RESUMEN
Type V collagen (Col V) is a "minor" component of normal lung extracellular matrix, which is subjected to decreased and abnormal synthesis in human lung infiltrating adenocarcinoma. We previously reported that a direct link between low amounts of Col V and decreased cell apoptosis may favor cancer cell growth in the mouse lung after chemical carcinogenesis. Moreover, this collagen species was able to trigger DNA fragmentation and impair survival of neoplastic cells. In this study, we have extended our investigation with the aim to obtain further evidence that the death induced by Col V-treatment is of the caspase-9 apoptotic type. We used (1) optical and electron microscopy, (2) quantitation of TUNEL-labeled cells and (3) analysis of the expression levels of Col V and selected genes coding for apoptosis-linked factors, by conventional RT-PCR. BALB/c mice were injected intraperitoneally with 1.5 g/kg body weight of urethane. After urethane injection, the animals received intranasal administration of 20 µg/20 µl of Col V every day during 2 months. We report here that Col V treatment was able to determine significant increase in Col V protein and gene expression and in the percentage of TUNEL-positive cells, to up-regulate caspase-9, resulting in low growth of tumor cells. Our data validate chemical carcinogenesis as a suitable "in vivo" model for further and more detailed studies on the molecular mechanisms of the death response induced by Col V in lung infiltrating adenocarcinoma opening new strategies for treatment.
Asunto(s)
Apoptosis , Carcinogénesis , Colágeno Tipo V/administración & dosificación , Células Endoteliales/citología , Células Epiteliales/citología , Neoplasias Pulmonares/patología , Administración Intranasal , Animales , Colágeno Tipo V/inmunología , Fragmentación del ADN , Epitelio/patología , Matriz Extracelular/metabolismo , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Uretano/efectos adversosRESUMEN
We have shown previously that collagen V (col(V)) autoimmunity is a consistent feature of atherosclerosis in human coronary artery disease and in the Apoe(-/-) mouse model. We have also shown sensitization of Apoe(-/-) mice with col(V) to markedly increase the atherosclerotic burden, providing evidence of a causative role for col(V) autoimmunity in atherosclerotic pathogenesis. Here we sought to determine whether induction of immune tolerance to col(V) might ameliorate atherosclerosis, providing further evidence for a causal role for col(V) autoimmunity in atherogenesis and providing insights into the potential for immunomodulatory therapeutic interventions. Mucosal inoculation successfully induced immune tolerance to col(V) with an accompanying reduction in plaque burden in Ldlr(-/-) mice on a high-cholesterol diet. The results therefore demonstrate that inoculation with col(V) can successfully ameliorate the atherosclerotic burden, suggesting novel approaches for therapeutic interventions. Surprisingly, tolerance and reduced atherosclerotic burden were both dependent on the recently described IL-35 and not on IL-10, the immunosuppressive cytokine usually studied in the context of induced tolerance and amelioration of atherosclerotic symptoms. In addition to the above, using recombinant protein fragments, we were able to localize two epitopes of the α1(V) chain involved in col(V) autoimmunity in atherosclerotic Ldlr(-/-) mice, suggesting future courses of experimentation for the characterization of such epitopes.
Asunto(s)
Aterosclerosis/prevención & control , Autoinmunidad , Colágeno Tipo V/uso terapéutico , Hipersensibilidad Tardía/prevención & control , Tolerancia Inmunológica , Interleucinas/metabolismo , Administración Intranasal , Animales , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/metabolismo , Aterosclerosis/etiología , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Bovinos , Células Cultivadas , Colágeno Tipo V/administración & dosificación , Colágeno Tipo V/química , Colágeno Tipo V/genética , Dieta Occidental/efectos adversos , Mapeo Epitopo , Humanos , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/metabolismo , Hipersensibilidad Tardía/fisiopatología , Inmunidad Mucosa , Interleucinas/antagonistas & inhibidores , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bazo/inmunología , Bazo/metabolismo , Bazo/patologíaRESUMEN
RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by progressive scarring and matrix deposition. Recent reports highlight an autoimmune component in IPF pathogenesis. We have reported anti-col(V) immunity in IPF patients. The objective of our study was to determine the specificity of col(V) expression profile and anti-col(V) immunity relative to col(I) in clinical IPF and the efficacy of nebulized col(V) in pre-clinical IPF models. METHODS: Col(V) and col(I) expression profile was analyzed in normal human and IPF tissues. C57-BL6 mice were intratracheally instilled with bleomycin (0.025 U) followed by col(V) nebulization at pre-/post-fibrotic stage and analyzed for systemic and local responses. RESULTS: Compared to normal lungs, IPF lungs had higher protein and transcript expression of the alpha 1 chain of col(V) and col(I). Systemic anti-col(V) antibody concentrations, but not of anti-col(I), were higher in IPF patients. Nebulized col(V), but not col(I), prevented bleomycin-induced fibrosis, collagen deposition, and myofibroblast differentiation. Col(V) treatment suppressed systemic levels of anti-col(V) antibodies, IL-6 and TNF-α; and local Il-17a transcripts. Compared to controls, nebulized col(V)-induced tolerance abrogated antigen-specific proliferation in mediastinal lymphocytes and production of IL-17A, IL-6, TNF-α and IFN-γ. In a clinically relevant established fibrosis model, nebulized col(V) decreased collagen deposition. mRNA array revealed downregulation of genes specific to fibrosis (Tgf-ß, Il-1ß, Pdgfb), matrix (Acta2, Col1a2, Col3a1, Lox, Itgb1/6, Itga2/3) and members of the TGF-ß superfamily (Tgfbr1/2, Smad2/3, Ltbp1, Serpine1, Nfkb/Sp1/Cebpb). CONCLUSIONS: Anti-col(V) immunity is pathogenic in IPF, and col(V)-induced tolerance abrogates bleomycin-induced fibrogenesis and down regulates TGF- ß-related signaling pathways.
