RESUMO
BACKGROUND: We previously demonstrated in mice that airway eosinophils traffic from the airway lumen into lung-draining paratracheal lymph nodes. However, mechanisms whereby eosinophils traverse from the lungs and home to paratracheal lymph nodes remain unclear. We investigated roles of cysteinyl leukotrienes in mediating eosinophil trafficking from lungs to paratracheal lymph nodes. METHODS: The expression of CCR7 was determined by flow cytometry. Transwell assays were used to test chemotactic responses of leukotriene C4 synthase-deficient and control airway eosinophils to the chemokine CCL19 ex vivo. Eosinophils from the spleens of IL-5 transgenic mice, fluorescently labeled ex vivo, were intratracheally injected into ovalbumin-sensitized and ovalbumin aerosol-challenged leukotriene C4 synthase-deficient and control mice. Eosinophils were identified by microscopy and flow cytometry in the lungs and paratracheal lymph nodes. RESULTS: Mouse eosinophils expressed CCR7, the receptor for CCL19, and responded chemotactically to CCL19. Leukotriene C4 synthase-deficient eosinophils exhibited impaired chemotaxis to CCL19 that was restored by exogenous leukotriene C4 . The migration of intratracheally injected eosinophils into paratracheal lymph nodes from distal alveolar lung was diminished in leukotriene C4 synthase-deficient mice compared with wild-type mice, with increased retention of eosinophils in the lungs of leukotriene C4 synthase-deficient mice. Exogenous administration of leukotriene C4 restored trafficking of eosinophils to paratracheal lymph nodes in leukotriene C4 synthase-deficient mice. CONCLUSIONS: Our findings that cysteinyl leukotrienes are involved in regulating airway and lung eosinophil migration into paratracheal lymph nodes identify previously unrecognized roles for the cysteinyl leukotrienes in regulating the pulmonary trafficking of eosinophils in experimental allergic asthma.
Assuntos
Quimiotaxia , Eosinófilos/citologia , Leucotrieno C4/imunologia , Linfonodos/citologia , Animais , Asma/patologia , Quimiocina CCL19/fisiologia , Eosinófilos/metabolismo , Leucotrieno C4/administração & dosagem , Leucotrieno C4/deficiência , Pulmão/citologia , Linfonodos/metabolismo , Camundongos , Receptores CCR7/fisiologiaRESUMO
BACKGROUND: Eosinophils have the capacity to secrete varied cytotoxic proteins. Among the proteins are the eosinophil-associated RNases (EARs): the human eosinophil-derived neurotoxin and eosinophilic cationic protein, and their murine ortholog EARs, which have been shown to be involved in host defense, tissue remodeling, and immunity regulation. However, the signal transduction that regulates EARs secretion in response to physiological stimuli, such as chemokines, has been little studied in human and scarcely in mouse eosinophils, the foremost animal model for eosinophil-associated human diseases. OBJECTIVE: In this study, we aimed to understand the signal transduction involved in the secretion of enzymatically active EARs following chemokine stimulation. METHODS: Fresh mouse and human eosinophils were stimulated with CCL11 and CCL24, and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of signaling factors or integrins was probed using specific inhibitors and blocking antibodies. Adhesion was evaluated by microscopy. RESULTS: We found that secretion of mouse EARs in response to CCL11 and CCL24 was Gαi -dependent. Both mouse and human eosinophils required the activation of PI3K, ERK, and p38 MAPK. In addition, the adhesion molecules ß1 and ß2 integrins were found to be crucial for EAR secretion, and we suggest a mechanism in which spreading is obligatory for EAR secretion. CONCLUSIONS: Collectively, these data suggest a common CCR3-mediated signaling pathway that leads to EAR secretion in both mouse and human eosinophils. These findings are applicable for eosinophil-mediated host defense and eosinophil-associated diseases.
