RESUMO
RATIONALE: Eosinophils are key effector cells in allergic diseases, including allergic rhinitis, eczema, and asthma. Their tissue presence is regulated by both recruitment and increased longevity at inflamed sites. OBJECTIVES: To investigate the ability of the flavone wogonin to induce eosinophil apoptosis in vitro and attenuate eosinophil-dominant allergic inflammation in vivo in mice. METHODS: Human and mouse eosinophil apoptosis in response to wogonin was investigated by cellular morphology, flow cytometry, mitochondrial membrane permeability, and pharmacological caspase inhibition. Allergic lung inflammation was modeled in mice sensitized and challenged with ovalbumin. Bronchoalveolar lavage (BAL) and lung tissue were examined for inflammation, mucus production, and inflammatory mediator production. Airway hyperresponsiveness to aerosolized methacholine was measured. MEASUREMENTS AND MAIN RESULTS: Wogonin induced time- and concentration-dependent human and mouse eosinophil apoptosis in vitro. Wogonin-induced eosinophil apoptosis occurred with activation of caspase-3 and was inhibited by pharmacological caspase inhibition. Wogonin administration attenuated allergic airway inflammation in vivo with reductions in BAL and interstitial eosinophil numbers, increased eosinophil apoptosis, reduced airway mucus production, and attenuated airway hyperresponsiveness. This wogonin-induced reduction in allergic airway inflammation was prevented by concurrent caspase inhibition in vivo. CONCLUSIONS: Wogonin induces eosinophil apoptosis and attenuates allergic airway inflammation, suggesting that it has therapeutic potential for the treatment of allergic inflammation in humans.
Assuntos
Apoptose/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Flavanonas/farmacologia , Hipersensibilidade/fisiopatologia , Animais , Lavagem Broncoalveolar , Eosinófilos/citologia , Feminino , Citometria de Fluxo , Humanos , Técnicas In Vitro , Inflamação , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologiaRESUMO
CCL3 is a protein of the CC chemokine family known to be important for T cell recruitment in inflammatory diseases. The aim of the current study was to evaluate the effects and putative mechanism of action of evasin-1, a novel CCL3-binding protein, in the pathogenesis of acute graft-versus-host disease (GVHD). GVHD was induced by the transplantation of splenocytes from C57BL/6J to B6D2F1 mice. Treatment of recipient mice with evasin-1 prevented mortality associated with GVHD. This was correlated with reduced weight loss and clinical disease severity. Analysis of the small intestine showed that evasin-1 treatment reduced the histopathological score and decreased levels of IFN-gamma and CCL5. Mechanistically, evasin-1 treatment reduced the number of CD4(+) and CD8(+) T cells infiltrating the small intestine, as assessed by immunohistochemistry, and the adhesion of leukocytes to intestinal venules of recipient mice, as assessed by intravital microscopy. Evasin-1 was also able to decrease liver damage, as seen by reduction of inflammatory infiltrate and IFN-gamma levels. Treatment with evasin-1 did not interfere with graft-versus-leukemia. Altogether, our studies demonstrate that CCL3 plays a major role in mediating GVHD, but not graft-versus-leukemia in mice and suggest that blockade of CCL3 with evasin-1 has potential therapeutic application in patients undergoing bone marrow transplantation.
