RESUMO
Hypertension is a major risk factor for the development of cardiovascular diseases. This study aimed to elucidate whether the natural product mixture No-ap (NA) containing Pine densiflora, Annona muricate, and Monordica charantia, or its single components have inhibitory effects on hypertension-related molecules in Angiotensin II (Ang II)-stimulated H9C2 cells. Individual functional components were isolated and purified from NA using various columns and solvents, and then their structures were analyzed using ESIâ»MS, ¹H-NMR, and 13H-NMR spectra. H9C2 cells were stimulated with 300 nM Ang II for 7 h. NA, telmisartan, ginsenoside, roseoside (Roseo), icariside E4 (IE4), or a combination of two components (Roseo and IE4) were administered to the cells 1 h before Ang II stimulation. The expression and activity of hypertension-related molecules or oxidative molecules were determined using RT-PCR, western blot, and ELISA. Ang II stimulation increased the expression of Ang II receptor 1 (AT1), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), tumor growth factor-ß (TGF-ß) mRNA, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and the levels of hydrogen peroxide (H2O2) and superoxide anion (â¢O2-) and reduced anti-oxidant enzyme activity. NA significantly improved the expression or activities of all hypertension-related molecules altered in Ang II-stimulated cells. Roseo or IE4 pretreatment either decreased or increased the expression or activities of all hypertension-related molecules similar to NA, but to a lesser extent. The pretreatment with a combination of Roseo and IE4 (1:1) either decreased or increased the expression of all hypertension-related molecules, compared to each single component, revealing a synergistic action of the two compounds. Thus, the combination of single components could exert promising anti-hypertensive effects similar to NA, which should be examined in future animal and clinical studies.
Assuntos
Glucosídeos/farmacologia , Glicosídeos/farmacologia , Lignanas/farmacologia , Norisoprenoides/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/genética , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Quimiocina CCL2/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosídeos/química , Glicosídeos/química , Humanos , Peróxido de Hidrogênio/química , Lignanas/química , Norisoprenoides/química , Oxirredução/efeitos dos fármacos , RNA Mensageiro , Ratos , Fator de Crescimento Transformador beta1/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
This study aimed to investigate the role of transglutaminase 2 (TG2) expressed in mast cells in substantia nigra (SN) in Parkinson's disease (PD) model or human PD patients. C57BL/6 mice received 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by ip injection to induce PD. Bone marrow-derived mast cells (BMMCs) were adoptively transferred to TG2 knockout (KO or TG2-/-) mice by iv injection 1â¯day before MPTP injection or stimulated by 1 methyl-4-phenylpyridinium (MMP+). KO-MPTP mice showed reduced expression of tyrosine hydroxylase (TH) and dopamine (DA) transporter (DAT) and loss of TH+ DA neurons, and expression of markers (c-kit, tryptase, FcεRI), mediators' release (histamine, leukotrienes, cytokines), and TG2 related to mast cells, and co-localization of DA neuronal cells and mast cells in SN tissues or release of mediators and TG2 activity in SN tissues and sera versus those in WT (wild type)-MPTP or BMâ¯+â¯KO-MPTP mice. KO-MPTP mice reversed the alterations of behavior. KO-BMMCs-transferred KO-MPTP (BMâ¯+â¯KO-MPTP) mice had restoration of all the responses versus the KO-MPTP mice. MPP+-stimulated BMMCs had increased mediators' release, which were inhibited by TG2 inhibitor (R2 peptide). All the mediators and TG2 activity were also increased in the sera of human PD patients. The data suggest that TG2 expressed in mast cells recruited into SN tissues might contribute to neuroinflammation, which is known as one of the important features in pathogenesis of PD, via up-regulating the release of various mediators.
