RESUMO
Galectin-1 (Gal-1) is a ß-galactoside-binding protein with diverse biological activities in the pathogenesis of inflammation, however the mechanisms by which Gal-1 modulates cellular responses in allergic inflammatory processes have not been fully determined. In this study, we evaluated the therapeutic potential of Gal-1 eye drops in an experimental model of conjunctivitis. Wistar rats received a topical application of compound (C)48/80 (100â¯mg/ml) into right eyes and a drop of vehicle into the contralateral eye. Another group of rats received Gal-1 (0.3 or 3⯵g/eye) or sodium cromoglycate (SCG; 40â¯mg/ml) in both eyes and, after 15â¯min, right eye was challenged with C48/80. Conjunctivitis-induced by C48/80 was characterized by severe eyelid oedema and tearing, but clinical signs were ameliorated by eye drop doses of both Gal-1 (0.3/3⯵g) and SCG. As expected, an increased proportion of degranulated mast cells (62%, Pâ¯<â¯0.01) and lower histamine levels were observed after 6â¯h of C48/80 challenge, compared to control (32%). This effect was abrogated by Gal-1 and SCG, which reduced mast cell degranulation (31-36%), eosinophil migration and eosinophil peroxidase levels in the eyes. Gal-1 (3⯵g) and SCG treatments also decreased IL-4 levels, as well as activation of mitogen activated protein kinases compared to untreated C48/80 eyes. Our findings suggest that Gal-1 eye drops represent a new therapeutic strategy for ocular allergic inflammation.
Assuntos
Antialérgicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Conjuntivite Alérgica/tratamento farmacológico , Galectina 1/uso terapêutico , Animais , Antialérgicos/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Degranulação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Conjuntivite Alérgica/induzido quimicamente , Conjuntivite Alérgica/imunologia , Conjuntivite Alérgica/patologia , Citocinas/imunologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/enzimologia , Eosinófilos/fisiologia , Olho/efeitos dos fármacos , Olho/imunologia , Olho/patologia , Galectina 1/administração & dosagem , Histamina/imunologia , Mastócitos/efeitos dos fármacos , Mastócitos/fisiologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Soluções Oftálmicas , Peroxidases/metabolismo , Ratos Wistar , p-Metoxi-N-metilfenetilaminaRESUMO
BACKGROUND AND AIM: Eosinophils are markers of the eosinophilic esophagitis (EoE) disease, and this work aimed to assess whether activation of eosinophils could be a noninvasive test to contribute for EoE diagnosis. METHODS: The activation state of peripheral blood eosinophils in EoE patients and control subjects was assessed based on the morphological aspects of the eosinophil after adherence to slide. Cyclooxygenase-2 and 5-lipoxygenase expressions were evaluated by means of immunofluorescence microscopy to verify if and which eicosanoid pathway is triggered in eosinophils in blood in EoE. RESULTS: The eosinophils of patients with EoE were significantly more activated than those of control individuals. The lowest percentage of normal eosinophils for control subjects was 40%, while the highest percentage of eosinophils of normal aspect for patients with EoE was 32%. Considering 36% as a cutoff for normal eosinophils, this value differentiated all individuals with EoE from individuals without the disease with a sensitivity of 100%, considering the diagnosis of EoE as currently defined. Eosinophils of EoE patients showed higher expression of cyclooxygenase-2 than those of control subjects. CONCLUSIONS: The quantification of morphological changes in eosinophils is a feasible, easy, and reliable manner to identify EoE patients. Therefore, patients with symptoms of esophageal dysfunction showing higher than 36% activated eosinophils in peripheral blood could be a useful way to help definition and diagnostic criterion for EoE.
