RESUMO
BACKGROUND: Allergic rhinitis (AR) and asthma are closely related conditions that often co-exist, and are characterized by a Th2 inflammatory response where eosinophils occupy a predominant role. Strategies aimed at blocking signaling through the CC chemokine receptor 3 (CCR3) and/or the common beta chain of the IL-3, IL-5 and GM-CSF receptor (ßc) efficiently reduced eosinophilic inflammation in both animal models and in asthmatic patients. This study was therefore aimed at characterizing the spatio-temporal expression pattern of ßc and CCR3 using a rat model of AR. METHODS: Sensitized rats were challenged with ovalbumin and sacrificed at 2h, 8h, 16h or 24h post-challenge. Nasal tissues were microdissected and used for mRNA quantification by QPCR, while histological evaluation determined the presence of eosinophils and mucosubstances. RESULTS: Allergen-induced recruitment of eosinophils in the distal septum and turbinates was maximal at 8h post-challenge, and was correlated with 2-4-fold increase in CCR3 and ßc mRNA. Recruitment of eosinophils was also accompanied by upregulated IL-5, IL-4Rα, TNF-α and IFN-γ mRNA at early time-points. In contrast, IL-13 and MUC5AC mRNA, as well as production of mucosubstances were maximal at 24h. CONCLUSIONS: ßc and CCR3 could play important roles in the modulation of the allergic response, and their inhibition may represent a promising therapeutic approach for AR.
Assuntos
Hipersensibilidade/metabolismo , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Tecido Linfoide/metabolismo , Mucosa Nasal/metabolismo , Receptores CCR3/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Rinite/metabolismo , Animais , Sequência de Bases , Primers do DNA , Modelos Animais de Doenças , Interleucina-3/genética , Interleucina-5/genética , Masculino , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Ratos , Receptores CCR3/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genéticaRESUMO
BACKGROUND: Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4) isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2'-deoxy-2'-Fluoro-beta-D-Arabinonucleic Acid (FANA)-containing antisense oligonucleotides (AON) targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both in vitro and in vivo. METHODS: Normal human bronchial epithelial (NHBE) cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs. RESULTS: In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation than roflumilast. Moreover, the protective effect of PDE4B/4D and 7A AON was maintained when a once-weekly treatment schedule was used. CONCLUSION: These results indicate that inhaled AON against PDE4B/4D and 7A have unique effects on biomarkers that are believed to be important in the pathophysiology of COPD, which supports further development as a potential therapy in this disease.
Assuntos
Arabinonucleotídeos/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/genética , Inflamação/fisiopatologia , Pulmão/fisiopatologia , Fumaça/efeitos adversos , Fumar/efeitos adversos , Animais , Brônquios/efeitos dos fármacos , Brônquios/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/efeitos dos fármacos , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Humanos , Inflamação/enzimologia , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Pulmão/enzimologia , Camundongos , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Asthma is characterized by airway hyperresponsiveness (AHR) and inflammation, consisting predominantly of eosinophils within the airway lumen and walls. Eosinophil recruitment to the airways is mediated mainly by eotaxin and other chemokines that bind to the CC-chemokine receptor-3 (CCR3), which is highly expressed on eosinophils. This study assessed whether topical inhibition of CCR3 mRNA expression by phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) modifies pulmonary eosinophilia and AHR in an antigen-induced allergic asthma model in Brown Norway (BN) rats. Results show that specific inhibition of CCR3 expression in the lungs by an AS-ODN (AS4) reduced total eosinophil infiltration and the percentage of eosinophils into the airways of ovalbumin challenged rats. Moreover, reduction in CCR3 mRNA levels was correlated with a decrease in CCR3 protein in lung tissue. In addition, AS4 treatment had no effect on circulating eosinophils or on eosinophils in the bone marrow. Finally, AHR was significantly decreased in AS4-treated rats when compared with rats treated with a mismatch AS-ODN. In conclusion, inhibition of the expression of CCR3 decreased pulmonary eosinophilia and reduced AHR after antigen challenge in rats. Topical inhibition of CCR3 expression, using an AS-ODN, could represent a novel approach for the treatment of asthma.
