Assuntos
Angioedema/terapia , Fibrose Endomiocárdica/terapia , Amarelo de Eosina-(YS)/análogos & derivados , Eosinofilia/terapia , Interferon-alfa/uso terapêutico , Anuloplastia da Valva Mitral , Prolapso da Valva Mitral/terapia , Tapsigargina/análogos & derivados , Adolescente , Adulto , Angioedema/imunologia , Angioedema/cirurgia , Criança , Fibrose Endomiocárdica/imunologia , Fibrose Endomiocárdica/cirurgia , Eosinofilia/imunologia , Eosinofilia/cirurgia , Feminino , Implante de Prótese de Valva Cardíaca , Humanos , Interferon alfa-2 , Pessoa de Meia-Idade , Prolapso da Valva Mitral/imunologia , Prolapso da Valva Mitral/cirurgia , Proteínas Recombinantes/uso terapêutico , Tapsigargina/imunologia , Adulto JovemRESUMO
Antimicrobial peptides (AMP) are ubiquitous innate immune elements in epithelial tissues. We recently discovered that a signaling lipid, the ceramide metabolite sphingosine-1-phosphate (S1P), regulates production of a major AMP, cathelicidin antimicrobial peptide (CAMP), in response to a subtoxic level of endoplasmic reticulum (ER) stress that can be induced by external perturbants in keratinocytes. We hypothesized that an ER stress-initiated signal could also regulate production of another major class of AMPs: i.e., the human beta-defensins 2 (hBD2) and 3 (hBD3). Keratinocytes stimulated with a pharmacological ER stressor, thapsigargin (Tg), increased hBD2/hBD3 as well as CAMP mRNA expression. While inhibition of sphingosine-1-phosphate production did not alter hBD expression following ER stress, blockade of ceramide-1-phosphate (C1P) suppressed Tg-induced hBD2/hBD3 but not CAMP expression. Exogenous C1P also increased hBD2/hBD3 production, indicating that C1P stimulates hBD expression. We showed further that C1P-induced hBD2/hBD3 expression is regulated by a novel pathway in which C1P stimulates downstream hBD via a cPLA2aâ15d-PGJ2âPPARα/PPARß/δâSrc kinaseâSTAT1/STAT3 transcriptional mechanism. Finally, conditioned medium from C1P-stimulated keratinocytes showed antimicrobial activity against Staphylococcus aureus. In summary, our present and recent studies discovered two new regulatory mechanisms of key epidermal AMP, hBD2/hBD3 and CAMP. The C1P and S1P pathways both signal to enhance innate immunity in response to ER stress.
Assuntos
Ceramidas/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Queratinócitos/imunologia , Lisofosfolipídeos/farmacologia , Esfingosina/análogos & derivados , Tapsigargina/farmacologia , Peptídeos Catiônicos Antimicrobianos , Catelicidinas/genética , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Estresse do Retículo Endoplasmático/imunologia , Regulação da Expressão Gênica , Células HeLa , Humanos , Transdução de Sinais/efeitos dos fármacos , Esfingosina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Tapsigargina/imunologia , beta-Defensinas/genética , beta-Defensinas/metabolismoRESUMO
To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Two Agilent DNA array designs were used. One contained 40,000 features using de Bruijn sequences where each 8-mer occurs 32 times in various positions in the DNA sequence. The second contained 180,000 features with each CG containing 8-mer occurring three times. The first design was better for identification of binding motifs, while the second was better for quantification. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD, and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. The electrophoretic mobility shift assay (EMSA) confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50× methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. This methodology can be used to identify new methylated DNA sequences preferentially bound by TFs, which may be functional in vivo.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ilhas de CpG , Metilação de DNA , Fator 4 Ativador da Transcrição/química , Animais , Sequência de Bases , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/química , Feminino , Fibroblastos , Camundongos , Motivos de Nucleotídeos , Matrizes de Pontuação de Posição Específica , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Tapsigargina/imunologia , Fatores de Transcrição/metabolismoRESUMO
We assessed the effects of Zizyphus lotus L. (Desf.) polyphenols (ZLP) on T-cell signaling and proliferation. Our results showed that ZLP exerted no effect on the increases in intracellular free calcium concentrations, [Ca(2+)]i, in human Jurkat T-cells. However, ZLP modulated the thapsigargin-induced increases in [Ca(2+)]i in these cells. ZLP treatment was found to decrease the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, ZLP induced a rapid (t1/2=33s) and dose-dependent decrease in intracellular pH (pHi) in human Jurkat T-cells. Furthermore, ZLP significantly curtailed T-cell proliferation by diminishing their progression from S to G2/M phase of cell cycle, and the expression of interleukin-2 (IL-2) mRNA. Taken together, the results of the present study demonstrate that ZLP modulate cell signaling and exert immunosuppressive effects in human T-cells.
Assuntos
Terapia de Imunossupressão , Inflamação/imunologia , Interleucina-2/metabolismo , Polifenóis/farmacologia , Linfócitos T/efeitos dos fármacos , Ziziphus/química , Sinalização do Cálcio/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Frutas/química , Regulação da Expressão Gênica , Humanos , Interleucina-2/genética , Células Jurkat , RNA Mensageiro/análise , Linfócitos T/imunologia , Tapsigargina/imunologiaRESUMO
Nucleic acid sensing by cells is a key feature of antiviral responses, which generally result in type-I Interferon production and tissue protection. However, detection of double-stranded RNAs in virus-infected cells promotes two concomitant and apparently conflicting events. The dsRNA-dependent protein kinase (PKR) phosphorylates translation initiation factor 2-alpha (eIF2α) and inhibits protein synthesis, whereas cytosolic DExD/H box RNA helicases induce expression of type I-IFN and other cytokines. We demonstrate that the phosphatase-1 cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), an important component of the unfolded protein response (UPR), is absolutely required for type I-IFN and IL-6 production by mouse embryonic fibroblasts (MEFs) in response to dsRNA. GADD34 expression in MEFs is dependent on PKR activation, linking cytosolic microbial sensing with the ATF4 branch of the UPR. The importance of this link for anti-viral immunity is underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection.
