RESUMEN
OBJECTIVE: To investigate the related risk factors for bronchiolitis obliterans (BO) in children with mycoplasma pneumonia (MP) bronchiolitis. METHOD: The clinical data of 227 children with MP bronchiolitis who were admitted to the II Department of Respiratory of Children's Hospital of Hebei Province from January 2018 to June 2020 were retrospectively analyzed. According to the sequelae of BO, they were divided into 32 cases in the BO group and 195 cases in the non-BO group. The univariate analysis was performed on the clinical and laboratory parameters of the two groups, and the multifactor logistic regression was performed further to determine the independent risk factors for the occurrence of BO in MP bronchiolitis, and then, the cut-off value with the maximum diagnostic value of indicators was found through the ROC curve analysis. RESULTS: The results of univariate and multivariate logistic regression analysis showed that the independent risk factors for the occurrence of BO in MP bronchioles were longer duration of moist rales (OR = 1.203, P = 0.003), higher levels of serum lactate dehydrogenase (LDH) (OR = 1.005, P = 0.036), hypoxemia (OR = 7.442, P = 0.035), and pleural effusion (OR = 4.437, P = 0.004). The area under the ROC curve was 78.2%, 72.0%, 68.2%, and 71.0%, respectively (P < 0.001). The cut-off value of duration of moist rales and levels of serum LDH are 7.5 d and 330 U/L, respectively. CONCLUSION: Children with MP bronchiolitis with high serum LDH level (≥330 U/L), combined with hypoxemia, pleural effusion, and lung wet rale duration (≥7.5 d), may be more prone to BO, in which lung wet rale duration prediction value is the largest. Among them, duration of pulmonary moist rales has the highest predictive value.
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Bronquiolitis Obliterante/etiología , Bronquiolitis/complicaciones , Neumonía por Mycoplasma/complicaciones , Adolescente , Bronquiolitis/enzimología , Bronquiolitis/microbiología , Bronquiolitis Obliterante/enzimología , Bronquiolitis Obliterante/microbiología , Niño , Preescolar , Biología Computacional , Femenino , Humanos , Hipoxia/complicaciones , Lactante , L-Lactato Deshidrogenasa/sangre , Modelos Logísticos , Masculino , Análisis Multivariante , Mycoplasma pneumoniae , Derrame Pleural/complicaciones , Neumonía por Mycoplasma/enzimología , Curva ROC , Estudios Retrospectivos , Factores de RiesgoRESUMEN
BACKGROUND: Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. METHODS: MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. RESULTS: MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. CONCLUSIONS: These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.
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Bronquiolitis Obliterante/enzimología , Fibroblastos/enzimología , Trasplante de Pulmón/efectos adversos , Pulmón/enzimología , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Adolescente , Adulto , Anciano , Animales , Bronquiolitis Obliterante/tratamiento farmacológico , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/patología , Proliferación Celular , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Francia , Humanos , Interleucina-6/metabolismo , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/cirugía , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/farmacología , Repitelización , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Regulación hacia Arriba , Adulto JovenRESUMEN
Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center (GC)-promoting T follicular helper (Tfh) cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ (PI3Kδ), a lipid kinase, is critical for activated T cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T cells that become Tfh cells is required for cGVHD in a nonsclerodermatous multiorgan system disease model that includes bronchiolitis obliterans (BO), dependent upon GC B cells, Tfhs, and counterbalanced by T follicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow-derived GC B cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443, that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of US Food and Drug Administration (FDA)-approved PI3Kδ inhibitors for cGVHD therapy in patients.
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Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Animales , Linfocitos B/inmunología , Trasplante de Médula Ósea/efectos adversos , Bronquiolitis Obliterante/tratamiento farmacológico , Bronquiolitis Obliterante/enzimología , Bronquiolitis Obliterante/etiología , Enfermedad Crónica , Fosfatidilinositol 3-Quinasa Clase I/deficiencia , Fosfatidilinositol 3-Quinasa Clase I/genética , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Esclerodermia Localizada/tratamiento farmacológico , Esclerodermia Localizada/enzimología , Esclerodermia Localizada/etiología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
BACKGROUND: Bronchiolitis obliterans syndrome (BOS), the major cause of death on lung transplantation, is characterized by bronchiolar inflammation and tissue remodeling. Matrix metalloproteinases (MMPs) have been implicated in these processes, although it is still unclear whether MMP activity and binding to their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), is abnormal in BOS. METHODS: We studied total MMP-1,-2,-3,-7,-8,-9,-12,-13 levels, their activity state using activity-based extraction and their binding to TIMP-1, -2, -3, and -4 in bronchoalveolar lavage (BAL) of lung transplant recipients with good outcome and BOS using a multiplex immunoassay. RESULTS: The BAL levels of TIMP-1 and -2 and MMP-2, -3, -7, -8, and -9 were significantly increased in BOS compared to good outcome recipients. Interestingly, activity of MMP-7, but none of the other MMPs, was detected in good outcome recipients, whereas no active MMPs were observed in BOS recipients. However, BAL levels of TIMP-bound MMP-8 and -9 were higher in BOS than in good outcome recipients, suggesting activity of these MMPs in an earlier stage. CONCLUSIONS: We demonstrate that development of BOS is associated with increased levels of TIMP-1 and -2 and total MMP-2, -3, -7, -8, and -9. Although active MMP-7 was only observed in good outcome recipients, levels of TIMP-bound MMP-8 and -9 were higher in BOS. By enabling profiling of active and TIMP-bound MMPs, our novel method may open opportunities for the screening of early predictors for BOS.