Assuntos
Esofagite Eosinofílica/diagnóstico , Eosinófilos/imunologia , Adulto , Araquidonato 5-Lipoxigenase/sangue , Biomarcadores/sangue , Estudos Transversais , Ciclo-Oxigenase 2/sangue , Esofagite Eosinofílica/imunologia , Eosinófilos/enzimologia , Eosinófilos/patologia , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Studies have shown that eosinophils are closely related to pathogenesis of bronchial asthma. Eosinophils release eosinophil cationic protein (ECP), which plays an important role in infection and allergic reactions. Serum ECP mRNA expression in children with bronchial asthma has not been adequately investigated. We analyzed serum ECP mRNA expression in 63 children with bronchial asthma and 21 healthy children by using reverse-transcriptase polymerase chain reaction to understand the role of ECP in children with bronchial asthma. The children with bronchial asthma were segregated into acute-phase and stable-phase groups, based on the severity of the illness. Serum ECP mRNA expression in children with bronchial asthma (0.375 ± 0.04) was significantly higher than that in healthy controls (0.20 ± 0.02; P < 0.05). Additionally, children in the acute-phase group showed higher ECP mRNA expression level (0.44 ± 0.06) than those in the stable-phase (0.31 ± 0.03) and healthy control groups (0.20 ± 0.02; P < 0.05), while the level in the stable-phase (0.31 ± 0.03) was markedly higher than that in the healthy control group (0.20 ± 0.02; P < 0.05). Detection of serum ECP mRNA expression level has possible applications in the diagnosis and treatment of children with bronchial asthma.
Assuntos
Asma/genética , Proteína Catiônica de Eosinófilo/genética , Eosinófilos/enzimologia , RNA Mensageiro/biossíntese , Asma/sangue , Asma/enzimologia , Hiper-Reatividade Brônquica/sangue , Hiper-Reatividade Brônquica/genética , Criança , Proteína Catiônica de Eosinófilo/biossíntese , Proteína Catiônica de Eosinófilo/sangue , Feminino , Humanos , Masculino , RNA Mensageiro/sangue , RNA Mensageiro/genéticaRESUMO
The roles of eosinophils in antimicrobial defense remain incompletely understood. In ovalbumin-sensitized mice, eosinophils are selectively recruited to the peritoneal cavity by antigen, eotaxin, or leukotriene(LT)B4, a 5-lipoxygenase (5-LO) metabolite. 5-LO blockade prevents responses to both antigen and eotaxin. We examined responses to eotaxin in the absence of sensitization and their dependence on 5-LO. BALB/c or PAS mice and their mutants (5-LO-deficient ALOX; eosinophil-deficient GATA-1) were injected i.p. with eotaxin, eosinophils, or both, and leukocyte accumulation was quantified up to 24 h. Significant recruitment of eosinophils by eotaxin in BALB/c, up to 24 h, was accompanied by much larger numbers of recruited neutrophils and monocytes/macrophages. These effects were abolished by eotaxin neutralization and 5-LO-activating protein inhibitor MK886. In ALOX (but not PAS) mice, eotaxin recruitment was abolished for eosinophils and halved for neutrophils. In GATA-1 mutants, eotaxin recruited neither neutrophils nor macrophages. Transfer of eosinophils cultured from bone-marrow of BALB/c donors, or from ALOX donors, into GATA-1 mutant recipients, i.p., restored eotaxin recruitment of neutrophils and showed that the critical step dependent on 5-LO is the initial recruitment of eosinophils by eotaxin, not the secondary neutrophil accumulation. Eosinophil-dependent recruitment of neutrophils in naive BALB/c mice was associated with increased binding of bacteria.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Quimiocina CCL11/química , Eosinófilos/enzimologia , Macrófagos/enzimologia , Neutrófilos/enzimologia , Animais , Eosinófilos/citologia , Escherichia coli/metabolismo , Feminino , Granulócitos/citologia , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/citologia , Mutação , Neutrófilos/citologia , FagocitoseRESUMO
Allergic asthma has emerged as an important public health problem of urban populations in developed countries. Very often herbal medicine is used to treat this widespread disease, due to the lack of efficacy and the important side effects related to the classical drugs in use. Along this line, Ocimum gratissimum (Og) is a plant widely used in Brazilian folk medicine to treat inflammatory disorders, such as asthma. In the present study we evaluated the immunomodulatory effects of Og and rosmarinic acid (RA, a polyphenolic compound) in a murine model of respiratory allergy induced by the Blomia tropicalis (Bt) mite. The respiratory allergy was induced in A/J mice by administration of Bt extract and the treatment was done using 25, 50 or 100mg/kg of an Og methanolic extract or using 2, 20 or 200mg/kg of RA. We then evaluated the changes induced by these drugs on immunological parameters related to the allergic process, which are up-regulated in this allergic model. The treatment of animals with 100mg/Kg Og and 200mg/Kg RA led to a significant reduction in the numbers of leukocytes/eosinophils in bronchoalveolar lavage (BAL); eosinophil peroxidase activity in BAL; presence of mucus in respiratory tract, histopathological changes in the lung, and IL-4 in BAL. These results suggest that the methanolic extract of Og and the polyphenol RA have therapeutic potential in this murine model of respiratory allergy to a clinically relevant human sensitizer allergen.