Assuntos
Eosinófilos/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Integrinas/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Ribonucleases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Western Blotting , Células Cultivadas , Quimiocina CCL11/imunologia , Quimiocina CCL11/metabolismo , Eosinófilos/citologia , Eosinófilos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Humanos , Integrinas/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores CCR3/imunologia , Receptores CCR3/metabolismo , Ribonucleases/imunologia , Sensibilidade e Especificidade , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Several lines of evidence suggest that deficiency of eosinophils is not associated with any characteristic abnormality. Patients lacking eosinophils, in the setting of immunodeficiency or as a consequence of IgG-mediated eosinophil precursor destruction, do not display any distinguishing abnormalities related to eosinophil reduction. The observation that eosinophil-deficient mice do not display any distinctive syndrome or failure of their health is evidence that, under ordinary laboratory conditions, the eosinophil does not play a critical role in the well-being of mammals. Observations that monoclonal antibodies to interleukin-5 (IL-5) are well tolerated appear unsurprising in light of these findings. For example, patients with the hypereosinophilic syndrome have received mepolizumab, an anti-IL-5 monoclonal antibody, for as long as 6 years and have not developed any characteristic set of adverse events. Safety data for reslizumab, another anti-IL-5 monoclonal antibody, and benralizumab, a monoclonal antibody to the IL-5 receptor α-chain, are comparatively limited, especially for benralizumab, although reports of administration of these antibodies to humans suggest that they are well tolerated. Thus, data to the present suggest that reduction of eosinophils appears to have no characteristic ill effects on normal health, and monoclonal antibodies that deplete eosinophils have the potential to be widely employed in the treatment of eosinophil-associated diseases.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Eosinófilos/imunologia , Síndromes de Imunodeficiência/sangue , Síndromes de Imunodeficiência/imunologia , Timoma/imunologia , Neoplasias do Timo/imunologia , Animais , Anticorpos Monoclonais Humanizados/efeitos adversos , Estudos de Coortes , Modelos Animais de Doenças , Eosinófilos/metabolismo , Feminino , Humanos , Síndromes de Imunodeficiência/tratamento farmacológico , Incidência , Interleucina-5/efeitos adversos , Interleucina-5/imunologia , Interleucina-5/uso terapêutico , Contagem de Leucócitos , Masculino , Camundongos , Prognóstico , Medição de Risco , Timoma/sangue , Timoma/epidemiologia , Neoplasias do Timo/sangue , Neoplasias do Timo/epidemiologiaRESUMO
The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Since prostanoids formed from arachidonic acid can modulate interactions among platelets, leukocytes, and endothelial cells, we examined whether intravascular nematode parasites can elaborate prostanoids. Microfilariae of Brugia malayi utilize exogenous and endogenous arachidonic acid to generate and release two predominant prostanoids, prostacyclin and prostaglandin E2. Filarial metabolism of host fatty acids to form these vasodilatory, antiaggregatory, and immunomodulatory eicosanoids provides a means by which these helminthic parasites may influence host immune and other cellular responses.
Assuntos
Brugia/metabolismo , Eicosanoides/biossíntese , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Brugia/isolamento & purificação , Cromatografia em Camada Fina , Prostaglandinas/biossíntese , Prostaglandinas/isolamento & purificação , Tromboxano B2/biossíntese , Tromboxano B2/isolamento & purificaçãoRESUMO
Lymphocyte chemoattractant factor (LCF) is a tetrameric glycoprotein of 56,000 relative molecular mass produced by activated T lymphocytes. LCF binds to CD4 and has previously been found to stimulate migration of CD4+ lymphocytes and monocytes. Because human eosinophils, like T cells and monocytes, express CD4, we examined functional responses of eosinophils to LCF. Recombinant LCF (rLCF) expressed in COS cells was purified on a CD4 affinity column. Migration of eosinophils was elicited by rLCF at low concentrations: the 50% effective dose (ED50) was 10(-12) to 10(-11) M, concentrations 100- to 1,000-fold lower than the ED50s for the recognized eosinophil chemoattractants C5a and platelet-activating factor. Two other ligands which bound to CD4, human immunodeficiency virus-1 envelope glycoprotein gp120 and monoclonal antibody OKT4, also stimulated eosinophil migration. Monovalent OKT4 Fab competitively inhibited eosinophil responses to rLCF. rLCF did not influence other functional responses of eosinophils tested, including degranulation, superoxide generation, leukotriene C4 production, in vitro survival, or surface expression of the adherence receptor CR3 (CD11b), human histocompatibility leukocyte antigen DR, or interleukin 2 receptor p55 (CD25). We conclude that CD4 on eosinophils is capable of transducing a migratory stimulus and serves as a receptor for a chemoattractant lymphokine LCF. T cell-derived LCF may contribute to recruitment of eosinophils and CD4+ mononuclear cells concomitantly at inflammatory reactions.
