RESUMO
Although the main regulators of leukocyte trafficking are chemokines, another family of chemotactic agents is cyclophilins. Intracellular cyclophilins function as peptidyl-prolyl cis-trans isomerases and are targets of the immunosuppressive drug cyclosporine A (CsA). Cyclophilins can also be secreted in response to stress factors, with elevated levels of extracellular cyclophilins detected in several inflammatory diseases. Extracellular cyclophilins are known to have potent chemotactic properties, suggesting that they might contribute to inflammatory responses by recruiting leukocytes into tissues. The objective of the present study was to determine the impact of blocking cyclophilin activity using a cell-impermeable derivative of CsA to specifically target extracellular pools of cyclophilins. In this study, we show that treatment with this compound in a mouse model of allergic lung inflammation demonstrates up to 80% reduction in inflammation, directly inhibits the recruitment of Ag-specific CD4(+) T cells, and works equally well when delivered at 100-fold lower doses directly to the airways. Our findings suggest that cell-impermeable analogs of CsA can effectively reduce inflammatory responses by targeting leukocyte recruitment mediated by extracellular cyclophilins. Specifically blocking the extracellular functions of cyclophilins may provide an approach for inhibiting the recruitment of one of the principal immune regulators of allergic lung inflammation, Ag-specific CD4(+) T cells, into inflamed airways and lungs.
Assuntos
Asma/tratamento farmacológico , Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Ciclofilinas/imunologia , Ciclosporinas/farmacologia , Imunossupressores/farmacologia , Animais , Antígenos/imunologia , Asma/patologia , Linfócitos T CD4-Positivos/patologia , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Allergic asthma is characterized by acute influxes of proinflammatory leukocytes in response to allergen stimulation, followed by quiescent (chronic) periods between allergen challenges, during which sustained, low-level inflammation is evident. These chronic phases of disease are thought to be mediated by populations of leukocytes persisting within airways and tissues. The lack of any in situ proliferation by these cells, along with their limited lifespan, suggests that a continual recruitment of leukocytes from the circulation is needed to maintain disease chronicity. The mechanisms regulating this persistent recruitment of leukocytes are unknown. Although classic leukocyte-attracting chemokines are highly elevated after acute allergen challenge, they return to baseline levels within 24 hours, and remain close to undetectable during the chronic phase. In the present study, we investigated whether an alternative family of chemoattractants, namely, extracellular cyclophilins, might instead play a role in regulating the recruitment and persistence of leukocytes during chronic asthma, because their production is known to be more sustained during inflammatory responses. Using a new murine model of chronic allergic asthma, elevated concentrations of extracellular cyclophilin A, but not classic chemokines, were indeed detected during the chronic phase of asthma. Furthermore, blocking the activity of cyclophilins during this phase reduced the number of persisting leukocytes by up to 80%. This reduction was also associated with a significant inhibition of acute disease reactivation upon subsequent allergen challenge. These findings suggest that blocking the function of cyclophilins during the chronic phase of asthma may provide a novel therapeutic strategy for regulating disease chronicity and severity.
Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Ciclofilinas/antagonistas & inibidores , Ciclosporina/uso terapêutico , Leucócitos/efeitos dos fármacos , Animais , Asma/prevenção & controle , Ciclofilinas/sangue , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Prevenção Secundária , Índice de Gravidade de DoençaRESUMO
Cyclophilins belong to the enzyme class of peptidyl prolyl cis-trans isomerases which catalyze the cis-trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family, CypA and CypB, exhibit specific cellular functions in several inflammatory diseases, cancer development, and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4(+) T cells by the inhibitor provided biological proof of discrimination in vivo.
Assuntos
Ciclofilinas/antagonistas & inibidores , Ciclofilinas/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Animais , Benzofuranos/química , Ligação Competitiva , Biocatálise , Linfócitos T CD4-Positivos/citologia , Domínio Catalítico , Quimiotaxia/efeitos dos fármacos , Ciclofilina A/antagonistas & inibidores , Ciclofilina A/química , Ciclofilina A/metabolismo , Ciclofilinas/metabolismo , Ciclosporina/química , Ciclosporina/metabolismo , Polarização de Fluorescência , Humanos , Indanos/química , Indanos/metabolismo , Indanos/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos , Estrutura Molecular , Ligação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinâmicaAssuntos
Ciclofilina A/metabolismo , Ciclosporina/química , Imunossupressores/química , Animais , Quimiotaxia/efeitos dos fármacos , Ciclofilina A/química , Ciclosporina/síntese química , Ciclosporina/farmacologia , Corantes Fluorescentes/química , Humanos , Imunossupressores/síntese química , Imunossupressores/farmacologia , Células Jurkat , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Peptídeos/química , Rodaminas/químicaRESUMO
Asthma is a chronic inflammatory condition of the lower respiratory tract associated with airway hyperreactivity and mucus obstruction in which a majority of cases are due to an allergic response to environmental allergens. Glucocorticoids such as prednisone have been standard treatment for many inflammatory diseases for the past 60 years. However, despite their effectiveness, long-term treatment is often limited by adverse side effects believed to be caused by glucocorticoid receptor-mediated gene transcription. This has led to the pursuit of compounds that retain the anti-inflammatory properties yet lack the adverse side effects associated with traditional glucocorticoids. We have developed a novel series of steroidal analogues (VBP compounds) that have been previously shown to maintain anti-inflammatory properties such as NFκB-inhibition without inducing glucocorticoid receptor-mediated gene transcription. This study was undertaken to determine the effectiveness of the lead compound, VBP15, in a mouse model of allergic lung inflammation. We show that VBP15 is as effective as the traditional glucocorticoid, prednisolone, at reducing three major hallmarks of lung inflammation--NFκB activity, leukocyte degranulation, and pro-inflammatory cytokine release from human bronchial epithelial cells obtained from patients with asthma. Moreover, we found that VBP15 is capable of reducing inflammation of the lung in vivo to an extent similar to that of prednisone. We found that prednisolone--but not VBP15 shortens the tibia in mice upon a 5 week treatment regimen suggesting effective dissociation of side effects from efficacy. These findings suggest that VBP15 may represent a potent and safer alternative to traditional glucocorticoids in the treatment of asthma and other inflammatory diseases.