Assuntos
Cinamatos/uso terapêutico , Depsídeos/uso terapêutico , Eosinófilos/imunologia , Fatores Imunológicos/uso terapêutico , Ocimum/química , Extratos Vegetais/uso terapêutico , Hipersensibilidade Respiratória/tratamento farmacológico , Sarcoptidae/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Cinamatos/administração & dosagem , Cinamatos/isolamento & purificação , Depsídeos/administração & dosagem , Depsídeos/isolamento & purificação , Modelos Animais de Doenças , Peroxidase de Eosinófilo/metabolismo , Eosinófilos/efeitos dos fármacos , Eosinófilos/enzimologia , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/isolamento & purificação , Interleucina-4/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/isolamento & purificação , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Mucosa Respiratória/imunologia , Ácido RosmarínicoRESUMO
Endocytic activity of phagocytic cells from armadillos infected with viruses, parasites or bacteria is unknown. This report shows that eosinophils from armadillos infected with microfilaria act against these helmintic parasites but have deficiencies in their oxygen-dependent bacteriocidal mechanisms and also in endocytic capacity against yeast.
Assuntos
Tatus/imunologia , Tatus/parasitologia , Eosinófilos/enzimologia , Eosinófilos/imunologia , Microfilárias/imunologia , NADPH Oxidases/sangue , Animais , Tatus/sangue , Endocitose , Eosinófilos/parasitologia , Feminino , Filariose/sangue , Filariose/imunologia , Filariose/veterinária , Citometria de Fluxo , Granulócitos/imunologia , Granulócitos/parasitologia , Masculino , Microfilárias/patogenicidade , Monócitos/imunologia , Monócitos/parasitologia , Nitroazul de TetrazólioRESUMO
In the present study, the distribution of eosinophils at different stages of the formation of hepatic granuloma in mice infected with Schistosoma mansoni was evaluated. From the results obtained, we suggest a new classification for the evolution of hepatic granuloma in mice, constructed from the phases described by other authors. In each phase, there is a different pattern of eosinophil distribution. In the exudative-necrotic phase, the eosinophils are concentrated in the periphery and center of the granuloma, and are scarce in the necrotic area; in the productive phase, the eosinophils are dispersed throughout the granuloma; and in the cure due to fibrosis phase, the eosinophils are concentrated in the periphery and center of the granuloma. Eosinophils were found in direct contact with the eggs at all stages of evolution of the granuloma. It was concluded that the dynamics of eosinophils have an important role in forming the granulomatous reaction of the host and in resolving the inflammatory process caused by the parasite egg, as well as adding new data regarding hepatic granuloma classification.
Assuntos
Eosinófilos/patologia , Granuloma/patologia , Hepatopatias Parasitárias/patologia , Esquistossomose mansoni/patologia , Animais , Modelos Animais de Doenças , Proteína Catiônica de Eosinófilo/análise , Peroxidase de Eosinófilo/análise , Eosinófilos/enzimologia , Granuloma/parasitologia , Imuno-Histoquímica , Hepatopatias Parasitárias/parasitologia , Masculino , Camundongos , Necrose , Esquistossomose mansoni/parasitologia , Fatores de TempoRESUMO
No presente estudo, avaliou-se a distribuição dos eosinófilos nas diferentes fases da formação do granuloma hepático de camundongos infectados pelo Schistosoma mansoni. A partir dos resultados obtidos sugerimos uma nova classificação para a evolução do granuloma hepático em camundongos montada a partir de fases descritas por outros autores. Em cada fase há um padrão diferente de distribuição dos eosinófilos. Na fase necrótico-exudativa os eosinófilos encontram-se concentrados na periferia e no centro do granuloma e na área de necrose eles são escassos; na "produtiva" os eosinófilos estão ainda distribuídos de maneira difusa por todo o granuloma; na de cura por fibrose se concentram na periferia e no centro do granuloma. Os eosinófilos estavam em contato direto com os ovos em todos os estágios de evolução dos granulomas. Conclui-se então que a dinâmica dos eosinófilos possui papel importante na formação da reação granulomatosa do hospedeiro e resolução do processo inflamatório causado pelo ovo do parasita, além de acrescentar novos dados na classificação dos granulomas hepáticos.
