed lamina propria of gingival tissue from patients with adult periodontitis. HGF express IL-2Rbeta and IL-2Rgamma at mRNA and protein levels, but the expression of IL-2Ralpha could not be detected, as assessed by reverse transcriptase-polymerase chain reaction and flow cytometry. IL-2Rbeta, and -gamma expressed on HGF were functionally active, as addition of neutralizing anti-IL-2Rbeta and -gamma antibodies caused inhibition of the IL-2-induced production of monocyte chemoattractant protein-1 (MCP-1), and addition of IL-2 induced phosphorylation of Janus tyrosine kinase 3, which is critical in signaling through IL-2Rgamma in HGF. The IL-2-induced MCP-1 production was significantly inhibited by pretreatment with neutralizing antibody to IL-15. Addition of IL-2 also induced a marked up-regulation of the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of HGF, which in turn, significantly augmented the adhesion of human neutrophils, which were inhibited by an anti-ICAM-1 antibody. These results suggest that HGF express functional IL-2Rbetagamma, respond to IL-2 from infiltrated T cells, and actively participate in the inflammatory process in the periodontal region and that IL-15 produced by HGF sustains IL-2-mediated signaling in HGF.
Assuntos
Adesão Celular/efeitos dos fármacos , Fibroblastos/metabolismo , Gengiva/metabolismo , Interleucina-2/farmacologia , Neutrófilos/metabolismo , Receptores de Interleucina-2/metabolismo , Adulto , Antígenos CD4/farmacologia , Células Cultivadas , Primers do DNA/química , Citometria de Fluxo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-15/metabolismo , Janus Quinase 3 , Lipopolissacarídeos/farmacologia , Ativação de Neutrófilo , Periodontite/metabolismo , Fosforilação , Isoformas de Proteínas , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Linfócitos T/metabolismo , Regulação para CimaAssuntos
Conservadores da Densidade Óssea/efeitos adversos , Ácido Etidrônico/análogos & derivados , Doenças Mandibulares/tratamento farmacológico , Osteomielite/tratamento farmacológico , Osteonecrose/tratamento farmacológico , Osteoporose Pós-Menopausa/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Ligação Competitiva , Conservadores da Densidade Óssea/metabolismo , Conservadores da Densidade Óssea/uso terapêutico , Difosfonatos , Ácido Etidrônico/efeitos adversos , Ácido Etidrônico/química , Ácido Etidrônico/metabolismo , Ácido Etidrônico/uso terapêutico , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/metabolismo , Doenças Mandibulares/induzido quimicamente , Doenças Mandibulares/diagnóstico por imagem , Doenças Mandibulares/metabolismo , Nitrogênio , Osteomielite/complicações , Osteomielite/metabolismo , Osteonecrose/induzido quimicamente , Osteonecrose/complicações , Osteoporose Pós-Menopausa/complicações , Cintilografia , Compostos Radiofarmacêuticos , Ácido Risedrônico , Compostos de Tecnécio , Extração Dentária/efeitos adversosRESUMO
It is speculated that more than 400 bacterial species reside in the oral cavity. Some cause inflammation (e.g. periodontitis), understanding of which requires examination of innate immunity in the oral cavity. Oral mucosal cells such as epithelial cells are thought to act as a physical barrier against the invasion of pathogenic organisms, but they have an ability to produce inflammatory cytokines and express adhesion molecules. Oral epithelial cells are refractory to many bacterial components although they express Toll-like receptors/MyD88, and acquire responsiveness after priming with IFN-gamma. When the cells are stimulated with lipopolysaccharide (LPS) and neutrophil protease (PR3) after IFN-gamma priming, the cells produce bio-active IL-18, which is critical to Th1 and Th2 responses. PR3 itself is able to activate the cells through G protein-coupled protease-activated receptor-2 on the cell surface. These results suggest that innate immune responses of oral epithelial cells to bacterial components are regulated in the inflammatory process. In addition, saliva contains abundant bio-active CD14 from salivary glands in a soluble form, although LPS-binding protein was below detectable levels, suggesting that saliva CD14 is important for the maintenance of oral health.
