RESUMO
OBJECTIVE: To investigate the effects of CysLT receptor agonist leukotriene D4(LTD4) and antagonists on activation of microglia BV2 cells. METHODS: The expression of CysLT1 and CysLT2 protein was determined by Western blotting and immunostaining in microglia BV2 cells. BV2 cells were pretreated with or without CysLT1 receptor selective antagonist montelukast, CysLT2 receptor selective antagonist HAMI 3379, or CysLT1/CysLT2 receptor dual antagonist BAY u9773 for 30 min, then the cells were treated with LTD4 for 24 h. Cell viability was detected by MTT reduction assay. Phagocytosis and mRNA expression of IL-6 were determined by fluorescent bead tracking and RT-PCR, respectively. RESULTS: In BV2 cells, LTD4 did not affect proliferation but significantly enhanced phagocytosis and increased IL-6 mRNA expression in a concentration-dependent manner. LTD4 at 100 nmol/L induced a 1.4-fold increase of phagocytic index and a 2-fold up-regulation of IL-6 mRNA expression (P<0.01). HAMI 3379 and BAY u9773 (100 nmol/L) further increased LTD4-induced phagocytosis; BAY u9773 and montelukast decreased LTD4-induced IL-6 mRNA expression, while HAMI 3379 had no effect on that. CONCLUSION: LTD4 activates BV2 cells in vitro and enhances IL-6 mRNA expression mediated by CysLT1 receptor, LTD4 induces phagocytosis which might be negatively regulated by CysLT2 receptor in BV2 cells.
Assuntos
Leucotrieno D4/farmacologia , Microglia/citologia , Receptores de Leucotrienos/metabolismo , Acetatos/farmacologia , Linhagem Celular , Proliferação de Células , Ácidos Cicloexanocarboxílicos/farmacologia , Ciclopropanos , Humanos , Interleucina-6/metabolismo , Antagonistas de Leucotrienos/farmacologia , Microglia/metabolismo , Fagocitose , Ácidos Ftálicos/farmacologia , Quinolinas/farmacologia , SRS-A/análogos & derivados , SRS-A/farmacologia , SulfetosRESUMO
RATIONALE: Biomarkers predicting development of atopic disease are needed for targeted preventive measures and to study if disease pathology may be active before onset of symptoms. OBJECTIVES: To investigate whether eosinophil protein X, leukotriene-C4/D4/E4, and 11ß-prostaglandin (PG) F2α (PGD2 metabolite) assessed in urine from healthy at-risk neonates precede development of atopic disease during the first 6 years of life. METHODS: We measured eosinophil protein X (n = 369), leukotriene-C4/D4/E4 (n = 367), and 11ß-PGF2α (n = 366) in urine from 1-month-old children participating in the Copenhagen Prospective Studies on Asthma in Childhood birth cohort. Clinical data on development of allergic sensitization, allergic rhinitis, nasal eosinophilia, blood eosinophilia, eczema, troublesome lung symptoms (significant cough or wheeze or dyspnea), and asthma were collected prospectively until age 6 years. Associations between urinary biomarkers and development of atopic traits were investigated using general estimating equations, logistic regression, and Cox regression. MEASUREMENTS AND MAIN RESULTS: Eosinophil protein X in the urine of the asymptomatic 1-month-old neonates was significantly associated with development of allergic sensitization (odds ratio, 1.49; 95% confidence interval [CI], 1.081.89), nasal eosinophilia (odds ratio, 3.2; 95% CI, 1.28.8), and eczema (hazard ratio, 1.4; 95% CI, 1.02.0), but not with allergic rhinitis, asthma, or blood eosinophilia. Neither leukotriene-C4/D4/E4 nor 11ß-PGF2α was associated with any of the atopic phenotypes. CONCLUSIONS: Eosinophil protein X in urine from asymptomatic neonates is a biomarker significantly associated with later development of allergic sensitization, nasal eosinophilia, and eczema during the first 6 years of life. These findings suggest activation of eosinophil granulocytes early in life before development of atopy-related symptoms.