Assuntos
Adjuvantes Imunológicos/farmacologia , Asma/tratamento farmacológico , Oligonucleotídeos Antissenso/farmacologia , Receptores de Quimiocinas/genética , Animais , Asma/metabolismo , Modelos Animais de Doenças , Ovalbumina/imunologia , Peritonite/tratamento farmacológico , Peritonite/imunologia , Peritonite/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos BN , Receptores CCR3 , Receptores de Quimiocinas/biossínteseRESUMO
Reliable assessment of cell death is now pivotal to many research programs aiming at generating new antitumor compounds or at screening cDNA libraries to identify genes with pro- or antiapoptotic functions. Such approaches need to rely on reproducible, easy handling, and rapid microplate-based cytotoxicity assays that are amenable to high-throughput screening technologies. We describe here a method for the direct measurement of cell death, based on the detection of a decrease in fluorescence observed following death induction in cells stably expressing enhanced green fluorescent protein (EGFP). Our data clearly show that such a decrease in EGFP fluorescence after cell death induction happens in various cell types, including those routinely used in anticancer drug screening (i.e., murine and human, lymphoid, fibroblastic, or epithelial cell lines). Moreover, the decrease in EGFP fluorescence is observed in cells induced to die by a variety of apoptosis-inducing agents, such as glucocorticoids (dexamethasone), DNA- damaging agents (etoposide, cisplatin), microtubule disorganizers (paclitaxel), protein kinase C inhibitors (staurosporine), or a caspase-independent apoptotic stimulus (CD45 crosslinking). A decrease in fluorescence can be assessed either by flow cytometry or with a fluorescence microplate reader. The kinetics and specificity of this EGFP-based assay were comparable with those of other conventional techniques used to detect cell death. This novel EGFP-based microplate assay combines sensitivity and rapidity and is amenable to high-throughput setups, making it an assay of choice for evaluation of cell cytotoxicity.
Assuntos
Morte Celular/efeitos dos fármacos , Dexametasona/farmacologia , Proteínas de Fluorescência Verde/biossíntese , Animais , Antineoplásicos/farmacologia , Apoptose , Células Cultivadas , Citometria de Fluxo , Fluorescência , Proteínas de Fluorescência Verde/genética , Humanos , Retroviridae/genética , Sensibilidade e Especificidade , TransfecçãoRESUMO
Endometriosis is a prevalent disease characterized by the estrogen-dependent ectopic growth of endometrial tissue. Most of the current medical therapies consist in inducing a hypoestrogenic state in patients, but these treatments are associated with severe side effects and high recurrence rates. The development of convenient and reliable endometriosis animal models would be instrumental to accelerate the emergence of new therapeutic alternatives. Recently, we developed an improved experimental model for endometriosis, relying on the infection of human endometrial fragments by an adenovirus carrying the green fluorescent protein. Following injection of fluorescent fragments into nude mice, the implantation and growth of endometriotic-like lesions could be followed noninvasively. In the present work, we demonstrate that this model can be used to quantify the size of fluorescent endometriotic lesions by in vivo imaging. To this end, we repeatedly measured lesion size over a 4-week period in mice supplemented or not with estradiol. The model was adequate to confirm previous results showing that estrogen is dispensable for the implantation phase of endometrial tissue, whereas it is required for lesion maintenance. As a proof of concept for inducing regression of established lesions, ganciclovir was used to treat animals implanted with human fluorescent endometrial fragments expressing thymidine kinase. A significant decrease in lesion size was observed by in vivo imaging in ganciclovir-treated mice. Together, the data indicate that the noninvasive animal model described here provides a tool for drug testing and/or gene target validation in endometriosis.
Assuntos
Endometriose/patologia , Endométrio/metabolismo , Adenoviridae/genética , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estradiol/metabolismo , Estrogênios/metabolismo , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Timidina Quinase/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: To test whether fragments of human endometrium transduced with the green fluorescent protein (GFP) cDNA and transplanted into nude mice can be noninvasively visualized. DESIGN: A murine experimental model for human endometriosis. SETTING: A biotechnology company. ANIMAL(S): Ovariectomized nude mice. INTERVENTION(S): Whole fragments of human endometrium were transduced in vitro by adenoviral infection with the GFP cDNA before transplantation into nude mice. Animals were noninvasively and repeatedly imaged before lesion collection. MAIN OUTCOME MEASURE(S): Fluorescence of GFP-expressing human endometrial fragments was evaluated before transplantation into animals. Development of endometriotic lesions was monitored through direct visualization of fluorescent tissue in the living animal or through conventional dissection. RESULT(S): GFP gene transfer into whole endometrial fragments can be performed, and a high proportion of cells express the reporter gene. Fluorescent endometrial fragments implant in nude mice and form endometriotic-like lesions, which can be directly visualized through the skin of living mice using a simple imaging device. CONCLUSION(S): This improved mouse model allows noninvasive and dynamic studies of lesion implantation and development to be conducted. In addition to helping better understand the pathophysiology of the disease, this model represents a valuable preclinical tool for testing the efficacy of new drugs targeting endometriosis, which should ultimately accelerate their development phase.