Assuntos
Proteínas de Ligação ao GTP/biossíntese , Regulação Enzimológica da Expressão Gênica , Mediadores da Inflamação/metabolismo , Mastócitos/metabolismo , Transtornos Parkinsonianos/metabolismo , Transglutaminases/biossíntese , Idoso , Animais , Sobrevivência Celular/fisiologia , Feminino , Proteínas de Ligação ao GTP/genética , Humanos , Masculino , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Transtornos Parkinsonianos/patologia , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/genéticaRESUMO
Osteoarthritis (OA) is the common form of arthritis and is characterized by disability and cartilage degradation. Although natural product extracts have been reported to have anti-osteoarthritic effects, the potential bioactivity of Ryupunghwan (RPH), a traditional Korean medicinal botanical formula that contains Astragalus membranaceus, Turnera diffusa, Achyranthes bidentata, Angelica gigas, Eclipta prostrata, Eucommia ulmoides, and Ilex paraguariensis, is not known well. Therefore, the inhibitory effects of single compounds isolated from RPH on the OA-related molecules were investigated using IL-1ß-stimulated chondrosarcoma SW1353 (SW1353) cell model. Two bioactive compounds, isomucronulatol 7-O-ß-d-glucoside (IMG) and ecliptasaponin A (ES) were isolated and purified from RPH using column chromatography, and then the structures were analyzed using ESI-MS, ¹H-NMR, and 13C-NMR spectrum. The expression or amount of matrix metalloproteinase 13 (MMP13), COX1/2, TNF-α, IL-1ß or p65 was determined by RT-PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA). RPH pretreatment reduced the expression and amounts of MMP13, and the expression of collagen II, COX1/2, TNF-α, IL-1ß or p65, which were increased in IL-1ß-stimulated SW1353 cells. IMG reduced the expression of all OA-related molecules, but the observed inhibitory effect was less than that of RPH extract. The other single compound ES showed the reduced expression of all OA-related molecules, and the effect was stronger than that in IMG (approximately 100 fold). Combination pretreatment of both single components remarkably reduced the expression of MMP13, compared to each single component. These synergic effects may provide potential molecular modes of action for the anti-osteoarthritic effects of RPH observed in clinical and animal studies.
Assuntos
Neoplasias Ósseas/metabolismo , Condrossarcoma/metabolismo , Glucosídeos/farmacologia , Osteoartrite/tratamento farmacológico , Preparações de Plantas/farmacologia , Saponinas/farmacologia , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Condrossarcoma/tratamento farmacológico , Humanos , Interleucina-1beta/farmacologia , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Osteoartrite/metabolismo , Preparações de Plantas/química , Saponinas/isolamento & purificaçãoRESUMO
BACKGROUND: Mast cells and their mediators play important roles in chronic spontaneous urticaria (CSU) pathogenesis. Transglutaminase 2 (TG2) is expressed in activated mast cells and contributes to airway inflammation in allergic asthma. OBJECTIVE: To investigate the role of TG2 in CSU. METHODS: Patients with CSU (n = 72) and healthy controls (n = 51) were evaluated. Skin biopsy specimens were obtained from 5 patients with CSU and 2 healthy controls. Cord blood-derived human mast cells and peripheral blood-derived human mast cells were activated with IgE. TG2 activity and inflammatory mediators, such as histamine, leukotriene C4, and cytokines, were measured in serum or supernatant from cultured mast cells by enzyme-linked immunosorbent assay. Colocalization of mast cells and TG2 was determined in skin tissues by immunofluorescence. RESULTS: TG2 activity was significantly higher in serum samples from patients with CSU than in serum samples from healthy controls (P < .001). Colocalization of mast cell surface marker c-kit and TG2 was significantly increased in the lesional skin of patients with CSU compared with that in healthy controls. The levels of histamine, leukotriene C4, tumor necrosis factor α, transforming growth factor ß, and interleukins 4, 5, and 6 were significantly higher in patients with CSU than in healthy controls (P < .001). Serum TG2 levels had positive correlations with each inflammatory mediator (P < .001). TG2 activity was increased in cord blood-derived human mast cells (CBMCs) and peripheral blood-derived human mast cells activated with IgE compared with those without activation (P < .05). CONCLUSION: Our findings suggest that TG2 expressed in and released from mast cells plays an important role in CSU pathogenesis.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Mastócitos/enzimologia , Transglutaminases/metabolismo , Urticária/metabolismo , Adulto , Citocinas/sangue , Feminino , Proteínas de Ligação ao GTP/sangue , Histamina/sangue , Humanos , Imunoglobulina E/sangue , Leucotrieno C4/sangue , Masculino , Pessoa de Meia-Idade , Proteína 2 Glutamina gama-Glutamiltransferase , Pele/citologia , Testes Cutâneos , Transglutaminases/sangue , Urticária/sangue , Adulto JovemRESUMO
BACKGROUND: Mastocytosis is characterized by a pathological increase in mast cells in organs such as skin and bone marrow. Transglutaminase 2 (TG2) expressed in mast cells contributes to allergic diseases, but its role in mastocytosis has not been investigated. This study aimed to investigate whether TG2 contributes to pediatric mastocytosis. METHODS: Serum, various skin tissues or bone marrow (BM) biopsy and aspirates were obtained from pediatric normal control or patients with indolent systemic mastocytosis (SM), mastocytoma, and urticaria pigmentosa (UP). Tryptase, individual cytokines, leukotriene C4 (LTC4 ), and TG2 activity in the serum were determined by enzyme-linked immunosorbent assay, mast cell population by May-Grünwald-Giemsa, CD 117 by immunofluorescence, cell surface molecules by Western blot, and colocalization of c-kit and TG2 or IL-10-expressing cells, CD25, and FOXP3 by immunohistochemistry. RESULTS: Infiltration of CD25(+) CD117(+) CD2(-) mast cells into BM and scalp/trunk/ear dermis; expression of FcεRI, tryptase, c-kit, FOXP3, CCL2/CCR2, and vascular cell adhesion molecule-1; and colocalization of c-kit and TG2 were enhanced in patient's skin tissues or BM, particularly SM, but colocalization of c-kit and IL-10-expressing cells was decreased vs. normal tissues. Amounts of LTC4 and inflammatory cytokines, expression of tryptase or TG2 activity were increased in patient's serum, BM aspirates, or ear/scalp skin tissues, respectively, vs. normal persons, but IL-10 level was decreased. CONCLUSION: The data suggest that mast cells, recruited in the skin and BM by CCL2/CCR, may induce the development of pediatric mastocytosis through reducing IL-10 due to upregulating TG2 activity via transcription factor nuclear factor-κB. Thus, TG2 may be used in diagnosis of pediatric mastocytosis, particularly SM.