Assuntos
Antígenos CD4/fisiologia , Fatores Quimiotáticos/fisiologia , Quimiotaxia de Leucócito , Eosinófilos/fisiologia , Linfocinas/fisiologia , Eosinófilos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Antígenos HLA-DR/análise , Humanos , Técnicas In Vitro , Interleucina-16 , Neutrófilos/fisiologia , Receptores de Interleucina-2/análise , Superóxidos/metabolismoRESUMO
The CD4 glycoprotein, expressed on leukocytes belonging to subsets of T lymphocytes and to cells of monocyte/macrophage lineage, participates in the functioning of T cells and serves as a receptor for HIV-1 and HIV-2. Human eosinophils, a class of granulocytic leukocytes, have been found to express CD4. With anti-CD4 mAbs CD4 was demonstrable on eosinophils from both normal and eosinophilic donors. Eosinophils synthesized a 55-kD CD4 polypeptide immunoprecipitable with two anti-CD4 mAbs. Eosinophil CD4 bound HIV-1 gp120 as assessed by competition for anti-OKT4A, but not anti-OKT4, mAb binding. Eosinophils, normally rich in gastrointestinal and genitourinary tract tissues, increase in numbers in patients with metazoan parasitic infections. In these sites and diseases, CD4 expression by eosinophils may be pertinent to their immunologic functions and could make these cells susceptible to HIV infection.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Eosinófilos/metabolismo , HIV/metabolismo , Proteínas dos Retroviridae/metabolismo , Proteínas do Envelope Viral/metabolismo , Citometria de Fluxo , HumanosRESUMO
Lipid bodies, lipid rich cytoplasmic inclusions, are characteristically abundant in vivo in leukocytes associated with inflammation. Because lipid bodies are potential reservoirs of esterified arachidonate and sites at which eicosanoid-forming enzymes may localize, we evaluated mechanisms of lipid body formation in neutrophils (PMN). Among receptor-mediated agonists, platelet activating factor (PAF), but not C5a, formyl-methyl-phenylalanine, interleukin 8, or leukotriene (LT) B4, induced the rapid formation of lipid bodies in PMN. This action of PAF was receptor mediated, as it was dose dependently inhibited by the PAF receptor antagonist WEB 2086 and blocked by pertussis toxin. Lipid body induction by PAF required 5-lipoxygenase (LO) activity and was inhibited by the 5-lipoxygenase-activating protein antagonist MK 886 and the 5-LO inhibitor zileuton, but not by cyclooxygenase inhibitors. Corroborating the dependency of PAF-induced lipid body formation on 5-LO, PMN and macrophages from wild-type mice, but not from 5-LO genetically deficient mice, formed lipid bodies on exposure to PAF both in vitro and in vivo within the pleural cavity. The 5-LO product inducing lipid body formation was not LTB4 but was 5(S)-hydroxyeicosatetraenoic acid [5(S)-HETE], which was active at 10-fold lower concentrations than PAF and was also inhibited by pertussis toxin but not by zileuton or WEB 2086. Furthermore, 5-HETE was equally effective in inducing lipid body formation in both wild-type and 5-LO genetically deficient mice. Both PAF- and 5(S)-HETE-induced lipid body formation were inhibited by protein kinase C (PKC) inhibitors staurosporine and chelerythrine, the phospholipase C (PLC) inhibitors D609 and U-73122, and by actinomycin D and cycloheximide. Prior stimulation of human PMN with PAF to form lipid bodies enhanced eicosanoid production in response to submaximal stimulation with the calcium ionophore A23187; and the levels of both prostaglandin (PG) E2 and