Asunto(s)
Bronquiolitis Obliterante/enzimología , Trasplante de Pulmón/efectos adversos , Metaloproteinasas de la Matriz/análisis , Proteómica , Adulto , Biomarcadores/análisis , Bronquiolitis Obliterante/diagnóstico , Bronquiolitis Obliterante/etiología , Líquido del Lavado Bronquioalveolar/química , Femenino , Humanos , Inmunoensayo , Masculino , Persona de Mediana Edad , Proteómica/métodos , Estudios Retrospectivos , Inhibidores Tisulares de Metaloproteinasas/análisis , Resultado del TratamientoRESUMEN
OBJECTIVE: Azithromycin has become a standard of care in therapy of bronchiolitis obliterans following lung transplantation. Matrix metalloprotease-9 broncho-alveolar lavage levels increase in airway neutrophilia and bronchiolitis obliterans. Interleukin-17 may play a role in lung allograft rejection, and interleukin-12 is downregulated in bronchiolitis obliterans. Whether these mechanisms can be targeted by azithromycin remains unclear. METHODS: Bronchiolitis obliterans was induced by transplantation of Fischer F344 rat left lungs to Wistar Kyoto rats. Allografts with azithromycin therapy from day 1 to 28 or 56 and mono- or combination therapy with the broad-spectrum matrix metalloprotease inhibitor tanomastat from day 1 to 56 were compared to control allografts and isografts. Graft histology was assessed, and tissue cytokine expression studied using Western blotting and immunofluorescence. RESULTS: The chronic airway rejection score in the azithromycin group did not change between 4 and 8 weeks after transplantation, whereas it significantly worsened in control allografts (P = .041). Azithromycin+tanomastat prevented complete allograft fibrosis, which occurred in 40% of control allografts. Azithromycin reduced interleukin-17 expression (P = .049) and the number of IL-17(+)/CD8(+) lymphocytes at 4 weeks, and active matrix metalloprotease-9 at 8 weeks (P = .017), and increased interleukin-12 expression (P = .025) at 8 weeks following transplantation versus control allografts. CONCLUSIONS: The expression of interleukin-17 and matrix metalloprotease-9 in bronchiolitis obliterans may be attenuated by azithromycin, and the decrease in interleukin-12 expression was prevented by azithromycin. Combination of azithromycin with a matrix metalloprotease inhibitor is worth studying further because it prevented complete allograft fibrosis in this study.
Asunto(s)
Azitromicina/farmacología , Compuestos de Bifenilo/farmacología , Bronquiolitis Obliterante/tratamiento farmacológico , Pulmón/efectos de los fármacos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Fenilbutiratos/farmacología , Animales , Bronquiolitis Obliterante/enzimología , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/patología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Fibrosis , Supervivencia de Injerto/efectos de los fármacos , Interleucina-12/metabolismo , Interleucina-17/metabolismo , Pulmón/enzimología , Pulmón/patología , Pulmón/cirugía , Trasplante de Pulmón , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratas Endogámicas F344 , Ratas Endogámicas WKY , Factores de TiempoRESUMEN
Obliterative bronchiolitis after lung transplantation is a chronic inflammatory and fibrotic condition of small airways. The fibrosis associated with obliterative bronchiolitis might be reversible. Matrix metalloproteinases (MMPs) participate in inflammation and tissue remodeling. MMP-2 localized to myofibroblasts in post-transplant human obliterative bronchiolitis lesions and to allograft fibrosis in a rat intrapulmonary tracheal transplant model. Small numbers of infiltrating T cells were also observed within the fibrosis. To modulate inflammation and tissue remodeling, the broad-spectrum MMP inhibitor SC080 was administered after the allograft was obliterated, starting at post-transplant day 21. The allograft lumen remained obliterated after treatment. Only low-dose (2.5 mg/kg per day) SC080 significantly reduced collagen deposition, reduced the number of myofibroblasts and the infiltration of T cells in association with increased collagenolytic activity, increased MMP-2 gene expression, and decreased MMP-8, MMP-9, and MMP-13 gene expression. In in vitro experiments using cultured myofibroblasts, a relatively low concentration of SC080 increased MMP-2 activity and degradation of type I collagen. Moreover, coculture with T cells facilitated persistence of myofibroblasts, suggesting a role for T-cell infiltration in myofibroblast persistence in fibrosis. By combining low-dose SC080 with cyclosporine in vivo at post-transplant day 28, partial reversal of obliterative fibrosis was observed at day 42. Thus, modulating MMP activity might reverse established allograft airway fibrosis by regulating inflammation and tissue remodeling.