Assuntos
Osso e Ossos/enzimologia , Quimiotaxia , Proteínas de Ligação ao GTP/metabolismo , Mastócitos/enzimologia , Mastocitose Sistêmica/enzimologia , Pele/enzimologia , Transglutaminases/metabolismo , Angioedema/enzimologia , Angioedema/imunologia , Biomarcadores/metabolismo , Osso e Ossos/imunologia , Criança , Pré-Escolar , Citocinas/imunologia , Citocinas/metabolismo , Diagnóstico Diferencial , Doenças do Nervo Facial/enzimologia , Doenças do Nervo Facial/imunologia , Feminino , Proteínas de Ligação ao GTP/sangue , Proteínas de Ligação ao GTP/imunologia , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Leucotrieno C4/imunologia , Leucotrieno C4/metabolismo , Masculino , Mastócitos/imunologia , Mastocitose Sistêmica/sangue , Mastocitose Sistêmica/diagnóstico , Mastocitose Sistêmica/imunologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fenótipo , Valor Preditivo dos Testes , Proteína 2 Glutamina gama-Glutamiltransferase , Transdução de Sinais , Pele/imunologia , Transglutaminases/sangue , Transglutaminases/imunologia , Triptases/imunologia , Triptases/metabolismoRESUMO
BACKGROUND: Astrocytes, which play an active role in chronic inflammatory diseases like multiple sclerosis, exist close to mast cells with which they share perivascular localization. We previously demonstrated the possibility that astrocytes and mast cells interact in vitro and in vivo. This study aimed to investigate the signaling pathways and the role for astrocytes in the interaction of astrocytes and mast cells. METHODS: We co-cultured human U87 glioblastoma (U87) and human mast cell-1 (HMC-1) cell lines, and mouse cerebral cortices-derived astrocytes and mouse bone marrow-derived mast cells (BMMCs). Intracellular Ca2+ ([Ca2+]i) was measured by confocal microscopy; CD40 siRNA by Silencer Express Kit; small GTPases by GTP-pull down assay; PKCs, MAPKs, CD40, CD40L, Jak1/2, STAT1, TNF receptor 1 (TNFR1) by Western blot; NF-κB and AP-1 by EMSA; cytokines by RT-PCR. An experimental allergic encephalomyelitis (EAE) model was induced using myelin oligodendrocyte glycoprotein (MOG) peptide and pertussis toxin in mice. Co-localization of TNFR1 and astrocytes in EAE brain tissues was determined by immunohistochemistry. RESULTS: Each astrocyte co-culture had increases in [Ca2+]i levels, release of cytokines and chemokines; activities of Rho-family GTPases, NF-κB/AP-1/STAT1727, and Jack1/2, STAT1701. These effects were inhibited by anti-CD40 antibody or CD40 siRNA, and signaling pathways for Jak1/2 were inhibited by anti-TNFR1 antibody. EAE score, expression of TNFR1, and co-localization of TNFR1 and astrocytes were enhanced in brain of the EAE model. Anti-CD40 antibody or 8-oxo-dG pretreatment reduced these effects in EAE model. CONCLUSIONS: These data suggest that astrocytes activated by the CD40-CD40L interaction in co-culture induce inflammatory cytokine production via small GTPases, and the secreted cytokines re-activate astrocytes via Jak/STAT1701 pathways, and then release more cytokines that contribute to exacerbating the development of EAE. These findings imply that the pro-inflammatory mediators produced by cell-to-cell cross-talk via interaction of CD40-CD40L may be as a promising therapeutic target for neurodegenerative diseases like MS.