LTB4 correlated with the number of lipid bodies. Furthermore, pretreatment of cells with actinomycin D or cycloheximide inhibited not only the induction of lipid body formation by PAF, but also the PAF-induced "priming" for enhanced PGE2 and LTB4 in PMN. Thus, the compartmentalization of lipids to form lipid bodies in PMN is dependent on specific cellular responses that can be PAF receptor mediated, involves signaling through 5-LO to form 5-HETE and then through PKC and PLC, and requires new protein synthesis. Since increases in lipid body numbers correlated with priming for enhanced PGE2 and LTB4 production in PMN, the induction of lipid bodies may have a role in the formation of eicosanoid mediators by leukocytes involved in inflammation.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Compartimento Celular , Corpos de Inclusão/efeitos dos fármacos , Metabolismo dos Lipídeos , Neutrófilos/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Animais , Araquidonato 5-Lipoxigenase/genética , Relação Dose-Resposta a Droga , Proteínas de Ligação ao GTP/metabolismo , Humanos , Inibidores de Lipoxigenase/farmacologia , Camundongos , Camundongos Knockout , Neutrófilos/enzimologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteína Quinase C/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transdução de Sinais , Fosfolipases Tipo C/metabolismoRESUMO
The specific intracellular sites at which enzymes act to generate arachidonate-derived eicosanoid mediators of inflammation are uncertain. We evaluated the formation and function of cytoplasmic lipid bodies. Lipid body formation in eosinophils was a rapidly (<1 h) inducible response which was platelet-activating factor (PAF) receptor-mediated, involved signaling through protein kinase C, and required new protein synthesis. In intact and enucleated eosinophils, the PAF-induced increases in lipid body numbers correlated with enhanced production of both lipoxygenase- and cyclooxygenase-derived eicosanoids. All principal eosinophil eicosanoid-forming enzymes, 5-lipoxygenase, leukotriene C4 synthase, and cyclooxygenase, were immunolocalized to native as well as newly induced lipid bodies in intact and enucleated eosinophils. Thus, lipid bodies are structurally distinct, inducible, nonnuclear sites for enhanced synthesis of paracrine eicosanoid mediators of inflammation.
Assuntos
Eicosanoides/biossíntese , Eosinófilos/metabolismo , Eosinófilos/ultraestrutura , Corpos de Inclusão/metabolismo , Corpos de Inclusão/ultraestrutura , Metabolismo dos Lipídeos , Adulto , Araquidonato 5-Lipoxigenase/metabolismo , Sítios de Ligação , Núcleo Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Eosinófilos/efeitos dos fármacos , Glutationa Transferase/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Corpos de Inclusão/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transdução de SinaisRESUMO
We report that the hypodense eosinophil population in three patients with corticosteroid-unresponsive IHES was uniquely long lived ex vivo in the absence of exogenous cytokines. Serum or plasma from these patients conferred prolonged viability ex vivo to normodense eosinophils from reference donors and converted them to a functionally activated hypodense phenotype. In that antibody against IL-5 neutralized this activity in IHES serum, excessive quantities of this cytokine may account for the characteristic eosinophilia and long-lived, functionally augmented eosinophil phenotype in this disorder.
