RESUMEN
Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.
Asunto(s)
Ácidos Araquidónicos/fisiología , Moduladores de Receptores de Cannabinoides/fisiología , Endocannabinoides , Glicéridos/fisiología , Leucotrieno B4/biosíntesis , Leucotrieno B4/fisiología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Antiinflamatorios no Esteroideos/sangre , Antiinflamatorios no Esteroideos/farmacología , Araquidonato 5-Lipooxigenasa/farmacología , Araquidonato 5-Lipooxigenasa/fisiología , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/sangre , Moduladores de Receptores de Cannabinoides/sangre , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Glicéridos/sangre , Humanos , Hidrólisis/efectos de los fármacos , Leucotrieno B4/sangre , Activación Neutrófila/efectos de los fármacos , Neutrófilos/metabolismoRESUMEN
Asthma is associated with an eosinophil infiltration into the bronchial mucosa. 5-Oxo-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemotactic factor, activates cell motility, adherence, and proteolysis, notably, by promoting CD11b expression, matrix metalloproteinase (MMP)-9 secretion, and plasmin generation. We investigated the intracellular signaling pathways implicated in these various steps by using different, selective inhibitors. Human eosinophil migration through a reconstituted basement membrane in response to 5-oxo-ETE was greatly inhibited (>or=72%) by the protein kinase C (PKC)-delta, PKC-zeta, ERK-1/2, and p38 inhibitors. Our findings indicate that PKC-delta mediates cell motility, CD11b expression, and MMP-9 granule release. PKC-zeta is also largely involved in eosinophil migration, although its specific targets remain undefined. ERK-1/2 and p38 modulate CD11b expression; ERK-1/2 is also involved in long-term MMP-9 secretion and p38 in the plasmin activation system. We demonstrated the crucial implication of PKC-delta, PKC-zeta, ERK-1/2, and p38 in human blood eosinophil migration through extracellular matrix components. Targeting specific pathways may have therapeutic potential for the treatment of allergic airway inflammation.
Asunto(s)
Asma/metabolismo , Asma/patología , Movimiento Celular , Eosinófilos/patología , Proteínas Serina-Treonina Quinasas/fisiología , Ácidos Araquidónicos/fisiología , Antígeno CD11b/biosíntesis , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteína Quinasa C , Proteína Quinasa C-delta , Transducción de Señal , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Nicotinic receptor agonists decreased the infiltration of eosinophils into the lung and airways in a mouse model of asthma. To better understand the mechanisms implicated in this anti-inflammatory phenomenon, the expression of nicotinic acetylcholine receptors (nAChRs) and the effect of dimethylphenylpiperazinium (DMPP), a nonselective nAChR agonist, on human blood eosinophils were studied. The expression of alpha-3, -4, and -7 nAChR subunits on human blood eosinophils was measured by cell ELISA and immunocytochemistry. mRNA expression for all three subunits was evaluated by quantitative RT-PCR. The effect of DMPP on leukotriene C4 (LTC4) and matrix metalloproteinase-9 (MMP-9) production, eosinophil migration, and intracellular calcium mobilization was measured. The results show that the alpha-3, -4, and -7 nAChR subunits and mRNAs are expressed by blood eosinophils. In vitro treatment of these cells with various concentrations of DMPP reduced platelet-activating factor (PAF)-induced LTC4 production significantly. DMPP (160 microM) decreased eotaxin, and 5-oxo-6,8,11,14-eicosatetranoic acid induced eosinophil migration through Matrigel by 40.9% and 55.5%, respectively. This effect was reversed by the nAChR antagonist mecamylamine. In addition, DMPP reduced MMP-9 release and the inositol 1,4,5-triphosphate-dependent intracellular calcium increase provoked by PAF. Taken together, these results indicate that functional nAChRs are expressed on eosinophils and that nAChR agonists down-regulate eosinophil function in vitro. These anti-inflammatory effects could be of interest in the treatment of allergic asthma.