Assuntos
Citocinas/biossíntese , Glicoproteínas de Membrana/fisiologia , Mucosa Bucal/imunologia , Receptores de Superfície Celular/fisiologia , Bactérias/patogenicidade , Moléculas de Adesão Celular , Técnicas de Cultura de Células , Citocinas/farmacologia , Células Epiteliais , Humanos , Inflamação , Receptores de Lipopolissacarídeos , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/imunologia , Saliva/imunologia , Células Th1/imunologia , Células Th2/imunologia , Receptores Toll-LikeRESUMO
OBJECTIVE: Nitrogen-containing bisphosphonates (NBPs), the first-choice drugs for diseases that cause enhanced bone resorption, may injure jawbones and gastrointestinal tissues. In rodents, NBPs cause necrosis at injection sites. Bisphosphonates accumulate within bones, especially where there is inflammation. We hypothesized that if jawbone-accumulated NBPs are released, they may directly injure cells around the jawbones. To examine this hypothesis, we compared the direct effects of zoledronate (NBP) and/or etidronate (non-NBP) on various cells, including periodontal cells. DESIGN: Various human tumour cells (such as squamous carcinoma cells and prostate adenocarcinoma cells) and periodontal cells (such as gingival fibroblasts and periodontal ligament cells) were incubated with or without zoledronate and/or etidronate. Cell viability and cytotoxicity were determined by tetrazolium dye assay and by FITC-Annexin V/propidium iodide assay, respectively. RESULTS: Zoledronate, at 100µM, was toxic to all types of cells tested, while its toxicity varied among cells at both 1 and 10µM. There was no clear difference between tumour cells and non-tumour cells in sensitivity to the cytotoxicity of zoledronate. In contrast, etidronate was not toxic at 1-100µM in any of the cells tested. Interestingly, etidronate reduced the cytotoxicity of zoledronate in many cell-types, including gingival fibroblasts. CONCLUSIONS: These results, together with those reported by others and those from our previous in vivo experiments, suggest that NBPs, upon release from jawbones (e.g., during dental surgery or bone infection), may directly injure various cells located around the jawbones, and that etidronate may be protective against the cytotoxicity of NBPs in periodontal tissues.
Assuntos
Conservadores da Densidade Óssea/toxicidade , Difosfonatos/toxicidade , Ácido Etidrônico/toxicidade , Imidazóis/toxicidade , Adenocarcinoma/patologia , Anexina A5 , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Corantes , Cemento Dentário/efeitos dos fármacos , Difosfonatos/antagonistas & inibidores , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Ácido Etidrônico/farmacologia , Fibroblastos/efeitos dos fármacos , Fluoresceína-5-Isotiocianato/análogos & derivados , Corantes Fluorescentes , Gengiva/citologia , Gengiva/efeitos dos fármacos , Humanos , Imidazóis/antagonistas & inibidores , Leucemia Monocítica Aguda/patologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Propídio , Sais de Tetrazólio , Veias Umbilicais/citologia , Ácido ZoledrônicoRESUMO
Microbial components activate the host's innate immunity via interactions with molecules including TLRs and NODs. We previously reported that in mice (i) Escherichia coli lipopolysaccharide (LPS; TLR4 agonist) promotes Ni-allergy even in T-cell-deficient mice, (ii) E. coli LPS reduces the minimum allergy-inducing concentrations of Ni at both the sensitization and elicitation steps, and (iii) various microbe-related substances promote sensitization to Ni. Here, we examined the effects of microbe-related substances at the elicitation step. Mice (except for TLR4-mutated C3H/HeJ mice) were sensitized to Ni by intraperitoneal injection of NiCl(2) + E. coli LPS. Ten days later their ear-pinnas were challenged with 1 µM NiCl(2) with or without a test substance. Although NiCl(2) alone at this concentration does not induce Ni-allergy, its combination with the following substances induced Ni-allergy in BALB/c mice: LPS preparations from oral gram-negative bacteria (Prevotella intermedia and Porphyromonas gingivalis), a mannan preparation from a fungus (Saccharomyces cerevisiae), and synthetic NOD2 and TLR2 agonists. The effect of the mannan preparation was small in C3H/HeJ mice (sensitized with NiCl(2) + the P. intermedia preparation). The P. intermedia preparation promoted Ni-allergy in C3H/HeJ and nude mice, but not in mice deficient in either TLR2 or histidine decarboxylase. Intragingival injection of the P. intermedia preparation and later challenge with NiCl(2) alone to ear-pinnas also promoted Ni-allergy. These results indicate that (i) in Ni-allergy, a microbial milieu or innate immunity is important at the elicitation step, too, and (ii) some oral bacteria may promote Ni-allergy via TLR2-stimulant(s) production.