Assuntos
Quimiocina CCL3/deficiência , Doença Enxerto-Hospedeiro/metabolismo , Proteínas Inflamatórias de Macrófagos/metabolismo , Receptores de Quimiocinas/metabolismo , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Transplante de Células , Quimiocina CCL3/genética , Quimiocina CCL5/metabolismo , Dexametasona/farmacologia , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia/efeitos dos fármacos , Imuno-Histoquímica , Interferon gama/metabolismo , Intestino Delgado/imunologia , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Proteínas Inflamatórias de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Camundongos Transgênicos , Baço/citologiaRESUMO
CC chemokines play an important role in the pathogenesis of idiopathic pulmonary fibrosis. Few studies have evaluated the efficacy of therapeutically targeting CC chemokines and their receptors during interstitial lung diseases. In the present study, the therapeutic effects of Evasin-1, a tick-derived chemokine-binding protein that has high affinity for CCL3/microphage inflammatory protein (MIP)-1α, was investigated in a murine model of bleomycin-induced lung fibrosis. CCL3/MIP-1α concentrations in lung homogenates increased significantly with time after bleomycin challenge, and this was accompanied by increased number of leukocytes and elevated levels of CCL2/monocyte chemoattractant protein (MCP)-1, CCL5/regulated upon activation, normal T cell expressed and secreted, TNF-α and transforming growth factor-ß(1), and pulmonary fibrosis. Administration of evasin-1 on a preventive (from the day of bleomycin administration) or therapeutic (from Day 8 after bleomycin) schedule decreased number of leukocytes in the lung, reduced levels of TNF-α and transforming growth factor-ß(1), and attenuated lung fibrosis. These protective effects were similar to those observed in CCL3/MIP-1α-deficient mice. In conclusion, targeting CCL3/MIP-1α by treatment with evasin-1 is beneficial in the context of bleomycin-induced lung injury, even when treatment is started after the fibrogenic insult. Mechanistically, evasin-1 treatment was associated with decreased recruitment of leukocytes and production of fibrogenic cytokines. Modulation of CCL3/MIP-1α function by evasin-1 could be useful for the treatment of idiopathic pulmonary fibrosis.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Bleomicina/efeitos adversos , Quimiocina CCL3/antagonistas & inibidores , Quimiocina CCL3/imunologia , Fibrose Pulmonar/tratamento farmacológico , Receptores de Quimiocinas/uso terapêutico , Animais , Antibióticos Antineoplásicos/administração & dosagem , Bleomicina/farmacologia , Quimiocina CCL3/genética , Quimiocina CCL3/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Camundongos , Camundongos Knockout , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Receptores de Quimiocinas/química , Rhipicephalus sanguineus/química , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Eosinophilic inflammation plays an important role in driving a variety of inflammatory and allergic conditions. Delineating the mechanisms by which these terminally differentiated granulocytes undergo programmed cell death (apoptosis) and their subsequent clearance by surrounding phagocytes are central to understanding disease pathogenesis and development of novel pharmacological agents. Dysregulation of the processes of either apoptosis or phagocytosis can result in chronic inflammation and disease progression due to either increased eosinophil life-span or cell necrosis with loss of cell membrane integrity and release of toxic intracellular mediators. A variety of in vitro methods have therefore been developed to understand these mechanisms in isolated primary human eosinophils. Here we describe the key assays used to study eosinophil apoptosis and the intracellular signalling pathways involved as well as phagocytic clearance of these cells.
Assuntos
Apoptose/fisiologia , Eosinófilos/citologia , Fagocitose/fisiologia , Animais , Eosinófilos/metabolismo , Humanos , Transdução de Sinais/fisiologiaRESUMO
Inflammation is a beneficial host reaction to tissue damage and has the essential primary purpose of restoring tissue homeostasis. Inflammation plays a major role in containing and resolving infection and may also occur under sterile conditions. The cardinal signs of inflammation dolor, calor, tumor and rubor are intrinsically associated with events including vasodilatation, edema and leukocyte trafficking into the site of inflammation. If uncontrolled or unresolved, inflammation itself can lead to further tissue damage and give rise to chronic inflammatory diseases and autoimmunity with eventual loss of organ function. It is now evident that the resolution of inflammation is an active continuous process that occurs during an acute inflammatory episode. Successful resolution requires activation of endogenous programs with switch from production of pro-inflammatory towards pro-resolving molecules, such as specific lipid mediators and annexin A1, and the non-phlogistic elimination of granulocytes by apoptosis with subsequent removal by surrounding macrophages. These processes ensure rapid restoration of tissue homeostasis. Here, we review recent advances in the understanding of resolution of inflammation, highlighting the pharmacological strategies that may interfere with the molecular pathways which control leukocyte survival and clearance. Such strategies have proved beneficial in several pre-clinical models of inflammatory diseases, suggesting that pharmacological modulation of the resolution process may be useful for the treatment of chronic inflammatory diseases in humans.