Assuntos
Eosinofilia/sangue , Eosinófilos/patologia , Interleucinas/sangue , Adulto , Sobrevivência Celular , Eosinofilia/imunologia , Eosinófilos/citologia , Humanos , Técnicas In Vitro , Interleucina-5 , Masculino , Pessoa de Meia-Idade , Fenótipo , Valores de Referência , SíndromeRESUMO
Transforming growth factor alpha (TGF-alpha) is a pleuripotential cytokine with diverse biological effects, including the ability to influence the proliferation of normal cells or neoplastic epithelial cells. Eosinophils are a subset of granulocytes that normally enter the peripheral tissues, particularly those beneath gastrointestinal, respiratory, and urogenital epithelium, where they reside in close proximity to the epithelial elements. In this study, we demonstrate that the great majority of eosinophils infiltrating the interstitial tissues adjacent to two colonic adenocarcinomas and two oral squamous cell carcinomas labeled specifically by in situ hybridization with a 35S-riboprobe for human TGF-alpha (hTGF-alpha). No other identifiable leukocytes in these lesions contained detectable hTGF-alpha mRNA. We also examined leukocytes purified from a patient with the idiopathic hypereosinophilic syndrome. 80% of these eosinophils, but none of the patient's neutrophils or mononuclear cells, were positive for hTGF-alpha mRNA by in situ hybridization, and 55% of these eosinophils were positive by immunohistochemistry with a monoclonal antibody directed against the COOH terminus of the mature hTGF-alpha peptide. Finally, the identification of the purified eosinophil-associated transcript as hTGF-alpha was confirmed by polymerase chain reaction product restriction enzyme analysis followed by Southern blot hybridization. In contrast to eosinophils from the patient with hypereosinophilic syndrome, the peripheral blood eosinophils from only two of seven normal donors had detectable TGF-alpha mRNA and none of these eosinophils contained immunohistochemically detectable TGF-alpha product. Taken together, these findings establish that human eosinophils can express TGF-alpha, but suggest that the expression of TGF-alpha by eosinophils may be under microenvironmental regulation. Demonstration of TGF-alpha production by tissue-infiltrating eosinophils and the eosinophils in the hypereosinophilic syndrome identifies a novel mechanism by which eosinophils might contribute to physiological, immunological, and pathological responses.
Assuntos
Adenocarcinoma/genética , Neoplasias do Colo/genética , Eosinófilos/metabolismo , Neoplasias Bucais/genética , Fatores de Crescimento Transformadores/genética , Sequência de Bases , Northern Blotting , Linhagem Celular , Eosinofilia/sangue , Eosinofilia/genética , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Síndrome , Fatores de Crescimento Transformadores/sangueRESUMO
Lipid bodies, nonmembrane-bound cytoplasmic inclusions, serve as repositories of esterified arachidonate and are increased in cells associated with inflammatory reactions. We have evaluated stimuli and mechanisms responsible for lipid body formation within human polymorphonuclear leukocytes (PMNs). Arachidonic acid and oleic acid stimulated dose-dependent formation of lipid bodies over 0.5-1 h. Other C20 and C18 fatty acids were less active and demonstrated rank orders as follows: cis-unsaturated fatty acids were much more active than trans-fatty acids, and activity diminished with decreasing numbers of double bonds. Lipid bodies elicited in vitro with cis-fatty acids were ultrastructurally identical to lipid bodies present in PMNs in vivo. Lipid body induction was not because of fatty acid-elicited oxidants or fatty acid-induced ATP depletion. Cis-fatty acid-induced activation of protein kinase C (PKC) was involved in lipid body formation as evidenced by the capacity of other PKC activators, 1-oleoyl-2-acetyl-glycerol and two active phorbol esters, phorbol myristate acetate, and phorbol 12,13 dibutyrate, but not an inactive phorbol, to induce lipid body formation. The PKC inhibitor, 1-O-hexadecyl-2-O-methyl-glycerol, inhibited PMN lipid body formation induced by oleic and arachidonic acids and by 1-oleoyl-2-acetyl-glycerol and phorbol myristate acetate. Other PKC inhibitors (staurosporine, H-7) also inhibited lipid body formation. Formation of lipid bodies in PMNs is a specific cellular response, stimulated by cis-fatty acids and diglycerides and apparently mediated by PKC, which results in the mobilization and deposition of lipids within discrete, ultrastructurally defined cytoplasmic domains.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Metabolismo dos Lipídeos , Neutrófilos/metabolismo , Trifosfato de Adenosina/fisiologia , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Citoplasma/metabolismo , Diglicerídeos/farmacologia , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Neutrófilos/ultraestrutura , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Maximum expiratory flow rate at 30 percent of vital capacity above residual volume served as an index of airway obstruction in comparing the effects of leukotriene C and histamine administered by aerosol to five normal persons. Leukotriene C was 600 to 9500 times more potent than histamine on a molar basis in producing an equivalent decrement in the residual volume. The leukotriene C response was slow in onset and prolonged, reminiscent of the effects of aerosol allergen challenge in asthmatic allergic subjects.