Assuntos
Astrócitos/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Mastócitos/metabolismo , Transdução de Sinais/fisiologia , Animais , Astrócitos/citologia , Biomarcadores/metabolismo , Antígenos CD40/genética , Ligante de CD40/genética , Cálcio/metabolismo , Células Cultivadas , Técnicas de Cocultura , Citocinas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Interferente Pequeno/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Transcrição STAT1/metabolismoRESUMO
BACKGROUND: Many studies have found that smoking reduces lung function, but the relationship between cigarette smoke and allergic asthma has not been clearly elucidated, particularly the role of mast cells. This study aimed to investigate the effects of smoke exposure on allergic asthma and its association with mast cells. METHODS: BALB/c mice were sensitized and challenged by OVA to induce asthma, and bone marrow-derived mast cells (BMMCs) were stimulated with antigen/antibody reaction. Mice or BMMCs were exposed to cigarette smoke or CSE solution for 1 mo or 6 h, respectively. The recruitment of inflammatory cells into BAL fluid or lung tissues was determined by Diff-Quik or H&E staining, collagen deposition by Sircol assay, penh values by a whole-body plethysmography, co-localization of tryptase and Smad3 by immunohistochemistry, IgE and TGF-ß level by ELISA, expressions of Smads proteins, activities of signaling molecules, or TGF-ß mRNA by immunoblotting and RT-PCR. RESULTS: Cigarette smoke enhanced OVA-specific IgE levels, penh values, recruitment of inflammatory cells including mast cells, expressions of smad family, TGF-ß mRNA and proteins, and cytokines, phosphorylations of Smad2 and 3, and MAP kinases, co-localization of tryptase and Smad3, and collagen deposition more than those of BAL cells and lung tissues of OVA-induced allergic mice. CSE solution pretreatment enhanced expressions of TGF-ß, Smad3, activities of MAP kinases, NF-κB/AP-1 or PAI-1 more than those of activated-BMMCs. CONCLUSIONS: The data suggest that smoke exposure enhances antigen-induced mast cell activation via TGF-ß/Smad signaling pathways in mouse allergic asthma, and that it exacerbates airway inflammation and remodeling.
Assuntos
Asma/metabolismo , Pulmão/metabolismo , Mastócitos/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Fumar/imunologia , Animais , Reações Antígeno-Anticorpo , Asma/genética , Asma/imunologia , Asma/fisiopatologia , Western Blotting , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/fisiopatologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/imunologia , Broncoconstrição , Células Cultivadas , Colágeno/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulina E/metabolismo , Imuno-Histoquímica , Pulmão/imunologia , Pulmão/fisiopatologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Ovalbumina , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Pletismografia Total , Interferência de RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Smad/genética , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Triptases/metabolismoRESUMO
Chronic spontaneous urticaria (CSU) is a complex idiopathic disease of the skin with various cellular infiltrations. Although mast cells are key effector cells in the pathogenesis of CSU, CD4+ T helper 2 cells also have particular roles in the development and maintenance of CSU. Periostin is known as a downstream molecule of interleukin (IL)-4 and IL-13, key cytokines of type 2 immune responses. In this study, we examined periostin and IL-13 levels in the sera of patients with CSU (n=84) and healthy normal controls (NCs, n=43). Periostin levels were significantly lower in the CSU group than in NCs (71.4±21.8 vs 85.1±22.4 ng/mL, P=0.04). Periostin levels were also lower in the severe CSU group than those in mild CSU (59.7±18.0 vs 73.4±22.0 ng/mL, P=0.04). However, IL-13 levels were significantly higher in patients with CSU than in NCs (508.5±51.2 vs 200.7±13.3 pg/mL, P=0.001). In conclusion, periostin and IL-13 may be independently related to the pathogenesis of CSU.