In the present study, the distribution of eosinophils at different stages of the formation of hepatic granuloma in mice infected with Schistosoma mansoni was evaluated. From the results obtained, we suggest a new classification for the evolution of hepatic granuloma in mice, constructed from the phases described by other authors. In each phase, there is a different pattern of eosinophil distribution. In the exudative-necrotic phase, the eosinophils are concentrated in the periphery and center of the granuloma, and are scarce in the necrotic area; in the productive phase, the eosinophils are dispersed throughout the granuloma; and in the cure due to fibrosis phase, the eosinophils are concentrated in the periphery and center of the granuloma. Eosinophils were found in direct contact with the eggs at all stages of evolution of the granuloma. It was concluded that the dynamics of eosinophils have an important role in forming the granulomatous reaction of the host and in resolving the inflammatory process caused by the parasite egg, as well as adding new data regarding hepatic granuloma classification.
Assuntos
Animais , Masculino , Camundongos , Eosinófilos/patologia , Granuloma/patologia , Hepatopatias Parasitárias/patologia , Esquistossomose mansoni/patologia , Modelos Animais de Doenças , Proteína Catiônica de Eosinófilo/análise , Peroxidase de Eosinófilo/análise , Eosinófilos/enzimologia , Granuloma/parasitologia , Imuno-Histoquímica , Hepatopatias Parasitárias/parasitologia , Necrose , Esquistossomose mansoni/parasitologia , Fatores de TempoRESUMO
We investigated the effects of substance P (SP) and neurokinin A (NKA) infusion and acute stimulation of capsaicin-sensitive sensory nerves fibers (CAP) on lung recruitment of neuronal nitric oxide synthase (nNOS)-positive inflammatory and respiratory epithelial (RE) cells in guinea-pigs. We evaluated if the effects of CAP stimulation were maintained until 14 days and had functional pulmonary repercussions. After 24h of CAP and 30 min after SP and NKA infusions there was an increase in nNOS-positive eosinophils and mononuclear cells compared to controls (P<0.05). SP group presented an increase in nNOS-positive RE (P<0.05). After 14 days of CAP stimulation, there was a reduction in resistance (R(rs)) and elastance (E(rs)) of respiratory system in capsaicin pre-treated animals. We noticed a correlation between nNOS-positive eosinophils (R=-0.644, P<0.05) and mononuclear cells (R=-0.88, P<0.001) and R(rs). Concluding, CAP and neurokinins increase nNOS expression by inflammatory and RE cells. The increase in nNOS expression induced by low and high doses stimulation of CAP is longstanding and correlated to pulmonary mechanical repercussions.
Assuntos
Capsaicina/farmacologia , Pulmão/enzimologia , Pulmão/fisiologia , Neurocinina A/fisiologia , Neurônios/fisiologia , Óxido Nítrico Sintase Tipo I/biossíntese , Resistência das Vias Respiratórias/fisiologia , Algoritmos , Animais , Eosinófilos/efeitos dos fármacos , Eosinófilos/enzimologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Cobaias , Imuno-Histoquímica , Pulmão/inervação , Masculino , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Neurônios/efeitos dos fármacos , Mecânica Respiratória/fisiologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/enzimologiaRESUMO
The precise role of each nitric oxide (NO) synthase (NOS) isoform in the pathobiology of asthma is not well established. Our objective was to investigate the contribution of constitutive NO synthase (cNOS) and inducible NOS (iNOS) isoforms to lung mechanics and inflammatory and remodeling responses in an experimental model of chronic allergic pulmonary inflammation. Guinea pigs were submitted to seven ovalbumin exposures with increasing doses (1 approximately 5 mg/ml) for 4 wk. The animals received either chronic L-NAME (N-nitro-L-arginine methyl ester, in drinking water) or 1,400 W (iNOS-specific inhibitor, intraperitoneal) treatments. At 72 h after the seventh inhalation of ovalbumin solution, animals were anesthetized, mechanically ventilated, exhaled NO was collected, and lung mechanical responses were evaluated before and after antigen challenge. Both L-NAME and 1,400 W treatments increased baseline resistance and decreased elastance of the respiratory system in nonsensitized animals. After challenge, L-NAME increased resistance of the respiratory system and collagen deposition on airways, and decreased peribronchial edema and mononuclear cell recruitment. Administration of 1,400 W reduced resistance of the respiratory system response, eosinophilic and mononuclear cell recruitment, and collagen and elastic fibers content in airways. L-NAME treatment reduced both iNOS- and neuronal NOS-positive eosinophils, and 1,400 W diminished only the number of eosinophils expressing iNOS. In this experimental model, inhibition of NOS-derived NO by L-NAME treatment amplifies bronchoconstriction and increases collagen deposition. However, blockage of only iNOS attenuates bronchoconstriction and inflammatory and remodeling processes.