Assuntos
Neurotoxina Derivada de Eosinófilo/urina , Hipersensibilidade Imediata/urina , Biomarcadores/urina , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Razão de Chances , Prostaglandinas/urina , SRS-A/urinaRESUMO
Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators that predominantly exert their effects by binding to cysteinyl leukotriene receptors of the G protein-coupled receptor family. CysLT receptor 2 (CysLT(2)R), expressed in endothelial cells of some vascular beds, has been implicated in a variety of cardiovascular functions. Endothelium-specific overexpression of human CysLT(2)R in transgenic mice (hEC-CysLT(2)R) greatly increases myocardial infarction damage. Investigation of this receptor, however, has been hindered by the lack of selective pharmacological antagonists. Here, we describe the characterization of 3-(((3-carboxycyclohexyl)amino)carbonyl)-4-(3-(4-(4-phenoxybutoxy)phenyl)-propoxy)benzoic acid (BayCysLT(2)) and explore the selective effects of this compound in attenuating myocardial ischemia/reperfusion damage and vascular leakage. Using a recently developed ß-galactosidase-ß-arrestin complementation assay for CysLT(2)R activity (Mol Pharmacol 79:270-278, 2011), we determined BayCysLT(2) to be â¼20-fold more potent than the nonselective dual CysLT receptor 1 (CysLT(1)R)/CysLT(2)R antagonist 4-(((1R,2E,4E,6Z,9Z)-1-((1S)-4-carboxy-1-hydroxybutyl)-2,4,6,9-pentadecatetraen-1-yl)thio)benzoic acid (Bay-u9773) (IC(50) 274 nM versus 4.6 µM, respectively). Intracellular calcium mobilization in response to cysteinyl leukotriene administration showed that BayCysLT(2) was >500-fold more selective for CysLT(2)R compared with CysLT(1)R. Intraperitoneal injection of BayCysLT(2) in mice significantly attenuated leukotriene D(4)-induced Evans blue dye leakage in the murine ear vasculature. BayCysLT(2) administration either before or after ischemia/reperfusion attenuated the aforementioned increased myocardial infarction damage in hEC-CysLT(2)R mice. Finally, decreased neutrophil infiltration and leukocyte adhesion molecule mRNA expression were observed in mice treated with antagonist compared with untreated controls. In conclusion, we present the characterization of a potent and selective antagonist for CysLT(2)R that is useful for discerning biological activities of this receptor.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Ácidos Cicloexanocarboxílicos/farmacologia , Antagonistas de Leucotrienos/farmacologia , Leucotrieno D4/antagonistas & inibidores , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Ácidos Ftálicos/farmacologia , Receptores de Leucotrienos/metabolismo , SRS-A/análogos & derivados , Animais , Arrestinas/análise , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Orelha/irrigação sanguínea , Humanos , Camundongos , Camundongos Transgênicos , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Peroxidase/metabolismo , SRS-A/farmacologia , beta-Arrestinas , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Little is known about the role of the cysteinyl leukotriene (cysLT) 2 receptor in the pathophysiology of asthma. The aim of this study is to investigate the effects of a cysLT1 receptor antagonist (montelukast) and a dual cysLT1/2 receptor antagonist (BAY-u9773) on airway hypersensitivity and airway inflammation induced by antigen challenge in ovalbumin (OVA)-sensitized guinea pigs. METHODS: Male Hartley guinea pigs sensitized with OVA were intraperitoneally administered 0.1, 1, or 10 mg/kg of montelukast or 0.1 mg/kg of BAY-u9773 and then challenged with inhaled OVA. Airway reactivity to acetylcholine, inflammatory cells in bronchoalveolar lavage (BAL) fluid, and eosinophil infiltration in airway walls after OVA challenge were evaluated. RESULTS: Pretreatment with 1 or 10 mg/kg, but not 0.1 mg/kg, of montelukast significantly suppressed airway hypersensitivity and eosinophil infiltration into the BAL fluid. Moreover, 0.1 mg/kg of BAY-u9773 significantly suppressed the development of these markers. The suppressive effects of BAY-u9773, although not significantly different, trended toward being greater than those of montelukast. Although all of the doses of montelukast tested and 0.1 mg/kg of BAY-u9773 significantly suppressed eosinophil infiltration in airway walls, the suppressive effect of BAY-u9773 was significantly greater than that of 0.1 mg/kg of montelukast. CONCLUSION: Signaling may contribute to the pathophysiology of asthma via the cysLT1/2 receptor.
Assuntos
Asma/tratamento farmacológico , Hiper-Reatividade Brônquica/prevenção & controle , Antagonistas de Leucotrienos/uso terapêutico , Receptores de Leucotrienos , Acetatos/farmacologia , Acetatos/uso terapêutico , Acetilcolina/farmacologia , Animais , Asma/induzido quimicamente , Asma/imunologia , Asma/patologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/fisiopatologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Ciclopropanos , Eosinófilos/patologia , Cobaias , Inflamação/patologia , Inflamação/prevenção & controle , Antagonistas de Leucotrienos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Quinolinas/farmacologia , Quinolinas/uso terapêutico , SRS-A/análogos & derivados , SRS-A/farmacologia , SRS-A/uso terapêutico , SulfetosRESUMO
The capacity to extract slow-reacting substance of anaphylaxis (SRS-A) from human lung tissue or cells after immunologic activation, together with the measurement of SRS-A in both the extract and the surrounding fluid, permits study of total SRS-A generation. That the material extracted is SRS-A was established by both differential bioassay and purification. SRS-A accumulation was entirely intracellular after limited IgE-dependent direct or reversed anaphylactic activation. Intracellular accumulation also generally preceded release, with generation of SRS-A continuing well beyond a plateau in the cellular SRS-A level and the release of preformed mediators. The quantity of SRS-A generated after immunologic activation was modulated by the introduction of exogenous cyclic nucleotides, revealing a site of cyclic nucleotide action distinct from that on mediator release. The capacity to determine not only the release of preformed mediators but also the generation of a newly formed mediator, the sum of SRS-A in cells and supernate, adds an additional dimension to the analysis of the cellular events of immediate hypersensitivity.