Assuntos
Infecções por Alphavirus/imunologia , Vírus Chikungunya/imunologia , Interferon beta/biossíntese , Proteína Fosfatase 1/metabolismo , RNA de Cadeia Dupla/imunologia , Células 3T3 , Fator 4 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Febre de Chikungunya , Fibroblastos/imunologia , Fibroblastos/virologia , Interferon Tipo I/biossíntese , Interleucina-6/biossíntese , Camundongos , Poli I-C/imunologia , Proteína Fosfatase 1/biossíntese , Proteína Fosfatase 1/genética , Tapsigargina/imunologia , Resposta a Proteínas não Dobradas , eIF-2 Quinase/biossíntese , eIF-2 Quinase/metabolismoRESUMO
SK&F 96365, 51-(beta-[3-(p-methoxyphenyl)-propyloxy]-p-methoxyphenethyl)-1H-imidazole hydrochloride, has emerged as a useful pharmacological tool in the study of store-operated Ca²âº entry (SOCE). But the precise molecular mechanism and effect of SK&F 96365 on mouse lymphocytes are still not well determined. This study investigated the pharmacological profile of SK&F 96365 on mouse lymphocytes stimulated by mitogen concanavalin A (Con A) or by a combination of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) and a calcium ionophore, ionomycin in vitro. Our results showed that SK&F 96365 pre-treatment diminished the cytosolic calcium rise on lymphocytes induced by ionomycin, PMA/ionomycin, and thapsigargin (TG), respectively. CFDA-SE staining results showed that SK&F 96365 (5-20 µM) inhibited both Con A- and PMA/ionomycin-induced lymphocytes proliferation in a time- and dose-dependent manner. Upon the same stimulation, SK&F 96365 inhibited the expression of CD69 and CD25 on CD3⺠T lymphocytes in a dose-dependent manner. The cell cycle analyzing results showed that SK&F 96365 caused a G0/G1 phase cell cycle arrest on both Con A- and PMA/ionomycin-activated lymphocytes in a dose-dependent manner. In addition, SK&F 96365 induced a decrease in mitochondrial membrane potential (ΔΨm) and promoted mitochondrial permeability transition (MPT) in both Con A- and PMA/ionomycin-activated lymphocytes. Furthermore, SK&F 96365 significantly inhibited the production of proinflammatory cytokines (interferon (IFN)-γ and tumor necrosis factor (TNF)), and the anti-inflammatory cytokine (IL-10) on both Con A- and PMA/ionomycin-activated lymphocytes. SK&F 96365 did not induce a statistically significant increase in levels of proinflammatory IL-6 and monocyte chemoattractant protein-1 (MCP-1) but of IL-12p70 upon the stimulation of Con A, whereas these three cytokines were markedly inhibited by it upon the stimulation of PMA/ionomycin. This finding revealed that SK&F 96365 exhibited an anti-inflammatory effect on mouse lymphocytes both upon the stimulation of Con A and PMA/ionomycin, and the precise mechanism of SK&F 96365 inhibiting Con A-activated lymphocytes proliferation is different from PMA/ionomycin.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Ciclo Celular/efeitos dos fármacos , Imidazóis/farmacologia , Inflamação/imunologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/farmacologia , Antígenos CD/biossíntese , Antígenos CD/imunologia , Cálcio/imunologia , Bloqueadores dos Canais de Cálcio/imunologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/imunologia , Ciclo Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Concanavalina A/imunologia , Concanavalina A/farmacologia , Citocinas/biossíntese , Citocinas/imunologia , Relação Dose-Resposta a Droga , Feminino , Fluoresceínas/análise , Corantes Fluorescentes/análise , Imidazóis/imunologia , Inflamação/tratamento farmacológico , Inflamação/patologia , Ionomicina/imunologia , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Contagem de Linfócitos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Transdução de Sinais/imunologia , Succinimidas/análise , Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Tapsigargina/imunologia , Tapsigargina/farmacologiaRESUMO
BACKGROUND: Human lung mast cells (HLMC) lie in close proximity to the bronchial epithelium in asthma and adhere with high affinity to bronchial epithelial monolayers in vitro. We investigated the consequences of this adhesive interaction on HLMC activation in response to Fc epsilon RI cross-linking. METHODS: Human lung mast cells were cultured with the bronchial epithelial cell line BEAS-2B or plastic control for either 30 min or 16 h and then activated with anti-IgE. Histamine was measured by radioenzymatic assay. RESULTS: After co-culture for 30 min, IgE-dependent histamine release from HLMC was identical on both BEAS-2B and plastic. After 16 h of co-culture, there was a marked decrease in constitutive and IgE-dependent histamine release from HLMC cultured on BEAS-2B compared with those cultured on plastic or fibronectin. In contrast, the Ca(2+)/ATPase inhibitor thapsigargin produced concentration-dependent histamine release that was significantly increased on BEAS-2B compared with plastic. IgE-dependent degranulation was not significantly affected by BEAS-2B-conditioned medium. CONCLUSIONS: BEAS-2B bronchial epithelial cells attenuate IgE-dependent but not thapsigargin-induced histamine release from HLMC. The differential effect with anti-IgE compared with thapsigargin suggests that the mechanism includes interference with the proximal Fc epsilon RI signalling pathway.