Asunto(s)
Yoduro de Dimetilfenilpiperazina/farmacología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , Ácidos Araquidónicos/antagonistas & inhibidores , Ácidos Araquidónicos/farmacología , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Quimiocina CCL11 , Quimiocinas CC/antagonistas & inhibidores , Quimiocinas CC/farmacología , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Humanos , Técnicas In Vitro , Leucotrieno C4/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Factor de Activación Plaquetaria/antagonistas & inhibidores , Factor de Activación Plaquetaria/inmunología , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Receptores Nicotínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Relación Estructura-ActividadRESUMEN
BACKGROUND: Migration of eosinophils into bronchial mucosa requires proteolysis. Montelukast, a cysteinyl leukotriene (CysLT) 1 receptor antagonist used in asthma treatment, decreases eosinophil infiltration into the asthmatic airways, suggesting that CysLTs modulate eosinophil protease activity. OBJECTIVE: We sought to determine whether CysLTs and montelukast regulate eosinophil protease activity. METHODS: Purified blood eosinophils were treated with or without montelukast; MK-0591, a 5-lipoxygenase-activating protein inhibitor; or leukotriene (LT) D(4). Migration assays through Matrigel were performed in the presence of 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemotactic factor, or LTD(4). Expression of molecules implicated in plasmin generation and matrix metalloproteinase (MMP) 9 release were also evaluated. RESULTS: Montelukast and MK-0591 decreased eosinophil migration promoted by 5-oxo-ETE, whereas LTD(4) failed to induce eosinophil migration. However, LTD(4) significantly boosted the migration rate obtained with a suboptimal concentration of 5-oxo-ETE and partially reversed the inhibition obtained with MK-0591. Montelukast significantly reduced the maximal rate of activation of plasminogen into plasmin by eosinophils obtained with 5-oxo-ETE. 5-Oxo-ETE increased the number of eosinophils expressing urokinase plasminogen activator receptor and stimulated secretion of MMP-9. Montelukast, but neither MK-0591 nor LTD(4), reduced the expression of urokinase plasminogen activator receptor and the secretion of MMP-9 and increased total cellular activity of urokinase plasminogen activator and the expression of plasminogen activator inhibitor 2 mRNA. CONCLUSION: Montelukast inhibits eosinophil protease activity in vitro through a mechanism that might be independent of its antagonist effect on CysLT 1 receptor. CLINICAL IMPLICATIONS: This could partially explain montelukast's anti-inflammatory effect in asthma and eventually amplify to improve its therapeutic efficacy.
Asunto(s)
Acetatos/farmacología , Cisteína/fisiología , Eosinófilos/efectos de los fármacos , Antagonistas de Leucotrieno/farmacología , Leucotrienos/fisiología , Quinolinas/farmacología , Adulto , Ácidos Araquidónicos/farmacología , Movimiento Celular/efectos de los fármacos , Ciclopropanos , Activación Enzimática , Eosinófilos/enzimología , Eosinófilos/fisiología , Femenino , Humanos , Indoles/farmacología , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Receptores de Superficie Celular/análisis , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Sulfuros , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
BACKGROUND: Animal and human studies demonstrated that interleukin (IL)-12, a Th1 cytokine, reduces blood and bronchial eosinophilia, and airway hyperreactivity. According to current concepts, these effects are mediated through the release of cytokines promoting eosinophil recruitment and activation. However, the presence of IL-12 receptors on eosinophils suggests that IL-12 also acts directly on eosinophils. We postulated that IL-12 directly modulates eosinophil functions and has the capacity to regulate eosinophil degranulation, migration and survival, in vitro. METHOD: Effects of IL- 12 on purified human blood eosinophils were evaluated for peroxidase (EPO) release, eotaxin-induced migration through a model of basement membrane (Matrigel), and survival. RESULTS: IL-12 inhibited 50% of PAF and secretory IgA-induced EPO release (n = 8, p < 0.001). IL-12 also reduced eotaxin-induced migration through Matrigel by 54 +/-6% (n = 6, p < 0.01). These effects were not explained by an IL-12-induced impaired viability or apoptosis. CONCLUSION: Our results demonstrate that IL-12 directly modulates eosinophil functions without promoting apoptosis and explain, at least in part, the effects of IL-12 on eosinophils observed in in vivo studies.