Assuntos
Pulmão/imunologia , SRS-A/biossíntese , Anafilaxia/imunologia , Animais , Antígenos , Bioensaio , Bucladesina/farmacologia , AMP Cíclico/farmacologia , GMP Cíclico/farmacologia , Cobaias , Humanos , Imunização , Imunoglobulina E , Imunoglobulina G , Ratos , SRS-A/isolamento & purificação , SRS-A/metabolismo , Extratos de TecidosRESUMO
Cultured endothelial cells from human umbilical cord labeled with [3H]20:4 release radiolabel when exposed to leukotrienes C or D (LTC or LTD). The major radiolabeled 20:4 metabolite recovered in the culture medium was prostacyclin. Both leukotrienes produced a dose-dependent synthesis of prostacyclin, with a maximal response at 10(-7) M leukotriene. LTC promoted a twofold greater response than did LTD at all concentrations tested (10(-9) to 10(-7) M). In contrast, no release of radiolabel above basal levels was evident with a challenge of LTE or LTB at the same concentrations. Endothelial cells metabolize approximately 40-50% of exogenously supplied LTC to LTD and LTE in 60 min. Levels of alpha-glutamyltranspeptidase (gamma-GTPase), the ectoenzyme reported to convert LTC or LTD, were detected in intact endothelial cells with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide at levels sufficient to account for the observed rate of LTC metabolism. High concentrations of the gamma-GTPase inhibitors, glutathione and AT-125, blocked the metabolism of LTC by endothelium. These results suggest that degradation of leukotrienes by endothelium may be one mechanism for inactivation of these lipid mediators.
Assuntos
Endotélio/metabolismo , Epoprostenol/biossíntese , SRS-A/farmacologia , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Células Cultivadas , Endotélio/citologia , Histamina/farmacologia , Humanos , Cinética , Leucotrieno B4/farmacologia , Leucotrieno E4 , SRS-A/análogos & derivados , SRS-A/metabolismo , Veias Umbilicais , gama-Glutamiltransferase/metabolismoRESUMO
Eicosanoids have been implicated in the pathogenesis of autoimmune diseases. In this study, peritoneal macrophages from autoimmune-prone mice were examined for their capacity to produce proinflammatory 5-lipoxygenase metabolites. The results indicate that enhanced production of leukotriene C4 is a common feature of murine autoimmunity and suggest further that aberrations in 5-lipoxygenase activity may play a role in the development of lupus.
Assuntos
Doenças Autoimunes/imunologia , Macrófagos/imunologia , SRS-A/biossíntese , Animais , Células Cultivadas , Lipoxigenase/metabolismo , Macrófagos/enzimologia , Camundongos , Camundongos MutantesRESUMO
The antigen-induced release of SRS-A and histamine was studied in the guinea pig and rat using whole and fractionated antiserum preparations. Guinea pig 7Sgamma(1)-antibody sensitized sliced guinea pig lung tissue for antigen-induced release of both SRS-A and histamine; neither substance was released from lung tissue prepared with 7Sgamma(1)-antibody. Rats injected intraperitoneally with hyperimmune rabbit or rat antiserum released only SRS-A in significant amounts when challenged with antigen by the same route. A definite time interval between the injection of antiserum and challenge with antigen was required for optimal release of SRS-A. Fractionation of rat antiserum demonstrated that the immunoglobulin responsible for most of the SRS-A release from rat peritoneal tissue was a gammaG-antibody or fraction thereof. Acting in this capacity, the gammaG-antibody or its subfraction may be considered a second type of homocytotropic antibody. Fractions of rat antisera containing the first type of homocytotropic antibody, i.e. antibody mediating release of histamine and serotonin, prepared peritoneal tissues for the release of large amounts of these pharmacological agents and only small amounts of SRS-A. Two different mechanisms for the production of PCA lesions in the rat were considered. One of these involves the antigen-induced release of histamine and serotonin from mast cells sensitized by homocytotropic antibody. This reaction has an optimal latent period of 24-72 hr. The second mechanism involves the local combination of antigen with "hyperimmune" heterologous or homologous antisera. This reaction can be elicited after a latent period of 4 but not 24 hr; host complement and leukocyte lysosomal enzymes, as well as SRS-A, may be involved.