Assuntos
Resistência das Vias Respiratórias/efeitos dos fármacos , Brônquios/efeitos dos fármacos , SRS-A/farmacologia , Adulto , Relação Dose-Resposta a Droga , Feminino , Histamina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Prostaglandinas F/farmacologia , Fatores de TempoRESUMO
Eosinophils are usually considered as end-stage degranulating effector cells of innate immunity. However, accumulating evidence has revealed additional roles for eosinophils that are immunoregulatory in nature in both the adaptive and innate arms of immunity. Specifically, eosinophils have key immunoregulatory roles as professional antigen-presenting cells and as modulators of CD4(+) T cell, dendritic cell, B cell, mast cell, neutrophil, and basophil functions. This review addresses the emerging immunoregulatory roles of eosinophils with a focus on recent data that support this new paradigm. Recognizing both the effector and immunoregulatory functions of eosinophils will enable a fuller understanding of the roles of eosinophils in allergic airways inflammation and may be pertinent to therapies that target eosinophils both for their acute and ongoing immunomodulatory functions.
Assuntos
Eosinófilos/imunologia , Imunidade Inata/imunologia , Animais , HumanosRESUMO
Arylsulfatase B from human eosinophils was purified free of contaminating proteins by gel filtration and sequential affinity chromatography on Affi-Gel Blue and zinc chelate Sepharose. 50 micrograms of the purified enzyme presented as a single stained band on alkaline disc gel electrophoresis. In both goats and rabbits, the purified enzyme elicited monospecific antisera that yielded single precipitation arcs on Ouchterlony analysis with a human eosinophil extract and the purified enzyme; the immunoprecipitation lines fused in a pattern of identity, providing immunochemical evidence for the homogeneity of the purified enzyme. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a dominant lower molecular weight protein and three other bands with molecular weights approximately two, three, and four times that of the major protein band were resolved. The prominence of the less rapidly migrating protein bands increased relative to the major band if the enzyme was maintained under acidic conditions or was reacted with the cross-linking agent dimethyl suberimidate under alkaline conditions before SDS-polyacrylamide gel electrophoresis, supporting the conclusion that the enzyme consists of four subunits. Two stained bands were present on acid disc gel electrophoresis; they were composed of oligomeric forms of enzyme on analysis by SDS-polyacrylamide gel electrophoresis in a second dimension. A minimum molecular weight of 70,190 was determined from amino acid composition analysis for the tetrameric form of the enzyme. The specific functional activity of the purified arylsulfatase B was concentration and time dependent, compatible with its association or dissociation into subunit forms with differing specific activities. Factors that govern subunit interactions of arylsulfatase B, including local enzyme concentration and pH, provide mechanisms for regulating the enzymatic activity of this lysosomal hydrolase.
Assuntos
Condro-4-Sulfatase/sangue , Eosinófilos/enzimologia , Sulfatases/sangue , Condro-4-Sulfatase/isolamento & purificação , Humanos , Lisossomos/enzimologia , Substâncias Macromoleculares , Ligação Proteica , Conformação ProteicaRESUMO
The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Platelets do not adhere to blood-borne microfilariae, and thrombo-occlusive phenomena are not observed in patients with circulating microfilariae. We studied the ability of microfilariae to inhibit human platelet aggregation in vitro. Brugia malayi microfilariae incubated with human platelets caused dose-dependent inhibition of agonist-induced platelet aggregation, thromboxane generation, and serotonin release. As few as one microfilaria per 10(4) platelets completely inhibited aggregation of platelets induced by thrombin, collagen, arachidonic acid, or ionophore A23187. Microfilariae also inhibited aggregation of platelets in platelet-rich plasma stimulated by ADP, compound U46619, or platelet-activating factor. The inhibition required intimate proximity but not direct contact between parasites and platelets, and was mediated by parasite-derived soluble factors of low (less than 1,000 Mr) molecular weight that were labile in aqueous media and caused an elevation of platelet cAMP. Prior treatment of microfilariae with pharmacologic inhibitors of cyclooxygenase decreased both parasite release of prostacyclin and PGE2 and microfilarial inhibition of platelet aggregation. These results indicate that microfilariae inhibit platelet aggregation, via mechanisms that may include the elaboration of anti-aggregatory eicosanoids.