Asunto(s)
Bronquiolitis Obliterante/enzimología , Trasplante de Pulmón , Metaloproteinasas de la Matriz/metabolismo , Complicaciones Posoperatorias/enzimología , Adulto , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Animales , Bronquiolitis Obliterante/patología , Bronquiolitis Obliterante/prevención & control , Ciclosporina/administración & dosificación , Quimioterapia Combinada , Inhibidores Enzimáticos/administración & dosificación , Femenino , Fibrosis , Humanos , Inmunosupresores/administración & dosificación , Pulmón/patología , Masculino , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/administración & dosificación , Metaloproteinasas de la Matriz/fisiología , Miofibroblastos/efectos de los fármacos , Complicaciones Posoperatorias/prevención & control , Ratas , Trasplante HomólogoRESUMEN
BACKGROUND: Long-term survival after lung transplantation is hindered by the development of bronchiolitis obliterans syndrome (BOS), and recent evidence suggests that dysregulated epithelial repair may underlie its development. Because matrix metalloproteinase (MMP) -2 and MMP-9 secretion is integral to repair, we hypothesized that airway epithelial cells from patients with BOS would over-express these matrix-degrading enzymes. METHODS: Cells obtained from bronchial and bronchiolar brushings from patients with and without BOS (without acute rejection or infection) were analyzed via quantitative polymerase chain reaction and immunocytochemistry for MMP-2, and MMP-9 gene and protein expression. The expression of tissue inhibitor of metalloproteinase (TIMP)2 and TIMP1 was also assessed. MMP activity in bronchoalveolar lavage was determined via gelatin zymography. RESULTS: MMP-2 and MMP-9 production was significantly higher in bronchoalveolar lavage (3.85- and 11.59-fold, p < 0.001) and airway epithelium (MMP-2 bronchial: 6.33-fold, bronchiolar: 3.57-fold, both p < 0.001; MMP-9 bronchial: 32.55-fold, p < 0.001; bronchiolar: 8.60-fold, p = 0.01) in patients with BOS, but expression in patients without BOS was not different from healthy controls. TIMP expression was similar in patients with and without BOS. Immunostaining confirmed that the airway epithelium was a direct source of MMP-2 and MMP-9 expression in patients with BOS. CONCLUSION: In patients with BOS, the airway epithelium over-expresses MMPs, even in the absence of acute rejection or infection. Dysregulated epithelial repair may be a key feature of BOS.
Asunto(s)
Bronquios/citología , Bronquiolitis Obliterante/enzimología , Trasplante de Pulmón , Adolescente , Adulto , Lavado Broncoalveolar , Broncoscopía , Epitelio/metabolismo , Femenino , Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/cirugía , Inmunohistoquímica , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/cirugía , Enfisema Pulmonar/cirugía , Reacción en Cadena en Tiempo Real de la Polimerasa , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Adulto JovenRESUMEN
Obliteration of the small airways is a largely unresolved challenge in pulmonary medicine. It represents either the irreversible cause of functional impairment or a morphologic disorder of limited importance in a multitude of diseases. Bronchiolitis obliterans is a key complication of lung transplantation. No predictive markers for the onset of obliterative remodeling are currently available. To further elucidate the molecular mechanisms of airway remodeling, compartment-specific expression patterns were analyzed in patients. For this purpose, remodeled and nonremodeled bronchioli were isolated from transplanted and nontransplanted lung explants using laser-assisted microdissection (n = 24). mRNA expression of 45 fibrosis-associated genes was measured using quantitative real-time RT-PCR. For 20 genes, protein expression was also analyzed by immunohistochemistry. Infiltrating cells were characterized at conventional histology and immunohistochemistry. Obliterative remodeling of the small airways in transplanted and nontransplanted lungs shared similar grades of chronic inflammation and pivotal fibrotic pathways such as transforming growth factor ß signaling and increased collagen expression. Bone morphogenetic protein and thrombospondin signaling, and also matrix metalloproteinases and tissue inhibitor of metalloproteinases, were primarily up-regulated in obliterative airway remodeling in nontransplanted lungs. In transplanted lungs, clinical remodeled bone morphogenetic protein but nonremodeled bronchioli were characterized by a concordant up-regulation of matrix metalloproteinase-9, RANTES, and tissue inhibitor of metalloproteinase-1. These distinct expression patterns warrant further investigation as potential markers of impending airway remodeling, especially for prospective longitudinal molecular profiling.