Assuntos
Anafilaxia , Anticorpos , Reações Antígeno-Anticorpo , Animais , Cromatografia , Fibrinogênio , Cobaias , Liberação de Histamina , Imunoeletroforese , Pulmão/fisiologia , Ovalbumina , Ratos , SRS-A , Soroalbumina Bovina , gama-Globulinas/análiseRESUMO
We examined changes in the levels of eicosanoids in blood and pulmonary lymph of anesthetized sheep undergoing acute anaphylaxis. Within 1-3 min of intravenous antigenic challenge of previously sensitized sheep, there were approximately 7-30-fold elevations in mean arterial plasma levels of thromboxane B2 and 6-ketoprostaglandin F1 alpha, respectively, as measured by RIA. Negligible changes in levels of these cyclooxygenase products were found in both nonsensitized sheep and in sensitized sheep treated with indomethacin before antigenic challenge. In contrast, no changes in levels of sulfidopeptide leukotrienes (SPLT) in pulmonary lymph were detectable by RIA during anaphylaxis in sensitized or nonsensitized sheep, but levels of SPLT in indomethacin-treated sensitized sheep increased more than fivefold above levels in lymph from both other groups of animals. The immunoreactive SPLT in lymph from indomethacin-treated sheep was accounted for as LTE4, as demonstrated by mobility on HPLC and absorbance at 280 nm. These results support the possibility that certain undesirable effects of nonsteroidal antiinflammatory drugs, such as cardiopulmonary reactions in aspirin-sensitive individuals, and impaired renal and cardiac function during therapy with these drugs, may be related in part to augmented synthesis of the 5-lipoxygenase pathway products, especially those of the sulfidopeptide class. Increased LT production could also limit the antiinflammatory effectiveness of these drugs in many disease states.
Assuntos
Anafilaxia/metabolismo , Inibidores de Ciclo-Oxigenase , SRS-A/análogos & derivados , 6-Cetoprostaglandina F1 alfa/biossíntese , Doença Aguda , Animais , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/farmacologia , Araquidonato Lipoxigenases , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Indometacina/farmacologia , Leucotrieno E4 , Lipoxigenase/metabolismo , Pulmão/imunologia , Linfa/análise , Prostaglandina-Endoperóxido Sintases/metabolismo , SRS-A/biossíntese , Ovinos , Tromboxano B2/biossínteseRESUMO
Horse eosinophils purified to greater than 98% generated slow reacting substance (SRS) when incubated with the calcium ionophore A23187. On a per cell basis, eosinophils generated four to five times the SRS produced by similarly treated horse neutrophils. Eosinophil SRS production was inhibited by 5,8,11,14-eicosatetraynoic acid and augmented by indomethacin and arachidonic acid, suggesting that it was a product(s) of the lipoxygenase pathway of arachidonic acid metabolism. Compounds with SRS activity were purified by high-pressure liquid chromatography (HPLC) and identified by ultraviolet spectra, spectral shift on treatment with lipoxygenase, incorporation of [14C]arachidonic acid, gas chromatography-mass spectrometry, and comparison of retention times on HPLC to authentic standards. The eosinophil products characterized were 5-(S), 12-(R)-dihydroxy-6-cis-8, 10-trans-14-cis-eicosatetraenoic acid (leukotriene B4) and its 5-(S), 12-(R)-6-trans and 5-(S), 12-(S)-6-trans isomers, 5-(S)-hydroxy-6-(R)-S-glutathionyl-7,9-trans-11, 14-cis-eicosatetraenoic acid (leukotriene C4) and its 11-trans isomer, and 5-(S)-hydroxy-6-(R)-S-cysteinylglycine-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4).
Assuntos
Ácidos Araquidônicos/biossíntese , Eosinófilos/metabolismo , Animais , Ácidos Araquidônicos/isolamento & purificação , Autacoides/antagonistas & inibidores , Autacoides/biossíntese , Autacoides/farmacologia , Calcimicina/farmacologia , Cromonas/farmacologia , Cobaias , Cavalos , Humanos , L-Lactato Desidrogenase/metabolismo , Leucotrieno B4 , Camundongos , Camundongos Endogâmicos , Neutrófilos/metabolismo , SRS-A/biossíntese , SRS-A/isolamento & purificaçãoRESUMO
We show that downregulation of arachidonic acid (20:4) metabolism which occurs following i.p. injection of C. parvum can occur in a single, localized macrophage population, and is therefore unlikely to be mediated solely by a systemic factor.