Assuntos
Inflamação/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Descoberta de Drogas , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Mediadores da Inflamação/metabolismoRESUMO
The technical limitations of isolating neutrophils without contaminating leukocytes, while concurrently minimizing neutrophil activation, is a barrier to determining specific neutrophil functions. We aimed to assess the use of FACS for generating highly pure quiescent neutrophil populations in an antibody-free environment. Peripheral blood human granulocytes and murine bone marrow-derived neutrophils were isolated by discontinuous Percoll gradient and flow-sorted using FSC/SSC profiles and differences in autofluorescence. Postsort purity was assessed by morphological analysis and flow cytometry. Neutrophil activation was measured in unstimulated-unsorted and sorted cells and in response to fMLF, LTB4, and PAF by measuring shape change, CD62L, and CD11b expression; intracellular calcium flux; and chemotaxis. Cytokine production by human neutrophils was also determined. Postsort human neutrophil purity was 99.95% (sem=0.03; n=11; morphological analysis), and 99.68% were CD16(+ve) (sem=0.06; n=11), with similar results achieved for murine neutrophils. Flow sorting did not alter neutrophil activation or chemotaxis, relative to presorted cells, and no differences in response to agonists were observed. Stimulated neutrophils produced IL-1ß, although to a lesser degree than CXCL8/IL-8. The exploitation of the difference in autofluorescence between neutrophils and eosinophils by FACS is a quick and effective method for generating highly purified populations for subsequent in vitro study.
Assuntos
Separação Celular/métodos , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Neutrófilos/metabolismo , Imagem Óptica/métodos , Animais , Medula Óssea/metabolismo , Cálcio/metabolismo , Células Cultivadas , Quimiotaxia , Ensaio de Imunoadsorção Enzimática , Eosinófilos/citologia , Eosinófilos/metabolismo , Granulócitos/citologia , Granulócitos/metabolismo , Humanos , Camundongos , Ativação de Neutrófilo , Neutrófilos/citologiaRESUMO
BACKGROUND: Eosinophils not only defend the body against parasitic infection but are also involved in pathological inflammatory allergic diseases such as asthma, allergic rhinitis and contact dermatitis. Clearance of apoptotic eosinophils by macrophages is a key process responsible for driving the resolution of eosinophilic inflammation and can be defective in allergic diseases. However, enhanced resolution of eosinophilic inflammation by deliberate induction of eosinophil apoptosis using pharmacological agents has not been previously demonstrated. Here we investigated the effect of a novel cyclin-dependent kinase inhibitor drug, AT7519, on human and mouse eosinophil apoptosis and examined whether it could enhance the resolution of a murine model of eosinophil-dominant inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Eosinophils from blood of healthy donors were treated with AT7519 and apoptosis assessed morphologically and by flow-cytometric detection of annexin-V/propidium iodide staining. AT7519 induced eosinophil apoptosis in a concentration dependent manner. Therapeutic administration of AT7519 in eosinophil-dominant allergic inflammation was investigated using an established ovalbumin-sensitised mouse model of allergic pleurisy. Following ovalbumin challenge AT7519 was administered systemically at the peak of pleural inflammation and inflammatory cell infiltrate, apoptosis and evidence of macrophage phagocytosis of apoptotic eosinophils assessed at appropriate time points. Administration of AT7519 dramatically enhanced the resolution of allergic pleurisy via direct induction of eosinophil apoptosis without detriment to macrophage clearance of these cells. This enhanced resolution of inflammation was shown to be caspase-dependent as the effects of AT7519 were reduced by treatment with a broad spectrum caspase inhibitor (z-vad-fmk). CONCLUSIONS: Our data show that AT7519 induces human eosinophil apoptosis and enhances the resolution of a murine model of allergic pleurisy by inducing caspase-dependent eosinophil apoptosis and enhancing macrophage ingestion of apoptotic eosinophils. These findings demonstrate the utility of cyclin-dependent kinase inhibitors such as AT7519 as potential therapeutic agents for the treatment of eosinophil dominant allergic disorders.