Asunto(s)
Colágeno Tipo V/inmunología , Tolerancia Inmunológica , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/inmunología , Transcripción Genética , Factor de Crecimiento Transformador beta/genética , Animales , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Bleomicina/efectos adversos , Colágeno Tipo I/inmunología , Colágeno Tipo V/administración & dosificación , Colágeno Tipo V/genética , Colágeno Tipo V/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/inmunología , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Mediadores de Inflamación/metabolismo , Activación de Linfocitos/inmunología , Ratones , Nebulizadores y Vaporizadores , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
BACKGROUND: Tolerance to collagen structures has been shown to inhibit the progression of autoimmune scleroderma and rheumatoid arthritis. More recently, tolerance induction to collagen type V (colV) in experimental models of lung transplantation was shown to ameliorate the complex pathology known as "chronic rejection." The link between colV autoimmunity and progressive graft dysfunction and subsequent development of bronchiolitis obliterans syndrome (BOS) has been established in human lung transplant recipients. We hypothesized that intravenous injection of colV inhibits development of lung fibrosis in a bleomycin-induced lung injury mouse model. METHODS: Experimental animals were injected intravenously with saline or colV 10 days before intratracheal instillation of bleomycin. Pulmonary inflammation was monitored and quantified for the presence of cells in the bronchoalveolar lavage (BAL) fluid by flow cytometry and histology of lung tissue. RESULTS: ColV-pre-treated animals showed a significant reduction in lung inflammation compared with non-treated animals, according to histology and morphometry. The number of inflammatory cells in the BAL fluid was significantly reduced and associated with a lower proportion of gammadelta T cells and CD4(+) T cells in the colV-pre-treated group. Matrix metalloproteinase-2 and -9 (MMP-2 and -9; also known as gelatinase A and gelatinase B, respectively) levels in the BAL fluid were significantly reduced in colV-pre-treated mice compared with the non-treated mice. In addition, intravenous injection of colV was associated with a significant reduction in the relative expression of interleukin (IL)-6, IL-17 and IL-22 in cells present in BAL fluid at 7 and 14 days after bleomycin instillation. CONCLUSIONS: Pre-treatment by intravenous injection of colV inhibits bleomycin-induced pulmonary fibrosis by inhibiting IL-6 and IL-17 production. Fibrosis treatment in this context therefore should target induction of colV tolerance and Th17 development.
Asunto(s)
Bleomicina/efectos adversos , Colágeno Tipo V/uso terapéutico , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/prevención & control , Animales , Autoinmunidad/fisiología , Colágeno Tipo V/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Inyecciones Intravenosas , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Trasplante de Pulmón , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Fibrosis Pulmonar/metabolismoRESUMEN
INTRODUCTION: The precise role of the remodeling process and possible therapies for bronchiolitis obliterans remain to be established. OBJECTIVE: [corrected] In the present study, we sought to validate the importance of nasal collagen V tolerance to verify whether bronchovascular axis remodeling could be reverted by this therapeutic approach when compared to steroid treatment. METHODS: Mice were randomly divided into 4 groups: control, bronchiolitis obliterans, collagen V tolerance, and prednisone groups. Morphometry was employed to evaluate bronchovascular axis dimensions, collagen density, and immune cell response. Collagen V nasal tolerance and steroid-treated mice showed significantly lower values of terminal bronchiole wall thickness and reduction in peribronchovascular cells; bronchioalveolar lymphoid tissue; and CD3+, CD4+, CD8+, and CD20+ lymphocytes. A significant decrease in CD68+ macrophage density was found in prednisone-treated mice. In addition, a strong quantitative relationship was found between collagen V tolerance, and reduction in density of immune cells and collagen. RESULTS: Our results indicate that bronchovascular axis remodeling in bronchiolitis obliterans can be reverted by collagen V nasal tolerance, possibly as the result of T-cell suppression. CONCLUSION: We concluded that the tolerance effects in this model were strongly related to the improvement in bronchovascular remodeling, and these may be an appropriate targets for further prospective studies on nasal collagen V tolerance.