Assuntos
Ácidos Araquidônicos/metabolismo , Macrófagos/metabolismo , 6-Cetoprostaglandina F1 alfa/metabolismo , Animais , Ácido Araquidônico , Dinoprostona , Feminino , Inflamação/metabolismo , Camundongos , Fosfolipídeos/metabolismo , Prostaglandinas E/metabolismo , SRS-A/metabolismo , Tromboxano B2/metabolismoRESUMO
Freshly isolated 2-h adherent normal human monocytes, when stimulated, degrade added leukotriene C4 (LTC4) by a myeloperoxidase (MPO) and H2O2-dependent mechanism. Among the stimuli effective in this regard are phorbol myristate acetate (PMA), the calcium ionophore A23187, opsonized zymosan, and N-formyl-methionine-leucine-phenylalanine (FMLP) when combined with cytochalasin B. The predominant products formed are the all-trans isomers of LTB4, 5-(S), 12-(R)-6-trans-LTB4 and 5-(S),12-(S)-6-trans-LTB4. Degradation is inhibited by azide and catalase, but not by superoxide dismutase. LTC4 degradation does not occur when MPO-deficient monocytes are used, unless MPO is added. Stimulated monocytes from patients with chronic granulomatous disease also are unable to degrade LTC4 under these conditions. Normal monocytes maintained in culture lose their ability to degrade LTC4. The addition of MPO to monocyte-derived macrophages increases degradation, particularly when the monolayers are pretreated with gamma-interferon. The oxidative degradation of LTC4 is a capacity shared by neutrophils, eosinophils, and mononuclear phagocytes, and may be an important mechanism for the modulation of leukotriene activity in inflammatory lesions.
Assuntos
Macrófagos/imunologia , Monócitos/imunologia , SRS-A/metabolismo , Calcimicina/farmacologia , Células Cultivadas , Humanos , Cinética , Monócitos/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Oxirredução , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The capacity of actively or passively sensitized guinea pig lung to react with antigen to release a factor specifically chemotactic for eosinophil leukocytes (ECF-A) has been demonstrated. The release of ECF-A was also accompanied by the elaboration of both histamine and SRS-A and the appearance of all these mediators exhibited a similar response in terms of the time course of passve sensitization, the effect of antigen dose, the time course of release, divalent cation dependence and enhancement by the presence of succinate or maleate. Decomplementation by the administration of purified cobra venom factor had no effect on the antigen-induced release of ECF-A from actively or passively sensitized lung fragments. When fragments of guinea pig lung were passively sensitized with fractions of guinea pig 7S IgG, only the IgG(1)-containing fractions prepared tissue for the antigen-induced release of ECF-A. Histamine, SRS-A, bradykinin, serotonin, and the prostaglandins PGE(1), PGE(2), and PGF(2alpha) were not eosinophilotactic per se; neither was ECF-A detected following the incubation of these agents with sensitized lung in the absence of antigen. Both eosinophilotactic activity and SRS-A survived extraction in 80% ethanol and evaporation to dryness. SRS-A, however, withstood boiling in alkaline solution for 20 min, whereas ECF-A activity was abolished by this procedure. SRS-A and ECF-A could also be separated by gel filtration. ECF-A activity was completely recovered following its passage through a column of Sephadex G-25 and had an estimated molecular weight of between 500 and 1000. On the basis of size and a formation mechanism independent of the complement system, ECF-A is distinguishable from a previously described complement-dependent eosinophilotactic factor (ECF-C). Thus, ECF-A represents a hitherto undescribed agent which selectively attracts eosinophil leukocytes.
Assuntos
Anafilaxia/imunologia , Quimiotaxia , Eosinófilos/imunologia , Pulmão/imunologia , Músculo Liso/fisiologia , Animais , Cromatografia DEAE-Celulose , Cromatografia em Gel , Proteínas do Sistema Complemento , Ácido Edético/farmacologia , Filtração , Cobaias , Liberação de Histamina , Imunoglobulina G , Técnicas In Vitro , Maleatos/farmacologia , SRS-A , Serpentes , Succinatos/farmacologia , PeçonhasRESUMO
Isolated dog mastocytoma cells sensitized with dog anti-ragweed IgE and challenged with ragweed antigen or incubated with ionophore A23187 or the carboxy-terminal dodecapeptide of platelet factor 4, PF4(59-70), release histamine and concurrently generate leukotrienes B4, C4, and D4. In contrast, the exposure of mastocytoma cells to 0.1-3 micrograms/ml of 15-hydroxyeicosatetraenoic acid (15-HETE) stimulates selectively the generation of leukotrienes, in the absence of histamine release, while 0.1-1 micrograms/ml of compound 48/80 releases histamine without enhancing the generation of leukotrienes. That natural stimuli are capable of selectively activating one synthetic or secretory compartment of mast cells suggests that separate subsets of receptors as well as different biochemical events may serve to mobilize each class of mediators.