Asunto(s)
Apoptosis/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Interleucina-12/farmacología , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Peroxidasa del Eosinófilo/metabolismo , Eosinófilos/metabolismo , Eosinófilos/fisiología , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina A Secretora/farmacología , Interleucina-5/farmacología , Leucotrieno C4/metabolismo , Factor de Activación Plaquetaria/farmacología , Receptores de Interleucina/análisis , Receptores de Interleucina/genética , Receptores de Interleucina-12 , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Increased eosinophil counts are a major feature of asthmatic airways. Eosinophil recruitment requires migration through epithelium and tissue extracellular matrix by activation of proteases. We assessed the capacity of IL-16, a CD4(+) cell chemotactic factor, to induce migration of eosinophils through a reconstituted basement membrane and evaluated the proteases, mediators, and receptors involved in this migration. IL-16 added to lower chambers of Invasion Chambers elicited eosinophil migration through Matrigel. This effect was decreased by inhibition of the plasminogen-plasmin system (Abs against urokinase plasminogen activator receptor or plasminogen depletion), but not by anti-matrix metalloproteinase-9 Abs. Abs against CD4 also inhibited IL-16-induced eosinophil migration. At the baseline level, few eosinophils (4.6% positive cells with a mean fluorescence of 0.9) expressed surface membrane CD4, while most permeabilized eosinophils (68% positive cells with a mean fluorescence of 18) express the CD4 Ag. TNF-pretreatment increased surface membrane CD4(+) expression by 6-fold as previously described, and increased IL-16-induced cell migration by 2.2-fold. Incubation of eosinophils with IL-16 also increased surface membrane CD4 expression by 5.4-fold, supporting the role of CD4 as receptor for IL-16. Abs against CCR3, eotaxin, or RANTES blocked IL-16-induced migration. In conclusion, IL-16 promotes eosinophil migration in vitro, by activating the plasminogen-plasmin system and increasing the membrane expression of its receptor. This effect is initiated via CD4 and mediated via the release of CCR3 ligand chemokines. Interestingly, most eosinophils express intracellular CD4. Hence, IL-16 may play an important role in the recruitment of blood eosinophils to the bronchial mucosa of asthmatics.
Asunto(s)
Antígenos CD4/biosíntesis , Factores Quimiotácticos Eosinófilos/fisiología , Quimiotaxis de Leucocito/inmunología , Eosinófilos/citología , Eosinófilos/inmunología , Interleucina-16/fisiología , Activadores Plasminogénicos/fisiología , Receptores de Quimiocina/fisiología , Adulto , Antígenos CD4/fisiología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Membrana Celular/fisiología , Quimiocina CCL11 , Quimiocina CCL5/metabolismo , Quimiocinas CC/metabolismo , Colágeno/inmunología , Colágeno/metabolismo , Colágeno/fisiología , Combinación de Medicamentos , Eosinófilos/enzimología , Eosinófilos/metabolismo , Matriz Extracelular/enzimología , Matriz Extracelular/inmunología , Femenino , Fibrinolisina/metabolismo , Fibrinolisina/fisiología , Humanos , Laminina/inmunología , Laminina/metabolismo , Laminina/fisiología , Masculino , Plasminógeno/metabolismo , Plasminógeno/fisiología , Proteoglicanos/inmunología , Proteoglicanos/metabolismo , Proteoglicanos/fisiología , Receptores CCR3 , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
BACKGROUND: Blood eosinophils express CD16 on their surface when stimulated in vitro with platelet-activating factor or IFNgamma. Transient expression of CD16 is also observed in vivo following aeroallergen challenge of asthmatic subjects. The present work is aimed at evaluating the possible mechanisms modulating eosinophil expression of CD16 and the biological functions of this receptor. METHODS: First, purified blood eosinophils were incubated with IL-1beta, IL-2, IL-4, IL-5, IL-9 or IL-16, GM-CSF, IFNgamma, eotaxin or 5-oxo-ETE and CD16 expression was measured. Second, the capacity of CD16 to mediate degranulation induced by IgG immune complexes (IC) was evaluated in eosinophils with low and high CD16 expression. Finally, serum allergen-specific IgE and IgG, and total IgE levels were measured at baseline in allergic asthmatics and correlated with changes observed in blood eosinophil CD16 expression (DeltaCD16) following allergen challenge. RESULTS: Only IFNgamma and IL-2 significantly increased the number of CD16+ eosinophils, respectively, 37 +/- 10% (p = 0.0038) and 38 +/- 8% (p = 0.0006), compared to control, 7 +/- 2%. IgG IC induced degranulation in eosinophils with low and high CD16 expression and monoclonal anti-CD16 and anti-CD32 antibodies inhibited this. IgG IC increased eosinophil CD16 expression (14 +/- 6%, p = 0.0008) and this effect was blocked by pretreatment with anti-CD32 antibodies. DeltaCD16 following allergen challenge correlated with the specific IgG/total IgE ratio (r(2) = 0.41, p = 0.036). CONCLUSION: These data suggest that formation of IgG IC is associated with surface eosinophil CD16 expression in asthma and that CD16 in cooperation with CD32 mediates IC-induced degranulation.