Assuntos
Brugia/patogenicidade , Agregação Plaquetária , Prostaglandinas/fisiologia , Animais , GMP Cíclico/sangue , Gerbillinae , Humanos , Microfilárias/patogenicidadeRESUMO
Because T cell-derived cytokines may affect the functioning of eosinophils, we have investigated the capacity of human eosinophils to respond to IL-2. IL-2 was a potent chemoattractant with ED50 of 10(-12) M with eosinophils from all normal and eosinophilic donors tested. The monoclonal antibodies anti-Tac and TU27 against p55 (Tac/CD25) and p75 receptor subunits, respectively, each inhibited IL-2-dependent eosinophil migration. The molar potency of IL-2 and the inhibitory activity of each of the above antibodies suggest that high affinity heterodimeric IL-2 receptor complexes mediated the migration responses of eosinophils to IL-2. Binding of monoclonal antibody against p75 was not detectable by flow cytometry, and high affinity binding sites for 125I-IL-2 were below the limits of quantitation on eosinophils from individuals with eosinophilia. Expression of p55 (Tac/CD25) by eosinophils, without requirement for in vitro activation, was demonstrable by flow cytometry, radioimmunoprecipitation, and Northern blotting for mRNA. Surface expression of p55 on eosinophils from normal or eosinophilic individuals increased during culture for 24-48 h with a biologic activity purified from stimulated U937 cells and to a lesser extent with granulocyte-macrophage CSF or lymphocyte chemoattractant factor but not with nine other cytokines. These studies indicate that blood eosinophils respond to IL-2 and identify one mechanism whereby activation of T lymphocytes may influence the function of eosinophils.
Assuntos
Eosinófilos/química , Receptores de Interleucina-2/análise , Movimento Celular/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Interleucina-5/farmacologia , RNA Mensageiro/análiseRESUMO
Because eosinophils recruited into the airways in allergic diseases are exposed to inhaled allergens, we evaluated whether eosinophils within the endobronchial lumen can function in vivo as antigen-presenting cells for inhaled antigens. We recovered eosinophils from the airways after aerosol antigen challenge in sensitized mice or from the peritoneal cavities of IL-5 transgenic mice and fluorescently labeled these cells ex vivo. These labeled cells, instilled intratracheally into normal mice, migrated into draining paratracheal lymph nodes and localized to T cell-rich paracortical areas. The homing of airway eosinophils to lymph nodes was not governed by eotaxin, because CCR3(-/-) and CCR3(+/+) eosinophils migrated identically. Airway eosinophils, recovered after inhalational antigen challenge in sensitized mice, expressed MHC class II and costimulatory CD80 and CD86 proteins and functioned in vitro as CD80- and CD86-dependent, antigen-specific, antigen-presenting cells. Moreover, when instilled into the airways of antigen-sensitized recipient mice, airway eosinophils recovered after inhalational antigen challenge stimulated antigen-specific CD4(+) T cell proliferation within paratracheal lymph nodes. Thus, eosinophils within the lumina of airways can process inhaled antigens, traffic to regional lymph nodes, and function in vivo as antigen-presenting cells to stimulate responses of CD4(+) T cells.