Assuntos
Transformação Celular Neoplásica/imunologia , Liberação de Histamina , Ácidos Hidroxieicosatetraenoicos , Sarcoma de Mastócitos/imunologia , SRS-A/biossíntese , Alérgenos/imunologia , Animais , Ácidos Araquidônicos/farmacologia , Calcimicina/farmacologia , Cães , Cinética , Leucotrieno B4/biossíntese , Fator Plaquetário 4/imunologia , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Resident mouse peritoneal macrophages release the slow-reacting substance leukotriene C (LTC) on exposure to particulate IgE immune complexes. Because these cells lose their responsiveness to an IgE stimulus after 4 h in culture, maximum release of 20:4 metabolites is observed before this time. However, a similar diminution in 20:4 metabolism was not observed with a zymosan stimulus. Freshly explanted cells are deficient in intracellular glutathione (GSH) (12.4 +/- 0.4 pmol/micrograms cell protein), but GSH increases to a steady state value of 30-35 pmol/micrograms of cell protein between 3 and 9 h of culture. Because GSH is required for the synthesis of LTC and prostaglandin (PG)E2, cultures challenged immediately after explanation have a diminished capacity to synthesize these 20:4 metabolites and release prostacyclin as the major product. By 4-5 h in culture, macrophages form significant amounts of LTC and PGE2. Under optimum conditions of maximum responsiveness to an IgE stimulus and GSH content (after 4 h of culture), macrophages challenged with latex beads coated with IgE immune complexes synthesize 1.0 +/- 0.3 pmol of LTC/microgram cell protein (60 +/- 18 pmol/10(6) cells) in addition to prostacyclin (8.2 +/- 0.8 pmol/micrograms cell protein) and PGE2 (4.7 +/- 1.5 pmol/micrograms cell protein). These amounts are quantitatively similar to the arachidonic acid metabolites produced by macrophages challenged with IgG immune complex-coated latex beads or zymosan. These data demonstrate that macrophages produce large quantities of LTC and other 20:4 metabolites in response to particle-bound IgE and antigen, provided that the appropriate in vitro conditions are met. The macrophage might, therefore, be a major source of slow-reacting substance and other 20:4 metabolites generated during IgE-mediated reactions in vivo.
Assuntos
Epoprostenol/metabolismo , Imunoglobulina E/imunologia , Macrófagos/metabolismo , Prostaglandinas E/metabolismo , Prostaglandinas/metabolismo , SRS-A/metabolismo , Animais , Complexo Antígeno-Anticorpo , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos ICR , Fatores de TempoRESUMO
Mouse bone marrow-derived mast cells differentiated in vitro and sensitized with monoclonal IgE respond to antigen-initiated activation with the release of histamine, beta-hexosaminidase, chondroitin sulfate E proteoglycan, and leukotriene C4 (LTC4). The chondroitin sulfate E nature of the glycosaminoglycan side chain was established by demonstrating that the chondroitinase ABC disaccharide digestion products were composed of equal quantities of 4-sulfated and 4,6-disulfated N-acetyl-galactosamine. The single immunoreactive sulfidopeptide leukotriene, released and quantitated with a class-specific antibody, was identified as LTC4 by its retention time on reverse-phase high-performance liquid chromatography and by its specific spasmogenic activity on the guinea pig ileum. The release of the preformed mediators, as well as of LTC4, was related in a dose-response fashion to the concentration of monoclonal IgE used during the sensitization step and to the concentration of specific antigen used to initiate the activation-secretion response. The optimal concentrations of IgE for sensitization and of antigen for challenge were the same for the release of preformed mediators and of LTC4. In addition, the time courses of their release were superimposable, with a plateau at 5 min after antigen challenge. The release of three preformed mediators and of LTC4 after fixation of IgE, washing of the sensitized cells, and antigen challenge unequivocally indicates a bone marrow-derived mast cell origin for these products. Linear regression analyses of the net percent release of beta-hexosaminidase to histamine and of 35S-chondroitin sulfate E to beta-hexosaminidase yielded straight lines that intersected at the origin, which indicates that the three preformed mediators are localized in the secretory granules of the bone marrow-derived mast cells. The concomitant generation of 23 ng of LTC4/10(6) sensitized bone marrow-derived mast cells represents the first example of IgE-dependent release of substantial amounts of LTC4, a component of slow reacting substance of anaphylaxis, from a mast cell population of greater than 95% purity. The IgE-dependent generation of LTC4, rather than prostaglandin D2, by the chondroitin sulfate E proteoglycan-containing bone marrow-derived mast cells contrasts with the predominant generation of prostaglandin D2 by heparin proteoglycan-containing mast cells. These differences together support the existence of two phenotypically different mast cell subclasses.