RESUMO
Mast cells are major effector cells of allergic diseases related to IgE. This study was undertaken to determine whether IgE or IgA, produced by CD40-CD40L or OX40-OX40L interactions between B cells and mast cells, re-activate FcεRI or FcαRI on mast cell surface. C57BL mice were sensitized and subjected to OVA challenge to induce asthma. Bone marrow-derived mast cells (BMMCs) and primary B cells were co-cultured. Mast cell recruitment into airways was stained by May-Grünwald Giemsa, the expression of markers or signaling molecules were determined by immunohistochemistry or Western blotting, and co-localization of B cells and mast cells by immunofluorescence. Anti-CD40 plus anti-OX40L Abs synergistically reduced IgE and IgA production, and mediators (histamine, LTs and cytokines) released in mast cells, and additively reduced other responses, such as, numbers of mast cells, the expression of markers (tryptase, mMCP5, B220 and CD19), surface molecules (CD40, CD40L, OX40 and OX40L), FcεRI or FcαRI and the co-localization of BMMCs and B cells, and IgE- or IgA-producing cells, as compared with individual blocking Ab treatment which reducedresponses in BAL cells or lung tissues of OVA-challenged mice or in co-culture of B and mast cells. The data suggest that IgE and IgA, produced by OX40-OX40L or CD40-CD40L interaction between B cells and mast cells, may re-activate receptors of FCεRI and FcαRI on mast cell surfaces, followed by more mediator release, and furthermore, that treatment with anti-CD40 plus anti-OX40L Abs offers a potential treatment for allergic asthma.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação/imunologia , Asma/imunologia , Linfócitos B/imunologia , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Imunoglobulina A/imunologia , Imunoglobulina E/imunologia , Mastócitos/imunologia , Ligante OX40/imunologia , Receptores Fc/metabolismo , Receptores de IgE/metabolismo , Animais , Asma/metabolismo , Linfócitos B/metabolismo , Movimento Celular/imunologia , Técnicas de Cocultura , Citocinas/metabolismo , Feminino , Histamina/metabolismo , Imunoglobulina A/sangue , Imunoglobulina E/sangue , Pulmão/imunologia , Mastócitos/metabolismo , CamundongosRESUMO
Allergic asthma is characterized by chronic airway remodeling, which is associated with the expression of extracellular matrix proteins (ECM) by TGF-ß. However, to date there are no reports demonstrating that structural proteins are directly expressed in mast cells. This study aimed to investigate whether ECM proteins are expressed in mast cells activated with antigen/antibody reaction, and whether the resolution effects of irradiation or 8-oxo-dG may contribute to allergic asthma prevention. Bone marrow-derived mast cells (BMMCs) were activated with DNP-HSA/anti-DNP IgE antibody (act-BMMCs). C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) to induce allergic asthma. Mice were treated orally with 8-oxo-dG or exposed to whole body irradiation (using (137)Cs gamma ray at a dose of 0.5 Gy) for three consecutive days 24 h after OVA challenge. Expression of extracellular matrix (ECM) proteins, TGF-ß signaling molecules and NF-κB/AP-1 was determined in the BMMCs, bronchoalveolar lavage (BAL) cells or lung tissues using Western blot, polymerase chain reaction (PCR) and electrophoretic mobility shift assay (EMSA), respectively. Act-BMMCs increased expression of ECM proteins, TGF-ß/TGF-ß receptor I, TGF-ß signaling molecules and cytokines; and increased both NF-κB and AP-1 activity. In addition, the population of mast cells; expression of mast cell markers, TGF-ß signaling molecules, ECM proteins/amounts; OVA-specific serum IgE level; numbers of goblet cells; airway hyperresponsiveness; cytokines/chemokines were increased in BAL cells and lung tissues of OVA-challenged mice. All of the above end points were reduced by irradiation or 8-oxo-dG in vitro and in vivo, respectively. The data suggest that mast cells induce expression of ECM proteins through TGF-ß produced in inflammatory cells of OVA mice and that post treatment of irradiation or 8-oxo-dG after OVA-challenge may reduce airway remodeling through down-regulating mast cell re-activation by TGF-ß/Smad signals.