Assuntos
Modelos Animais de Doenças , Endometriose/patologia , Endométrio/metabolismo , Proteínas Luminescentes/genética , Animais , Endometriose/metabolismo , Feminino , Fluorescência , Proteínas de Fluorescência Verde , Histocitoquímica , Humanos , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Nus , Microscopia Confocal , Organismos Livres de Patógenos Específicos , TransfecçãoRESUMO
The protein tyrosine phosphatase CD45 is a highly expressed glycoprotein present on all nucleated cells of hematopoietic origin. To date, all the functions attributed to CD45 are inherently coupled to its phosphatase activity. For instance, the regulation of lymphocyte antigen receptor signaling is mediated through the dephosphorylation, and hence activation, of Src-family kinases by CD45. Moreover, signaling via cytokine receptors is negatively modulated by CD45 by dephosphorylation of Janus kinase family members. Recently, another function for CD45, unrelated to regulation of surface receptor signaling, has been unraveled. Specific engagement of CD45 by monoclonal antibodies at the surface of lymphocytes induced their death, through an alternative caspase-independent pathway. In striking contrast to all other previously reported functions for CD45, its phosphatase activity is completely dispensable for the induction of cell death. This article reviews the current knowledge on the death pathway triggered by CD45 ligation on lymphocytes. In an attempt to better elucidate the mechanism of cell death induction through CD45, we also provide original data regarding the susceptibility of various subsets of immature and mature T and B cells to death induced by CD45 engagement. The physiological significance and therapeutic potential of CD45-induced death are also discussed.
Assuntos
Apoptose/imunologia , Antígenos Comuns de Leucócito/metabolismo , Linfócitos/citologia , Linfócitos/imunologia , Animais , Anticorpos Monoclonais , Reagentes de Ligações Cruzadas , Galectina 1/metabolismo , Humanos , Antígenos Comuns de Leucócito/química , Antígenos Comuns de Leucócito/imunologia , Ligantes , Linfócitos/metabolismo , Camundongos , Transdução de SinaisRESUMO
We have recently observed that monoglycerides (MGs), a family of lipids consisting of a single fatty acid moiety attached to a glycerol backbone, induce rapid dose-dependent apoptosis in murine thymocytes. In this work, we evaluated the sensitivity of various normal and malignant immune and non-immune cells to MGs. We demonstrate that the propensity to MG-induced death displayed by both T and B lymphocytes is clearly modulated according to their differentiation and activation status. For instance, the earliest T and B cell precursors are refractory to MG-mediated cell death. In the T-cell lineage, immature thymocytes are the most susceptible to MG treatment, while B cells from peripheral lymphoid organs appear more sensitive than B-cell subsets from the bone marrow. On the other hand, both activated T and B cells are more resistant to MG exposure than their non-activated counterparts. In addition, other hematopoietic lineages such as natural killer cells, macrophages, and erythroid cells are quite resistant to MG-induced death. Furthermore, using various immortalized cell lines from different tissues, we found that lymphomas and thymomas are the most sensitive among all lineages tested, while epithelial cells and fibroblasts are unaffected by MG treatment. Finally, MG-induced death was shown to be independent of Fas/Fas ligand (FasL) interactions. Altogether, our findings indicate that there is a cellular specificity related to MG-mediated cell death biased towards T and B lymphocytes. This suggests that MGs could potentially be used in the treatment of specific lymphoid disorders by bypassing the requirement for the Fas/FasL system.
Assuntos
Linfócitos B/efeitos dos fármacos , Diferenciação Celular , Glicerídeos/farmacologia , Ativação Linfocitária , Linfócitos T/efeitos dos fármacos , Células 3T3/patologia , Adenocarcinoma/patologia , Animais , Apoptose , Linfócitos B/imunologia , Células Epiteliais/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Proteína Ligante Fas , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Linfoma/patologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Timoma/patologia , Células Tumorais CultivadasRESUMO
Besides the well-recognized role of CD45 as a major player in TCR signaling, we and others have demonstrated that cross-linking of CD45 with mAbs can induce cell death in T lymphocytes. To investigate the role of CD45 phosphatase activity in apoptosis induction, we expressed either wild-type or phosphatase-dead CD45 molecules in a CD45-deficient BW5147 T cell line. We show here that the phosphatase activity of CD45 was not required for apoptosis triggering after cross-linking of the molecule. It is noteworthy that a revertant of the CD45-negative BW5147 cell line, expressing a truncated form of CD45 lacking most of the cytoplasmic domain, was also susceptible to CD45-mediated death. Moreover, we also demonstrate that leukocyte phosphatase-associated phosphoprotein expression is totally dispensable for CD45-mediated apoptosis to occur. Taken together, these results strongly suggest a role for the extracellular and/or the transmembrane portion of CD45 in apoptosis signaling, which contrasts with the previously reported functions for CD45 in T lymphocytes.