Assuntos
Hiper-Reatividade Brônquica/enzimologia , Pulmão/fisiologia , Óxido Nítrico Sintase Tipo II/fisiologia , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico/metabolismo , Pneumonia/enzimologia , Traqueia/imunologia , Administração por Inalação , Animais , Doença Crônica , Inibidores Enzimáticos/administração & dosagem , Eosinófilos/enzimologia , Cobaias , Inalação , Isoenzimas/metabolismo , Pulmão/enzimologia , Pulmão/patologia , Masculino , NG-Nitroarginina Metil Éster/administração & dosagem , Óxido Nítrico Sintase/antagonistas & inibidores , Ovalbumina/administração & dosagem , Pneumonia/imunologia , Pneumonia/patologia , Mecânica Respiratória , Fatores de Tempo , Traqueia/metabolismoRESUMO
We investigated the effects of the Th2-like cytokines IL-4 and IL-13 and of IL-10 on the induction of iNOS and NO production in rat eosinophils. Addition of mIL-4 to the eosinophil culture increased iNOS activity and nitrite production but did not improve the stimulatory effect of IFN-gamma and LPS. In contrast to eosinophils, addition of mIL-4 to macrophage cultures inhibited the iNOS expression and nitrite production induced by IFN-gamma plus LPS. Addition of mIL-13 to the eosinophil cultures did not significantly change iNOS activity and nitrite production in cells stimulated or not with IFN-gamma plus LPS. On the other hand, IL-13 inhibited iNOS activity in IFN-gamma plus LPS-stimulated macrophages. In the presence of IL-10, iNOS activity in non-stimulated eosinophil or macrophage cultures was not significantly altered, but the enzyme expression was inhibited in IFN-gamma plus LPS-stimulated eosinophils or macrophages. The production of nitrite by eosinophils stimulated by IFN-gamma plus LPS was inhibited by the presence of IL-10 in the medium. In conclusion, eosinophils might exhibit differential modulation of the L-arginine/iNOS pathway depending on the profile of Th2 cytokines produced during allergic diseases. IL-4 appears to be an important Th2 cytokine involved in the induction of the L-arginine/iNOS pathway in eosinophils.
Assuntos
Eosinófilos/efeitos dos fármacos , Eosinófilos/enzimologia , Interleucina-4/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Interleucina-10/metabolismo , Interleucina-13/metabolismo , RatosRESUMO
The aim of this study was to investigate the role of immunoglobulin E (IgE) in the late phase reaction (LPR) of murine experimental asthma. Our model consisted of an implant of DNP-conjugated, heat-coagulated hen's egg white (DNP-EWI), followed 14 days later by an intratracheal challenge with aggregated DNP-ovalbumin. Airway inflammation was analyzed 48 h after challenge and compared with a similarly immunized group of mice with highly suppressed humoral response due to anti-micro and anti-delta antibody treatment. Total number of cells in the bronchoalveolar lavage (BAL) (with predominance of eosinophils) and EPO activity in the lung homogenate were increased in the DNP-EWI-immunized group compared with immunosuppressed or nonimmunized mice. However, the cellular infiltration and EPO activity observed in the immunosuppressed group were still significantly above those obtained in the nonimmunized group, indicating that inhibition of antibody production did not completely prevent the inflammatory manifestations in BAL and lung. Airway hyperresponsiveness to methacoline was obtained in DNP-EWI-immunized mice, but the respiratory mechanical parameters returned to normal levels in the immunosuppressed group. When these mice were reconstituted with monoclonal anti-DNP antibodies, only IgE, but not IgG1, restored lung inflammation and decreased the conductance of the respiratory system, therefore, increasing hyperresponsiveness. These results indicate that antibodies are not essential for induction of LPR in the lung. However, IgE enhances pulmonary inflammation and hyperresponsiveness.