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Desoxiguanosina/análogos & derivados , Hipersensibilidade/imunologia , Mastócitos/imunologia , Ovalbumina/administração & dosagem , Fator de Crescimento Transformador beta/fisiologia , 8-Hidroxi-2'-Desoxiguanosina , Remodelação das Vias Aéreas/efeitos dos fármacos , Remodelação das Vias Aéreas/efeitos da radiação , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/efeitos da radiação , Líquido da Lavagem Broncoalveolar , Desoxiguanosina/farmacologia , Relação Dose-Resposta à Radiação , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mast cells play important roles via FcεRI-mediated activation in allergic asthma. A nonpolymorphic MHC I-like molecule CD1d, which is mainly expressed in APCs, presents glycolipid Ag to iTCR on iNKT cells and modulates allergic responses. This study aimed to investigate the role of CD1d on IgE production and mast cell activation related to allergic asthma. Bone marrow-derived mast cells (BMMCs) from C57BL/6 Wild type (WT) or KO (CD1d(-/-)) mice were activated with Ag/Ab (refer to WT-act-BMMCs and KO-act-BMMCs, respectively) or α-Galactosylceramide (WT-αGal-BMMCs, KO-αGal-BMMCs) in the presence of iNKT cells. WT, KO or BMMC-transferred KO mice were sensitized and/or challenged by OVA or α-Gal to induce asthma. KO-act-BMMCs reduced intracellular Ca(2+) levels, expression of signaling molecules (Ras, Rac1/2, PLA2, COX-2, NF-κB/AP-1), mediator release (histamines, leukotrienes and cytokines/chemokines), and total IgE levels versus the corresponding WT-BMMCs. KO mice reduced total and OVA-specific serum IgE levels, number of mast cells, recruiting molecules (CCR2/CCL2, VCAM-1, PECAM-1), expression of tryptase, c-kit, CD40L and cytokine mRNA, co-localization of c-kit and CD1d or iNKT cells in BAL cells or lung tissues, and PCA responses, compared with the corresponding WT mice. BMMC-transferred KO-both mice showed the restoration of all allergic responses versus KO-both mice (Ag/Ab reaction plus α-Gal). KO-αGal-BMMCs or KO-αGal mice did not show any responses. Our data suggest that CD1d-expressed mast cells may function as APC cells for iNKT cells and exacerbate airway inflammation and remodeling through up-regulating IgE production via B cell Ig class switching and mediator release in mast cells of OVA-challenged mice.
Assuntos
Antígenos CD1d/metabolismo , Linfócitos B/metabolismo , Ligante de CD40/metabolismo , Imunoglobulina E/metabolismo , Mastócitos/metabolismo , Ovalbumina/toxicidade , Regulação para Cima , Animais , Antígenos CD1d/genética , Asma/induzido quimicamente , Asma/metabolismo , Asma/patologia , Linfócitos B/citologia , Células da Medula Óssea/citologia , Cálcio/metabolismo , Células Cultivadas , Técnicas de Cocultura , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Pulmão/citologia , Pulmão/patologia , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Gamma radiation is used for several therapeutic indications such as cancers and autoimmune diseases. Low-dose whole-body γ irradiation has been shown to activate immune responses in several ways, however, the effect and mechanism of irradiation on allergic asthma remains poorly understood. This study investigated whether or not irradiation exacerbates allergic asthma responses and its potential mechanism. C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) to induce asthma. The mice received whole-body irradiation once daily for 3 consecutive days with a dose of 0.667 Gy using (137)Cs γ rays 24 h before every OVA challenge. Repeated low-dose irradiation reduced OVA-specific IgE levels, the number of inflammatory cells including mast cells, goblet cell hyperplasia, collagen deposition, airway hyperresponsiveness, expression of inflammatory cytokines, CCL2/CCR2, as well as nuclear factor kappa B (NF-κB) and activator protein-1 activities. All of these factors were increased in BAL cells and lung tissue of OVA-challenged mice. Irradiation increased the number of Treg cells, expression of interleukin (IL)-10, IL-2 and IL-35 in BAL cells and lung tissue. Irradiation also increased Treg cell-expressed Foxp3 and IL-10 by NF-κB and RUNX1 in OVA-challenged mice. Furthermore, while Treg cell-expressing OX40 and IL-10 were enhanced in lung tissue or act-bone marrow-derived mast cells (BMMCs) with Treg cells, but BMMCs-expressing OX40L and TGF-ß were decreased. The data suggest that irradiation enhances Foxp3(+)- and IL-10-producing Treg cells, which reduce OVA-induced allergic airway inflammation and tissue remodeling through the down-regulation of migration by the CCL2/CCR2 axis and activation of mast cells via OX40/OX40L in lung tissue of OVA-challenged mice.