Asunto(s)
Bronquios/efectos de los fármacos , Bronquiolitis Obliterante/inmunología , Colágeno Tipo V/administración & dosificación , Linfocitos T/inmunología , Animales , Bronquios/inmunología , Bronquiolitis Obliterante/patología , Colágeno Tipo V/inmunología , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Instilación de Medicamentos , Ratones , Ratones Endogámicos BALB C , Distribución AleatoriaRESUMEN
Our aim was to study skin remodeling and autoantibody production in an experimental model of scleroderma (SSc), following nasal tolerance with human type V collagen (Col V). Female New Zealand rabbits (n = 12) were immunized with two doses of 1 mg/ml of Col V in complete Freund's adjuvant and additional two boosters in incomplete Freund's adjuvant to induce SSc. After 150 days, half of these immunized rabbits were submitted to type V collagen-induced tolerance receiving a daily nasal administration of 25 mug of Col V. Control animals (n = 6) were only submitted to type V collagen-induced tolerance. Serial skin biopsies were performed on days 0, 150 and 210, and stained with H&E, Masson's trichrome and Picrosirius for morphological and morphometric analysis. Types I, III and V collagen were identified by immunofluorescence. The animals' serum samples were collected to determine anti types I, III, IV and V collagen and antinuclear antibodies (ANA). Skin biopsies from immunized animals confirmed SSc morphology as previously described, such as progressive decrease of papillary dermis, appendages atrophy, increased type I, III and V collagen deposition. Rabbits with Col V-induced nasal tolerance showed reduction of skin involvement, with significant decrease of collagen amount. Humoral immune response did not change with nasal tolerance. Collagen V nasal tolerance promotes regression of skin remodeling process in an experimental model of SSc. We suggest that nasal tolerance with type V collagen can be a promising therapeutic option to treat scleroderma patients.
Asunto(s)
Colágeno Tipo V/efectos adversos , Colágeno Tipo V/uso terapéutico , Esclerodermia Sistémica/tratamiento farmacológico , Administración Intranasal , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Anticuerpos Antinucleares/inmunología , Anticuerpos Antinucleares/metabolismo , Biopsia , Colágeno/inmunología , Colágeno/metabolismo , Colágeno Tipo V/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Conejos , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Piel/metabolismo , Piel/patologíaRESUMEN
INTRODUCTION: The precise role of the remodeling process and possible therapies for bronchiolitis obliterans remain to be established. OBJETIVE: In the present study, we sought to validate the importance of nasal collagen V tolerance to verify whether bronchovascular axis remodeling could be reverted by this therapeutic approach when compared to steroid treatment. METHODS: Mice were randomly divided into 4 groups: control, bronchiolitis obliterans, collagen V tolerance, and prednisone groups. Morphometry was employed to evaluate bronchovascular axis dimensions, collagen density, and immune cell response. Collagen V nasal tolerance and steroid-treated mice showed significantly lower values of terminal bronchiole wall thickness and reduction in peribronchovascular cells; bronchioalveolar lymphoid tissue; and CD3+, CD4+, CD8+, and CD20+ lymphocytes. A significant decrease in CD68+ macrophage density was found in prednisone-treated mice. In addition, a strong quantitative relationship was found between collagen V tolerance, and reduction in density of immune cells and collagen. RESULTS: Our results indicate that bronchovascular axis remodeling in bronchiolitis obliterans can be reverted by collagen V nasal tolerance, possibly as the result of T-cell suppression. CONCLUSION: We concluded that the tolerance effects in this model were strongly related to the improvement in bronchovascular remodeling, and these may be an appropriate targets for further prospective studies on nasal collagen V tolerance.
INTRODUÇÃO: A participação precisa do processo de remodelamento e possíveis implicações no tratamento da bronquiolite obliterante ainda não está estabelecida. OBJETIVOS: Estabelecer a importância da tolerância nasal induzida pelo colágeno do tipo V e verificar se o processo de remodelamento do eixo broncovascular pode ser revertido com esta estratégia terapêutica comparada ao efeito do tratamento com esteróides. MATERIAL E MÉTODO: Camundongos foram divididos em quatro grupos: controle, bronquiolite obliterante, tolerância nasal com colágeno do tipo V e prednisona. Morfometria foi realizada para avaliar as dimensões do eixo broncovascular, densidade de colágeno e resposta imunocelular. Camundongos submetidos à tolerância nasal com colágeno do tipo V e tratados com prednisona exibiram significativas reduções da espessura da parede de bronquíolos terminais, da densidade de células inflamatórias ao redor do eixo peribroncovascular e da resposta imunocelular às custas de linfócitos CD3, CD4, CD8 e CD20. Houve também significativa redução da densidade de macrófagos CD68 nos camundongos tratados com prednisona. Adicionalmente, houve uma forte associação entre tolerância nasal induzida pelo colágeno do tipo V, resposta imunocelular e redução do conteúdo de colágeno peribroncovascular. RESULTADOS: O remodelamento do eixo broncovascular na bronquiolite obliterante pode ser revertido pela indução de tolerância nasal com o colágeno do tipo V, possivelmente como resultado de supressão de linfócitos T. CONCLUSÃO: Os efeitos da tolerância nasal no presente modelo estiveram fortemente relacionados à melhora no remodelamento do eixo broncovascular, despontando como um alvo promissor para estudos prospectivos.