Assuntos
Formação de Anticorpos , Asma/fisiopatologia , Hiper-Reatividade Brônquica/imunologia , Imunoglobulina E/imunologia , Animais , Asma/imunologia , Asma/patologia , Hiper-Reatividade Brônquica/fisiopatologia , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Dinitrofenóis/imunologia , Clara de Ovo , Peroxidase de Eosinófilo , Eosinófilos/enzimologia , Eosinófilos/patologia , Imunoglobulinas/sangue , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Peroxidases/metabolismoRESUMO
Eosinophils and neutrophils are granulocytic leukocytes that are present in the blood of most vertebrates. Studies have been performed on lower vertebrates to understand the biological roles of the cells in defense mechanisms and to establish phylogenetic studies and new experimental models. Whether these 2 cell types exist in reptiles is a matter of controversy. In the blood of turtles there are 2 types of granulocytes that exhibit eosinophilia, one of them with round cytoplasmic granules and the other with elongated cytoplasmic granules. It has been suggested that these cells may be eosinophils in different stages of maturation but they also may be distinct cell types, i.e. eosinophils and neutrophils. In the present study, we characterized the 2 types of granulocytes that are present in the blood of Chrysemys dorbignih, using cytochemical techniques. Type I eosinophils showed activity of nonspecific esterase, peroxidase activity that is resistant to KCN, and basic proteins. Type II eosinophils exhibited activity of trimetaphosphatase, alkaline phosphatase, nonspecific esterase, peroxidase that is sensitive to KCN, and basic proteins. These observations indicate the existence of 2 distinct cell types in the blood of Chrysemys dorbignih, type I and type II eosinophils, that correspond to eosinophils and heterophils (neutrophils) of mammals and other vertebrates.
Assuntos
Eosinófilos/enzimologia , Histocitoquímica/métodos , Tartarugas/sangue , Hidrolases Anidrido Ácido/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Carboxilesterase , Hidrolases de Éster Carboxílico/metabolismo , Grânulos Citoplasmáticos/enzimologia , Eosinófilos/classificação , Eosinófilos/ultraestrutura , Peroxidases/metabolismo , Proteínas/metabolismoRESUMO
The aim of this study was to investigate the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in HL-60 clone 15 cells as they differentiate along the eosinophilic lineage. The results were compared to the eosoniphilic inducers interleukin-5 (IL-5) and butyric acid. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) or IL-5 (200 pM) caused a significant increase in the expression of the eosinophil peroxidase (EPO) and the major basic protein (MBP) genes. Similar results were observed when the cells were cultured with 0.5 mM butyric acid for 5 days. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) also caused a significant increase in superoxide release by HL-60 clone 15 cells after 2 days compared with control or with butyric acid-induced cells. After 5 days, these cytokines and butyric acid induced an even stronger release of superoxide. HL-60 clone 15 cells cultured with IFN-gamma and TNF-alpha for 2 days showed a significant increase in gp91-phox gene expression. We conclude that IFN-gamma and TNF-alpha are sufficient to induce the differentiation of HL-60 clone 15 cells to the eosinophilic lineage and to upregulate gp91-phox gene expression and activity of the NADPH oxidase system.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Interferon gama/farmacologia , NADPH Oxidases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Biomarcadores/análise , Ácido Butírico/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem da Célula , Células Clonais/efeitos dos fármacos , Células Clonais/enzimologia , Células Clonais/metabolismo , Ativação Enzimática/efeitos dos fármacos , Eosinófilos/citologia , Eosinófilos/enzimologia , Eosinófilos/metabolismo , Eritropoetina/genética , Eritropoetina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Interleucina-5/farmacologia , Proteínas Ligantes de Maltose , Glicoproteínas de Membrana/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , Peroxidase/genética , Peroxidase/metabolismoRESUMO
The studies on the characterization of eosinophils and neutrophils/heterophils of turtles are contradictory. Some authors have pointed out the existence of two distinct cell types: eosinophils and heterophils. Other authors have proposed that eosinophils and heterophils may be the same cells in different stages of maturation. These interpretations are based only on a morphological analysis. In the blood of the turtle (Chrysemys dorbignih), a South American freshwater species, there are two types of granulocytes with eosinophilic staining pattern: the first with round cytoplasmic granules and the second with ellipsoidal cytoplasmic granules. In the present study by using histoenzymological methods for the analyses of enzymological cellular content, we found that the cells with round cytoplasmic granules were positive for nonspecific esterase and the cells with ellipsoidal granules were positives for acid phosphatase, alkaline phosphatase, nonspecific esterase and peroxidase. The results show that these cells are distinct cells and that the cells with ellipsoidal cytoplasmic granules have the same histoenzymological characteristics as the neutrophils/heterophils of mammalians and other vertebrates.