Assuntos
Apoptose/efeitos dos fármacos , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Hipersensibilidade/metabolismo , Piperidinas/farmacologia , Pirazóis/farmacologia , Animais , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Piperidinas/uso terapêutico , Pirazóis/uso terapêuticoRESUMO
PDE4 inhibitors are effective anti-inflammatory drugs whose effects and putative mechanisms on resolution of inflammation and neutrophil apoptosis in vivo are still unclear. Here, we examined the effects of specific PDE4 inhibition on the resolution of neutrophilic inflammation in the pleural cavity of LPS-challenged mice. LPS induced neutrophil recruitment that was increased at 4 h, peaked at 8-24 h, and declined thereafter. Such an event in the pleural cavity was preceded by increased levels of KC and MIP-2 at 1 and 2 h. Treatment with the PDE4 inhibitor rolipram, at 4 h after LPS administration, decreased the number of neutrophils and increased the percentage of apoptotic cells in the pleural cavity in a PKA-dependent manner. Conversely, delayed treatment with a CXCR2 antagonist failed to prevent neutrophil recruitment. Forskolin and db-cAMP also decreased the number of neutrophils and increased apoptosis in the pleural cavity. The proapoptotic effect of rolipram was associated with decreased levels of the prosurvival protein Mcl-1 and increased caspase-3 cleavage. The pan-caspase inhibitor zVAD-fmk prevented rolipram-induced resolution of inflammation. LPS resulted in a time-dependent activation of Akt, which was blocked by treatment with rolipram or PI3K and Akt inhibitors, and PI3K and Akt inhibitors also enhanced apoptosis and promoted neutrophil clearance. Although LPS induced NF-kappaB activation, which was blocked by rolipram, NF-kappaB inhibitors did not promote resolution of neutrophil accumulation in this model. In conclusion, our data show that PDE4 inhibition resolves neutrophilic inflammation by promoting caspase-dependent apoptosis of inflammatory cells by targeting a PKA/PI3K/Akt-dependent survival pathway.
Assuntos
Apoptose/imunologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Inflamação/imunologia , Neutrófilos/imunologia , Transdução de Sinais/imunologia , Animais , Western Blotting , Quimiotaxia de Leucócito , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/imunologia , Fragmentação do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Inflamação/induzido quimicamente , Inflamação/enzimologia , Lipopolissacarídeos/imunologia , Camundongos , NF-kappa B/metabolismo , Neutrófilos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Cavidade Pleural/imunologia , Cavidade Pleural/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Eosinophils are major players in inflammatory allergic diseases such as asthma, hay fever and eczema. Here we show that the cyclin-dependent kinase inhibitor (CDKi) R-roscovitine efficiently and rapidly induces human eosinophil apoptosis using flow cytometric analysis of annexin-V/propidium iodide staining, morphological analysis by light microscopy, transmission electron microscopy and Western immunoblotting for caspase-3 cleavage. We further dissect these observations by demonstrating that eosinophils treated with R-roscovitine lose mitochondrial membrane potential and the key survival protein Mcl-1 is down-regulated. This novel finding of efficacious induction of eosinophil apoptosis by CDKi drugs has potential as a strategy for driving resolution of eosinophilic inflammation.
Assuntos
Apoptose/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Eosinófilos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Purinas/farmacologia , Inibidores de Caspase , Caspases/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Eosinófilos/fisiologia , Eosinófilos/ultraestrutura , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/genética , RoscovitinaRESUMO
Selective and timely induction of apoptosis is an effective means of resolving inflammation. The effects and putative mechanisms by which cyclic AMP (cAMP) modulates leukocyte apoptosis in vivo are still unclear. The present study aims at identifying intracellular pathways underlying the ability of cAMP elevating agents to resolve eosinophilic inflammation in a model of allergic pleurisy in mice. Ovalbumin (OVA) challenge of immunized mice induced eosinophil recruitment that peaked at 24h and persisted till 48h. Treatment with the PDE4 inhibitor rolipram, cAMP mimetic db-cAMP or adenylate cyclase activator forskolin, at 24h after antigen-challenge resulted in profound resolution of eosinophilic inflammation, without a decrease of mononuclear cell numbers. There was a concomitant increase in number of apoptotic cells in the pleural cavity. The effects of rolipram and db-cAMP were inhibited by the PKA inhibitor H89. Inhibition of PI3K/Akt or NF-kappaB induced resolution of inflammation that was associated with increased apoptosis. OVA-challenge resulted in a time-dependent activation of Akt and NF-kappaB, which was blocked by treatment with rolipram or PI3K/Akt pathway inhibitors. Thus, cAMP elevating agents resolve established eosinophilic inflammation by inducing leukocyte apoptosis. Mechanistically, the actions of cAMP are dependent on PKA and target a PI3K/Akt-dependent NF-kappaB survival pathway.
Assuntos
Apoptose/efeitos dos fármacos , AMP Cíclico/efeitos adversos , Eosinófilos/fisiologia , Hipersensibilidade/patologia , NF-kappa B/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Pleurisia/induzido quimicamente , Animais , Células Cultivadas , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and -3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P.J. Hudson. 2005. Nat. Biotechnol. 23:1126-1136), and may be therapeutically useful as novel antiinflammatory agents in the future.