Assuntos
Asma/imunologia , Fatores de Transcrição Forkhead/metabolismo , Raios gama/efeitos adversos , Hipersensibilidade/complicações , Ovalbumina/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/efeitos da radiação , Animais , Especificidade de Anticorpos , Asma/induzido quimicamente , Asma/complicações , Células da Medula Óssea/citologia , Líquido da Lavagem Broncoalveolar , Tamanho Celular/efeitos da radiação , Quimiocinas/metabolismo , Relação Dose-Resposta à Radiação , Feminino , Regulação da Expressão Gênica/imunologia , Regulação da Expressão Gênica/efeitos da radiação , Histonas/genética , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Pulmão/diagnóstico por imagem , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Mastócitos/imunologia , Mastócitos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Ligante OX40/metabolismo , Especificidade de Órgãos , Transporte Proteico/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Radiografia , Receptores de Quimiocinas/metabolismo , Receptores OX40/metabolismo , Baço/imunologia , Baço/efeitos da radiação , Linfócitos T Reguladores/imunologia , Fator de Transcrição AP-1/metabolismoRESUMO
TGase 2 is over-expressed in a variety of inflammatory diseases including allergic asthma. This study aimed to investigate the role of TGase 2 on IgE production and signaling pathways in mast cell activation related to OVA-induced allergic asthma. Bone marrow-derived mast cells (BMMCs) isolated from WT or TGase 2(-/-) mice were activated with Ag/Ab (refer to act-WT-BMMCs and act-KO-BMMCs, respectively). B cells isolated from splenocytes were activated with anti-mouse IgM (act-B cells), and B cells were co-cultured with BMMCs. WT and TGase 2(-/-) mice were sensitized and challenged with OVA adsorbed in alum hydroxide. Intracellular Ca(2+) ([Ca(2+)]i) levels were determined by fluorescence intensity; IgE, mediators and TGase 2 activity by ELISA; the CD138 expression by FACS analyzer; cell surface markers and signal molecules by Western blot; NF-κB by EMSA; co-localization of mast cells and B cells by immunohistochemistry; Fcε RI-mediated mast cell activation by PCA test; expression of cytokines, MMPs, TIMPs, TLR2 and FcεRI by RT-PCR. In vitro, act-KO-BMMCs reduced the [Ca(2+)]i levels, NF-κB activity, expression of CD40/CD40L, plasma cells, total IgE levels and TGase 2 activity in act-B cells co-cultured with act-BMMCs, expression of inflammatory cytokines and MMPs2/9, release of mediators (TNF-α, LTs and cytokines), and activities of signal molecules (PKCs, MAP kinases, I-κB and PLA2), which were all increased in act-WT-BMMCs. TGase 2 siRNA transfected/activated-BMMCs reduced all responses as same as those in act-KO-BMMCs. In allergic asthma model, TGase 2(-/-) mice protected against PCA reaction, OVA-specific IgE production and AHR, and they reduced co-localization of mast cells and B cells or IgE in lung tissues, expression and co-localization of surface molecules in mast cells (c-kit and CD40L) and B cells (CD23 and CD40), inflammatory cells including mast cells, goblet cells, amounts of collagen and mediator release in BAL fluid and/or lung tissues, which were all increased in WT mice. TLR expression in TGase 2(-/-) mice did not differ from those in WT mice. Our data suggest that TGase 2 expression and Ca(2+) influx required by bidirectional events in mast cell activation facilitate IgE production in B cells via up-regulating mast cell CD40L expression, and induce the expression of numerous signaling molecules associated with airway inflammation and remodeling in allergic asthma.
Assuntos
Asma/metabolismo , Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Imunoglobulina E/metabolismo , Mastócitos/metabolismo , Transglutaminases/metabolismo , Animais , Asma/imunologia , Asma/patologia , Linfócitos B/citologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Cálcio/metabolismo , Técnicas de Cocultura , Citocinas/genética , Citocinas/metabolismo , Feminino , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Pulmão/metabolismo , Mastócitos/citologia , Mastócitos/patologia , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Inibidores Teciduais de Metaloproteinases/genética , Inibidores Teciduais de Metaloproteinases/metabolismo , Transglutaminases/antagonistas & inibidores , Transglutaminases/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This study investigated whether anti-CD40 Ab and 8-oxo-dG attenuate mast cell migration and EAE development. Anti-CD40 Ab and 8-oxo-dG reduced EAE scores, mast cell numbers, expression of adhesion molecules, OX40L and Act1, levels of TNF-α, LTs, expression of cytokines, and co-localization of Treg cells and mast cells, all of which are increased in EAE-brain tissues. Each treatment enhanced Treg cells, expression of OX40, and cytokines related to suppressive function of Treg cells in EAE brain tissues. Act-BMMCs with Treg cells reduced expression of OX40L and CCL2/CCR2, VCAM-1, PECAM-1, [Ca²âº]i levels, release of mediators, various signaling molecules, Act1 related to IL-17a signals versus those in act-BMMCs without Treg cells. The data suggest that IL-10- and IL-35-producing Foxp3âº-Treg cells, enhanced by anti-CD40 Ab or 8-oxo-dG, suppress migration of mast cells through down-regulating the expression of adhesion molecules, and suppress mast cell activation through cell-to-cell cross-talk via OX40/OX40L in EAE development.