Assuntos
Eosinófilos/citologia , Eosinófilos/enzimologia , Tartarugas/fisiologia , Fosfatase Ácida/análise , Fosfatase Alcalina/análise , Animais , Carboxilesterase , Hidrolases de Éster Carboxílico/análise , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/ultraestrutura , Eosinófilos/química , Histocitoquímica , Microscopia Eletrônica , Peroxidase/análiseRESUMO
Tachyzoites of Toxoplasma gondii are ingested by neutrophils and eosinophils through a process which can be significantly inhibited by previous incubation of the host cells with cytochalasin D. Although dividing zoites within the leukocytes could be observed, after 3 h of infection, killing of parasites within the parasitophorous vacuole was detected. Cytochemical studies showed that both in neutrophils and eosinophils there is a process of NADP(H) oxidase activation, which was higher in the latter.
Assuntos
Eosinófilos/parasitologia , Eosinófilos/ultraestrutura , Neutrófilos/parasitologia , Neutrófilos/ultraestrutura , Toxoplasma/patogenicidade , Toxoplasma/ultraestrutura , Animais , Células Cultivadas , Citocalasina D/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/enzimologia , Interações Hospedeiro-Parasita , Camundongos , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Ratos , Ratos Wistar , Toxoplasma/efeitos dos fármacosRESUMO
The expression of nitric oxide (NO) synthases and the role of the NO cyclic GMP pathway on the migration of eosinophils from untreated patients with allergic rhinitis were investigated. Inducible NO synthase was strongly expressed in eosinophils from healthy individuals, but not in eosinophils from allergic rhinitis patients. The neuronal isoform was observed in eosinophils from each group studied, whereas no staining for the endothelial isoform was detected in either group. The chemotaxis to N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 x 10(-7) M) and eotaxin (100 ng/ml) was significantly potentiated in allergic rhinitis eosinophils. In both groups, N(omega)-nitro-L-arginine methyl ester (L-NAME, 1.0 mM) or 1H(1,2,4)-oxadiazolo(4,3,-a)quinoxalin-1-one (ODQ, 0.2 mM) markedly reduced the chemotaxis. The selective iNOS inhibitor N-(3-(aminomethyl)benzyl)acetamidine (1400 W, 0.1-1.0 mM) significantly reduced the chemotaxis of eosinophils from healthy but not from allergic rhinitis subjects. The inhibition by L-NAME was restored by 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetyl-penicillamine, whereas the inhibition by ODQ was restored by dibutyryl cyclic GMP. In conclusion, both endothelial and inducible NO synthase isoforms are absent in allergic rhinitis eosinophils, suggesting that the NO cyclic GMP pathway in this cell type is maintained through the activity of a neuronal isoform.
Assuntos
Eosinófilos/enzimologia , Óxido Nítrico Sintase/biossíntese , Rinite Alérgica Perene/sangue , Movimento Celular/efeitos dos fármacos , Quimiocina CCL11 , Quimiocinas CC/farmacologia , Quimiotaxia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/patologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Humanos , Isoenzimas/biossíntese , Molsidomina/análogos & derivados , Molsidomina/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Oxidiazóis/farmacologia , Quinoxalinas/farmacologia , Rinite Alérgica Perene/patologia , S-Nitroso-N-Acetilpenicilamina/farmacologia , SolubilidadeRESUMO
Eosinophils purified from the rat peritoneal cavity have been found to contain nitric oxide synthase (NOS) functionally coupled to a cyclic GMP transduction pathway that is involved in in vitro eosinophil migration, but no studies on cell locomotion have been done with purified human eosinophils. Therefore, this study was carried out to investigate the effects of N(omega) -nitro-L-arginine methyl ester (L-NAME; a non-selective NOS inhibitor), 1-(2-trifluoromethylphenyl) imidazole (TRIM; a type I/type II NOS inhibitor), 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; a selective type II NOS inhibitor), and 1H-[1,2,4]-oxidiazolo[4,3-a] quinoxalin-1-one (ODQ; a soluble guanylate cyclase inhibitor) on human eosinophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP). Human eosinophils were purified from peripheral blood of healthy volunteers using a Percoll gradient followed by an immunomagnetic cell separator. Chemotaxis was evaluated using a 48-well microchemotaxis chamber. The fMLP (1.0 x 10(-7) M)-induced eosinophil migration was reduced significantly by l-NAME (0.1 and 1.0 mM), whereas the inactive enantiomer N(omega)-nitro-D-arginine methyl ester (D-NAME) had no effect. The inhibition by l-NAME was restored by sodium nitroprusside (0.25 mM). The NOS inhibitors AMT and TRIM (0.05 to 0.25 mM each) also markedly attenuated fMLP-induced chemotaxis. Additionally, ODQ (0.01 to 0.5 mM) concentration-dependently inhibited fMLP-induced migration, and the inhibition was restored by 2.0 mM dibutyryl cyclic GMP. In conclusion, this study demonstrates that human eosinophils present a nitric oxide-cyclic GMP pathway that is involved in the in vitro locomotion of this cell type.