Assuntos
Apresentação de Antígeno/imunologia , Movimento Celular , Eosinófilos/imunologia , Linfonodos/imunologia , Animais , Antígenos CD/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-2 , Brônquios/citologia , Eosinófilos/fisiologia , Feminino , Antígenos de Histocompatibilidade Classe II/biossíntese , Linfonodos/citologia , Masculino , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Knockout , Receptores CCR3 , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Traqueia/citologiaRESUMO
By in situ hybridization, 44-100% of the blood eosinophils from five patients with hypereosinophilia and four normal subjects exhibited intense hybridization signals for TNF-alpha mRNA. TNF-alpha protein was detectable by immunohistochemistry in blood eosinophils of hypereosinophilic subjects, and purified blood eosinophils from three atopic donors exhibited cycloheximide-inhibitable spontaneous release of TNF-alpha in vitro. Many blood eosinophils (39-91%) from hypereosinophilic donors exhibited intense labeling for macrophage inflammatory protein-1 alpha (MIP-1 alpha) mRNA, whereas eosinophils of normal donors demonstrated only weak or undetectable hybridization signals for MIP-1 alpha mRNA. Most tissue eosinophils infiltrating nasal polyps were strongly positive for both TNF-alpha and MIP-1 alpha mRNA. By Northern blot analysis, highly enriched blood eosinophils from a patient with the idiopathic hypereosinophilic syndrome exhibited differential expression of TNF-alpha and MIP-1 alpha mRNA. These findings indicate that human eosinophils represent a potential source of TNF-alpha and MIP-1 alpha, that levels of expression of mRNA for both cytokines are high in the blood eosinophils of hypereosinophilic donors and in eosinophils infiltrating nasal polyps, that the eosinophils of normal subjects express higher levels of TNF-alpha than MIP-1 alpha mRNA, and that eosinophils purified from the blood of atopic donors can release TNF-alpha in vitro.
Assuntos
Citocinas/análise , Eosinofilia/metabolismo , Eosinófilos/química , Monocinas/análise , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/análise , Células Cultivadas , Quimiocina CCL4 , Citocinas/genética , Eosinófilos/citologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Proteínas Inflamatórias de Macrófagos , Masculino , Monocinas/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
In this study, we have investigated CD40 expression in human peripheral blood eosinophils and in human chronically inflamed nasal tissues, i.e., nasal polyps. We show by both reverse transcriptase-PCR and Northern blot analysis that eosinophils from allergic subjects express human CD40 mRNA. We also show that constitutive CD40 mRNA expression in eosinophils could be upregulated by exposure to IgA immune complexes and downregulated by IL-10 and the synthetic steroid budesonide. In addition, we demonstrate that eosinophils express CD40 protein by flow cytometry. Such expression is biologically functional as cross-linking CD40 with CD40 mAbs enhances eosinophil survival in a dose-dependent fashion; in addition, CD40 ligation stimulates eosinophils to release GM-CSF. CD40-mediated eosinophil survival was largely inhibited by an anti-GM-CSF neutralizing antibody suggesting GM-CSF involvement in the survival enhancing mechanism. CD40 mRNA was also detected in total RNA extracted from nasal polyp tissues but not in RNA isolated from normal nasal mucosa (inferior turbinate); by immunohistochemistry, we were able to detect immunoreactive CD40 protein in a variety of cell types in the polyp stroma, but primarily in eosinophils. These observations suggest previously unforeseen interactions between eosinophils and cells expressing the CD40 ligand and, thus, novel pathways by which eosinophils may contribute to the regulation of airway inflammation.
Assuntos
Antígenos CD40/sangue , Eosinófilos/imunologia , Anticorpos Monoclonais/farmacologia , Sequência de Bases , Antígenos CD40/química , Antígenos CD40/genética , Reagentes de Ligações Cruzadas , Primers do DNA/genética , Eosinófilos/metabolismo , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Imuno-Histoquímica , Inflamação/genética , Inflamação/imunologia , Dados de Sequência Molecular , Pólipos Nasais/genética , Pólipos Nasais/imunologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Although much has been learned about the basic contents and capabilities of eosinophils, some of the roles eosinophils play in host defense and the immunopathogenesis of diseases remain enigmatic. In addition to containing four notable cationic granule proteins and their ability to synthesize lipid mediators of inflammation, eosinophils have recently been shown to be able to elaborate a range of cytokines that may exert autocrine as well as paracrine effects. The roles of eosinophils within tissues are modulated by interactions with the extracellular matrix and other cells during eosinophil recruitment and activation, and eosinophils may engage in cooperative interactions with other cells.