Assuntos
Antígenos CD40/imunologia , Movimento Celular/imunologia , Desoxiguanosina/análogos & derivados , Encefalomielite Autoimune Experimental , Mastócitos/imunologia , Linfócitos T Reguladores/imunologia , 8-Hidroxi-2'-Desoxiguanosina , Animais , Autoanticorpos/imunologia , Autoanticorpos/farmacologia , Cálcio/metabolismo , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Movimento Celular/efeitos dos fármacos , Desoxiguanosina/farmacologia , Regulação para Baixo/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Interleucina-17/imunologia , Interleucina-17/farmacologia , Leucotrienos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
It has become widely accepted that the role of mast cells is not restricted to allergic processes. Thus, mast cells play an important role in innate and adaptive immune responses, but study of proteins related to differentiation of mast cells has not been done yet. Enolase 1 is a glycolytic enzyme expressed in most tissues and calreticulin, known as endoplasmic reticulum (ER) resident chaperon, has multifunctional responses. This study aimed to investigate the effects of these proteins on the differentiation and functions of mouse bone marrow-derived mast cells (BMMCs). To identify the target proteins related to the differentiation of BMMCs, we examined the protein expression pattern of BMMCs using 2-dimensional electrophoresis (2-DE) and MALDI-TOF analysis. Expressions of FcεRIα, surface molecules (c-kit, CD40, CD40L, VCAM-1), tryptase, and cytokines were examined in BMMCs using FACS analysis, Western blot, and RT-PCR respectively. Enolase 1 and calreticulin were transfected into BMMCs, and [Ca(2+)]i levels were determined by confocal microscope, while amounts of TNF-α and LTs were measured by ELISA. Eight proteins were identified by proteomic analysis. Enolase and calreticulin siRNA transfection inhibited the expressions of FcεRIα, surface molecules, tryptase, and cytokine mRNA, which are gradually enhanced during culture periods of BMMCs. Enolase 1 and calreticulin siRNA reduced the [Ca(2+)]i levels, amounts of total TNF-α, and the release of TNF-α and leukotrienes, all of which are increased in the BMMCs activated with antigen/antibody reaction. The data suggest that enolase 1 and calreticulin are important proteins in regulating the differentiation and functions of BMMCs.
Assuntos
Células da Medula Óssea/fisiologia , Calreticulina/metabolismo , Diferenciação Celular , Mastócitos/fisiologia , Fosfopiruvato Hidratase/metabolismo , Animais , Células da Medula Óssea/metabolismo , Antígenos CD40/metabolismo , Cálcio/metabolismo , Calreticulina/genética , Linhagem Celular , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosfopiruvato Hidratase/genética , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Interferência de RNA , Receptores de IgE/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND AND PURPOSE: Transglutaminase 2 (TGase 2) expression is increased in inflammatory diseases, and TGase 2 inhibitors block these increases. We examined whether the R2 peptide inhibited the expression of TGase 2 in a mouse model of inflammatory allergic asthma. EXPERIMENTAL APPROACH: C57BL/6 mice were sensitized and challenged by ovalbumin (OVA) to induce asthma. OVA-specific serum IgE and leukotrienes (LTs) levels were measured by enzyme-linked immunosorbent assay. Recruitment of inflammatory cells into bronchoalveolar lavage (BAL) fluid or lung tissues and goblet cell hyperplasia were assessed histologically. Airway hyperresponsiveness was determined in a barometric plethysmographic chamber. Expression of TGase 2, eosinophil major basic protein (EMBP), the adhesion molecule vascular cell adhesion molecule-1, Muc5ac and phospholipase A(2) (PLA(2) ) protein were determined by Western blot. Expression of mRNAs of Muc5ac, cytokines, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were measured by reverse transcriptase-polymerase chain reaction and nuclear factor-κB (NF-κB) by electrophoretic mobility shift assay. KEY RESULTS: R2 peptide reduced OVA-specific IgE levels; the number of total inflammatory cells, macrophages, neutrophils, lymphocytes and eosinophils in BAL fluid and the number of goblet cells. Airway hyperresponsiveness, TGase 2 and EMBP levels, mRNA levels of interleukin (IL)-4, IL-5, IL-6, IL-8, IL-13, RANTES, tumour necrosis factor-α, and MMP2/9, Muc5ac, NF-κB activity, PLA(2) activity and expressions, and LT levels in BAL cells and lung tissues were all reduced by R2 peptide. R2 peptide also restored expression of TIMP1/2. CONCLUSION AND IMPLICATIONS: R2 peptide reduced allergic responses by regulating NF-κB/TGase 2 activity in a mouse model of allergic asthma. This peptide may be useful in the treatment of allergic asthma.