Assuntos
Movimento Celular/fisiologia , Eosinófilos/citologia , Óxido Nítrico/fisiologia , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/enzimologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Humanos , Imidazóis/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Oxidiazóis/farmacologia , Quinoxalinas/farmacologiaRESUMO
Airway inflammation plays a major role in human asthma. Increasing evidence points to a close correlation between eosinophil infiltration and allergic lung disease. A new murine model of eosinophilic lung inflammation has recently been developed; it consists of immunizing mice with small fragments of solidified hen egg white implanted (EWI) into the subcutaneous tissue. In this model, which is further characterized here, mice challenged with ovalbumin (OVA) present an intense and persistent lung eosinophilia, as well as histopathological findings that resemble human asthma. In the present work, the effect of oral tolerance on the development of allergic lung inflammation in B6 mice immunized with antigen plus adjuvant or with EWI is investigated. It was found that in mice rendered orally tolerant by previous exposure to antigen in the drinking water, the T-helper type 2 cell (Th2)-associated allergic responses in both protocols of immunization were almost completely abolished. The allergic responses were assessed by pulmonary and bone marrow eosinophilia, lung histopathology and antigen-specific IgE and IgG1 production. These findings provide the first indication that Th2-associated lung pathology can be prevented by oral tolerance.
Assuntos
Tolerância Imunológica/imunologia , Imunoterapia , Eosinofilia Pulmonar/prevenção & controle , Administração Oral , Adsorção , Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/imunologia , Animais , Clara de Ovo , Peroxidase de Eosinófilo , Eosinófilos/enzimologia , Feminino , Hipersensibilidade/imunologia , Hipersensibilidade/prevenção & controle , Imunização , Isotipos de Imunoglobulinas/análise , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Peroxidases/imunologia , Peroxidases/metabolismo , Eosinofilia Pulmonar/imunologia , Células Th2/imunologiaRESUMO
Eosinophil migration in vivo is markedly attenuated in rats treated chronically with the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). In this study, we investigated the existence of a NOS system in eosinophils. Our results demonstrated that rat peritoneal eosinophils strongly express both type II (30.2 +/- 11.6% of counted cells) and type III (24.7 +/- 7.4% of counted cells) NOS, as detected by immunohistochemistry using affinity purified mouse mAbs. Eosinophil migration in vitro was evaluated by using 48-well microchemotaxis chambers and the chemotactic agents used were N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 x 10(-8) M) and leukotriene B4 (LTB4, 10(-8) M). L-NAME (but not D-NAME) significantly inhibited the eosinophil migration induced by both fMLP (54% reduction for 1.0 mM; P < 0.05) and LTB4 (61% reduction for 1.0 mM; P < 0.05). In addition, the type II NOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and the type I/II NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole also markedly (P < 0. 05) attenuated fMLP- (52% and 38% reduction for 1.0 mM, respectively) and LTB4- (52% and 51% reduction for 1.0 mM, respectively) induced migration. The inhibition of eosinophil migration by L-NAME was mimicked by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (0.01 and 0.1 mM) and reversed by either sodium nitroprusside (0.1 mM) or dibutyryl cyclic GMP (1 mM). We conclude that eosinophils do express NO synthase(s) and that nitric oxide plays an essential role in eosinophil locomotion by acting through a cyclic GMP transduction mechanism.