Assuntos
Hexosaminidases/metabolismo , Liberação de Histamina , Imunoglobulina E/imunologia , Mastócitos/imunologia , Proteoglicanas/metabolismo , SRS-A/metabolismo , Animais , Células da Medula Óssea , Células Cultivadas , Sulfatos de Condroitina/metabolismo , Exocitose , Mastócitos/metabolismo , CamundongosRESUMO
The cellular infiltrates of certain inflammatory processes found in parasitic infection or in allergic diseases consist predominantly of eosinophilic granulocytes, often in association with activated T cells. This suggests the existence of chemotactic agonists specific for eosinophils and lymphocyte subsets devoid of neutrophil-activating properties. We therefore examined four members of the intercrine/chemokine superfamily of cytokines (monocyte chemotactic peptide 1 [MCP-1], RANTES, macrophage inflammatory protein 1 alpha [MIP-1 alpha], and MIP-1 beta), which do not activate neutrophils, for their ability to affect different eosinophil effector functions. RANTES strongly attracted normal human eosinophils by a chemotactic rather than a chemokinetic mechanism with a similar efficacy as the most potent chemotactic myeloid cell agonist, C5a. MIP-1 alpha also induced eosinophil migration, however, with lower efficacy. RANTES and MIP-1 alpha induced eosinophil cationic protein release in cytochalasin B-treated eosinophils, but did not promote leukotriene C4 formation by eosinophils, even after preincubation with interleukin 3 (IL-3), in contrast to other chemotactic agonists such as C5a and formyl-methionyl-leucyl-phenylalanine (FMLP). RANTES, but not MIP-1 alpha, induced a biphasic chemiluminescence response, however, of lower magnitude than C5a. RANTES and MIP-1 alpha both promoted identical transient changes in intracellular free calcium concentration ([Ca2+]i), with kinetics similar to those induced by chemotactic peptides known to interact with G protein-coupled receptors. No cross-desensitization towards other peptide agonists (e.g., C5a, IL-8, FMLP) was observed, suggesting the presence of specific receptors. Despite its weaker eosinophil-activating properties, MIP-1 alpha was at least 10 times more potent on a molar basis than RANTES at inducing [Ca2+]i changes. Interestingly, RANTES deactivated the MIP-1 alpha-induced [Ca2+]i changes, while the RANTES response was preserved after MIP-1 alpha stimulation. MCP-1, a potent monocyte chemoattractant and basophil agonist, as well as MIP-1 beta, a peptide with pronounced homology to MIP-1 alpha, did not activate the eosinophil functions tested. Our results indicate that RANTES and MIP-1 alpha are crucial mediators of inflammatory processes in which eosinophils predominate.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Citocinas/farmacologia , Eosinófilos/fisiologia , Linfocinas/farmacologia , Monocinas/farmacologia , Ribonucleases , Proteínas Sanguíneas/biossíntese , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5 , Complemento C5a/farmacologia , Proteínas Granulares de Eosinófilos , Eosinófilos/efeitos dos fármacos , Escherichia coli/genética , Humanos , Técnicas In Vitro , Cinética , Medições Luminescentes , Proteínas Inflamatórias de Macrófagos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Proteínas Recombinantes/farmacologia , SRS-A/biossíntese , SRS-A/sangue , Superóxidos/sangueRESUMO
Normodense human peripheral blood eosinophils were isolated under sterile conditions from the 22/23 and 23/24% interfaces and the cell pellet of metrizamide gradients. After culture for 7 d in RPMI media in the presence of 50 pM biosynthetic (recombinant) human granulocyte/macrophage colony-stimulating factor (rH GM-CSF), 43 +/- 7% (mean +/- SEM, n = 8) of the cells were viable; in the absence of rH GM-CSF, no eosinophils survived. The rH GM-CSF-mediated viability was concentration dependent; increased survival began at a concentration of 1 pM, a 50% maximal response was attained at approximately 3 pM, and a maximal effect was reached at concentrations of greater than or equal to 10 pM rH GM-CSF. In the presence of rH GM-CSF and mouse 3T3 fibroblasts, 67 +/- 6% (mean +/- SEM, n = 8) of the eosinophils survived for 7 d. In a comparative analysis, there was no difference in eosinophil viability after 7 and 14 d (n = 3) in the presence of 50 pM GM-CSF and fibroblasts. Culture with fibroblasts alone did not support eosinophil survival. The addition of fibroblast-conditioned media to rH GM-CSF did not further improve eosinophil viability, indicating a primary role for GM-CSF in supporting these eosinophil cell suspensions ex vivo and a supplementary role for 3T3 fibroblasts. Eosinophils cultured for 7 d localized on density gradient sedimentation at the medium/18, 18/20, and 20/21 interfaces of metrizamide gradients, indicating a change to the hypodense phenotype from their original normodense condition. In addition, the cultured eosinophils generated approximately 2.5-fold more LTC4 than freshly isolated cells when stimulated with the calcium ionophore A23187 and manifested sevenfold greater antibody-dependent killing of S. mansoni larvae than the freshly isolated, normodense cells from the same donor. Thus we demonstrate the rH GM-CSF dependent conversion in vitro of normodense human eosinophils to hypodense cells possessing the augmented biochemical and biological properties characteristic of the hypodense eosinophils associated with a variety of hypereosinophilic syndromes. In addition, these studies provide a culture model of at least 14 d suitable for the further characterization of hypodense eosinophils.