Asunto(s)
Complejo Antígeno-Anticuerpo/farmacología , Modulación Antigénica/fisiología , Citocinas/farmacología , Eosinófilos/metabolismo , Receptores de IgG/biosíntesis , Especificidad de Anticuerpos/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Antígenos Dermatofagoides/efectos adversos , Antígenos Dermatofagoides/farmacología , Asma/inmunología , Asma/metabolismo , Biomarcadores/sangre , Pruebas de Provocación Bronquial , Degranulación de la Célula/efectos de los fármacos , Citocinas/metabolismo , Eosinófilos/citología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Interferón gamma/metabolismo , Interferón gamma/farmacología , Interleucinas/metabolismo , Interleucinas/farmacología , Poaceae/efectos adversos , Estadística como AsuntoRESUMEN
Circulating eosinophils need proteinases to mediate a spatially limited and orientated digestion of the extracellular matrix and to migrate into tissue. Moreover, proteinases are likely involved in tissue remodeling, a crucial feature of chronic diseases including asthma. Eosinophils express matrix metalloproteinase (MMP)-9, which is increased upon stimulation with TNF-alpha. Other MMPs, the membrane type (MT)-MMPs, likely play a major role in cell invasion and tissue remodeling. MT4-MMP was identified in peripheral blood leukocyte preparations, but it is not known whether eosinophils express MT4-MMP. We investigated the expression of MT4-MMP and its modulation by TNF-alpha in purified human blood eosinophils. The constitutive expression of MT4-MMP mRNA was detected by RT-PCR in unstimulated eosinophils, lymphocytes, and monocytes, but not neutrophils. Stimulation of eosinophils with TNF-alpha increased MT4-MMP mRNA expression. This effect appeared at 4h and reached a maximum at 8h of incubation. MT4-MMP protein was detected in freshly isolated blood eosinophils by Western blotting and immunocytochemistry. TNF-alpha increased expression of the MT4-MMP protein. MT4-MMP protein was also detected in nasal polyp eosinophils by immunohistochemistry. In conclusion, eosinophils constitutively express MT4-MMP, which is increased upon stimulation with TNF-alpha. Consequently, MT4-MMP may be directly involved in the degradation of extracellular matrix components and/or modulate the activity of other proteins implicated in eosinophil migration and tissue remodeling.
Asunto(s)
Eosinófilos/enzimología , Metaloproteinasas de la Matriz , Metaloendopeptidasas/metabolismo , Células Cultivadas , Eosinófilos/citología , Eosinófilos/efectos de los fármacos , Humanos , Inmunohistoquímica , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Pólipos Nasales/metabolismo , Pólipos Nasales/patología , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Blood eosinophils have mRNA for FcgammaRIIIB (CD16) but no or minimal spontaneous CD16 expression. Because IFN-gamma and chemotactic factors induce eosinophil CD16 expression in vitro, we postulated that blood eosinophils could express CD16. OBJECTIVE: Blood of nonallergic controls and subjects with allergic rhinitis, allergic and nonallergic asthma, or hypereosinophilia of various etiologies were analyzed for leukocyte CD16 surface expression. METHODS: CD16(+) eosinophils were identified on the basis of physico-optic characteristics, major basic protein, CD49b expression, and sorting by flow cytometry and microscope examination. RESULTS: Subjects with allergic rhinitis and subjects with asthma had higher median percentages of CD16(+) eosinophils (8.1% [1% to 48.6%] and 7.3% [1.4% to 31.1%], respectively) than nonallergic controls and nonallergic asthmatics (3% [0% to 11%] and 4.6% [2.9% to 5.1%], respectively). In subjects with hypereosinophilia, CD16(+) eosinophils were increased only in a case of drug allergy. When subjects with mild allergic asthma were challenged with a relevant aeroallergen, blood CD16(+) eosinophils further increased during or after the late-phase response (6 to 48 hours after challenge; mean +/- SEM, 9.4% +/- 2.5% to 20.0% +/- 3.0%). CD16(+) eosinophils expressed more IL-5 receptor but less CD11b and IL-12p35 than did CD16(-) eosinophils. CONCLUSION: Upregulation of blood CD16(+) eosinophils in allergic conditions and its association with a modified phenotype suggest that CD16 receptor could play a role in eosinophil activation in allergy.