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Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Bronquiolitis Obliterante/fisiopatología , Trasplante de Pulmón , Pulmón/metabolismo , Pulmón/fisiopatología , Transducción de Señal , Adulto , Remodelación de las Vías Aéreas (Respiratorias)/genética , Biomarcadores/metabolismo , Biopsia , Bronquiolos/patología , Bronquiolos/fisiopatología , Bronquiolitis Obliterante/enzimología , Bronquiolitis Obliterante/genética , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Pulmón/patología , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Smad/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismoAsunto(s)
Bronquiolitis Obliterante/inmunología , Quimiotaxis de Leucocito , Metaloproteinasa 8 de la Matriz/fisiología , Animales , Bronquiolitis Obliterante/enzimología , Células Cultivadas/fisiología , Colágeno , Geles , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/fisiología , Tráquea/trasplante , Trasplante HeterotópicoRESUMEN
Increased levels of MMP-8 (neutrophil collagenase) have been reported in OB, but the biological role of MMP-8 in OB is not known. MMP-8 is an interstitial collagenase highly expressed by polymorphonuclear leukocytes, which are prominent in early OB. Here, we show that MMP-8 promotes migration of PMNs through the collagen-rich matrix in a mouse heterotopic airway transplant model of OB. Overall, MMP-8(-/-) mice had significantly fewer PMNs in the airway lumen 2 and 14 days post-transplantation, and the percentage of PMNs traversing the matrix to the lumen was decreased markedly in the MMP-8(-/-) compared with WT mice at 14 days. There were significantly more PMNs outside of the lumen in the ECM in the MMP-8(-/-) mice compared with WT mice. In vitro, significantly fewer MMP-8(-/-) PMNs migrated through 3D cross-linked collagen gels than WT PMNs. MMP inhibitor GM6001 was also able to impede migration of WT PMNs through collagen gels. The decreased migration was likely a result of pericollagenase activity of MMP-8, as WT PMNs expressing MMP-8 were not able to migrate effectively through collagen that was resistant to the collagenase. Protection from OB was seen in the MMP-8(-/-) mice, as the airway lumen had significantly less obliteration and collagen deposition, suggesting that MMP-8 plays an important role in the pathogenesis of OB.
Asunto(s)
Bronquiolitis Obliterante/inmunología , Quimiotaxis de Leucocito/fisiología , Metaloproteinasa 8 de la Matriz/fisiología , Animales , Células Sanguíneas/fisiología , Células de la Médula Ósea/fisiología , Bronquiolitis Obliterante/enzimología , Células Cultivadas/fisiología , Colágeno , Matriz Extracelular , Femenino , Geles , Metaloproteinasa 8 de la Matriz/deficiencia , Metaloproteinasa 8 de la Matriz/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/fisiología , Tráquea/trasplante , Trasplante HeterotópicoRESUMEN
BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), an enzyme involved in the degradation of tryptophan (Try) to kynurenine (Kyn), is thought to suppress T-cell activity. Although a few experimental studies have suggested a role for IDO in graft acceptance, human data are scarce and inconclusive. We sought to establish whether, in lung transplant recipients (LTRs), plasma IDO activity mirrors the level of graft acceptance. METHODS: We measured the plasma Kyn/Try ratio, reflecting IDO activity, by high-performance liquid chromatography (HPLC) in 90 LTRs, including 26 patients who were still functionally/clinically stable for >36 post-transplant months (stable LTRs) and 64 LTRs with bronchiolitis obliterans syndrome (BOS, Grades 0-p to 3). Twenty-four normal healthy controls (NHCs) were also included. RESULTS: The Kyn/Try ratio in stable LTRs resembled that observed in NHCs, whereas, unexpectedly, patients with BOS, who had lower counts of peripheral CD4(+) T-regulatory cells and tolerogenic plasmacytoid dendritic cells than stable LTRs, showed an increased plasma Kyn/Try ratio compared with both NHCs and stable LTRs. IDO expression by in vitro-stimulated peripheral blood mononuclear cells (PBMC) did not vary between BOS and stable LTRs. Furthermore, BOS patients displayed signs of chronic systemic inflammation (increased plasma levels of interleukin-8 and tumor necrosis factor-alpha) and higher T-cell activation (increased frequency of peripheral interferon-gamma-producing clones). CONCLUSIONS: Our results suggest that, in vivo, in lung transplantation, plasma IDO activity does not reflect the degree of lung graft acceptance, but instead is correlated with the degree of chronic inflammation.