Assuntos
Eosinófilos/imunologia , Fibroblastos/fisiologia , Interleucina-3/fisiologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Calcimicina/farmacologia , Contagem de Células , Sobrevivência Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Humanos , Camundongos , Neutrófilos/fisiologia , Proteínas Recombinantes/fisiologia , SRS-A/biossíntese , Schistosoma mansoni/imunologia , Fatores de TempoRESUMO
Leukotriene C4 (LTC4) underwent rapid elimination from the circulating blood and was extensively converted to LTD4 within the vascular space of the guinea pig. To mimic the elimination and metabolism of endogenous LTC4 generated during anaphylaxis, 14,15-3H-labeled LTC4 was infused intravenously over a period of 15 min, leading to a recovery in bile of 85% of the infused LT radioactivity within 2 h. Corresponding to the tracer studies, LTD4 and, to a lesser extent, LTC4 were the predominant endogenous cysteinyl LTs in guinea pig bile. The biliary production rate of endogenous LTD4 increased from 0.3 +/- 0.1 to 6.2 +/- 1.8 pmol x min-1 x kg-1 (p less than 0.001) during anaphylactic shock induced by intravenous injection of OVA (0.2 mg/kg) into sensitized guinea pigs. A novel LT biosynthesis inhibitor (MK-886; 10 mg/kg, i.v., 15 min before antigen challenge) suppressed the antigen-induced cysteinyl LT production by greater than 92% (p less than 0.001). This inhibition of systemic LTC4 formation was associated with a complete protection against lethal anaphylactic shock in animals pretreated in addition with the H1 receptor antagonist pyrilamine. Pretreatment with either the inhibitor of LT synthesis or the histamine receptor antagonist reduced the lethality during anaphylactic shock from 100 to 60 and 78%, respectively. In artificially ventilated, pyrilamine-pretreated animals, the antigen-induced decrease in dynamic lung compliance and the rise in hematocrit were significantly reduced (p less than 0.05) by pretreatment with the inhibitor of LT synthesis. Dexamethasone at high doses (10 mg/kg, i.p., once daily for 7 d, or in a single dose of 10 mg/kg, i.v., 3.5 h before challenge) had no inhibitory effect on LT generation during anaphylaxis in vivo. However, in resident peritoneal macrophages, harvested from these dexamethasone-treated sensitized guinea pigs and stimulated with zymosan, both cysteinyl LT and 6-keto-PGF1 alpha formation were strongly suppressed. These studies indicate an important role of cysteinyl LTs in systemic anaphylaxis in vivo and demonstrate the blockade of anaphylactic LT generation by a novel inhibitor of LT biosynthesis (MK-886) but not by dexamethasone.
Assuntos
Anafilaxia/metabolismo , Dexametasona/farmacologia , Indóis/farmacologia , Antagonistas de Leucotrienos , SRS-A/biossíntese , Animais , Cobaias , Hemodinâmica/efeitos dos fármacos , Masculino , Ovalbumina/imunologia , Respiração/efeitos dos fármacosRESUMO
IgE-independent mediator release from basophils is considered an important event in inflammation, particularly in nonallergic immediate hypersensitivity and in allergic late-phase reactions. This study demonstrates that after exposure to IL-3, basophils release histamine and leukotrienes in response to the neutrophil-activating peptide NAF/NAP-1. Thus, the sequential action of two pure cytokines can promote basophils mediator release. In the presence of IL-3, NAF/NAP-1 functions like a "histamine-releasing factor" and may therefore not only induce cellular infiltration but also provoke symptoms of hypersensitivity reactions.