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Indolamina-Pirrol 2,3,-Dioxigenasa/sangre , Trasplante de Pulmón/fisiología , Tolerancia al Trasplante/fisiología , Trasplante Homólogo/fisiología , Biomarcadores/sangre , Bronquiolitis Obliterante/sangre , Bronquiolitis Obliterante/enzimología , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Estudios de Seguimiento , Humanos , Interleucina-8/sangre , Quinurenina/sangre , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/enzimología , Valores de Referencia , Subgrupos de Linfocitos T/enzimología , Factores de Tiempo , Triptófano/sangreRESUMEN
Bronchiolitis obliterans syndrome (BOS) is characterized by persistent alloreactive, infective and non-specific epithelial injury, loss of epithelial integrity and dysregulated repair. We have reported increased apoptosis of epithelial cells collected from the large airway in lung transplant recipients. As part of the alloreactive response, T cells induce apoptosis of target epithelial cells by secreting granzyme b. We hypothesized that granzyme b would be increased in lung transplant patients with acute rejection and BOS and that commonly used immunosuppressive agents would fail to suppress this serine protease adequately. We investigated intracellular T cell granzyme b in blood, bronchoalveolar lavage (BAL) and large airway brushing (23 controls, 29 stable transplant, 23 BOS, 28 acute rejection, 31 infection) using flow cytometry and assessed the effect of clinically relevant concentrations of cyclosporin A, tacrolimus, methylprednisolone and a protease inhibitor, gabexate mesilate, on in vitro granzyme b production. Granzyme b was increased significantly in all compartments of all transplant groups compared to controls. Surprisingly, granzyme b was even higher in patients with BOS than in patients with acute rejection. In longitudinal analysis in three patients, blood granzyme b increased prior to or at the onset of BOS. In vitro, methylprednisolone and gabexate mesilate had no effect and cyclosporin A and tacrolimus only a moderate effect on production of granzyme b by CD8(+) T cells. Increased T cell granzyme b production may contribute to BOS pathogenesis and is not curtailed by current immunosuppressants. Longitudinal investigation of granzyme b in blood may provide an adjunctive non-invasive method for predicting BOS/OB.
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Bronquiolitis Obliterante/enzimología , Granzimas/metabolismo , Inmunosupresores/uso terapéutico , Trasplante de Pulmón/inmunología , Subgrupos de Linfocitos T/enzimología , Adulto , Anciano , Bronquiolitis Obliterante/tratamiento farmacológico , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Broncoscopía/métodos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo/métodos , Rechazo de Injerto/enzimología , Rechazo de Injerto/inmunología , Granzimas/biosíntesis , Humanos , Inmunosupresores/farmacología , Estudios Longitudinales , Trasplante de Pulmón/efectos adversos , Masculino , Persona de Mediana Edad , Subgrupos de Linfocitos T/efectos de los fármacosRESUMEN
Obliterative bronchiolitis (OB) is the histopathological finding in chronic lung allograft rejection. Mounting evidence suggests that epithelial damage drives the development of airway fibrosis in OB. Tissue inhibitor of metalloproteinase (TIMP)-1 expression increases in lung allografts and is associated with the onset of allograft rejection. Furthermore, in a mouse model of OB, airway obliteration is reduced in TIMP-1-deficient mice. Matrilysin (matrix metallproteinase-7) is essential for airway epithelial repair and is required for the re-epithelialization of airway wounds by facilitating cell migration; therefore, the goal of this study was to determine whether TIMP-1 inhibits re-epithelialization through matrilysin. We found that TIMP-1 and matrilysin co-localized in the epithelium of human lungs with OB and both co-localized and co-immunoprecipitated in wounded primary airway epithelial cultures. TIMP-1-deficient cultures migrated faster, and epithelial cells spread to a greater extent compared with wild-type cultures. TIMP-1 also inhibited matrilysin-mediated cell migration and spreading in vitro. In vivo, TIMP-1 deficiency enhanced airway re-epithelialization after naphthalene injury. Furthermore, TIMP-1 and matrilysin co-localized in airway epithelial cells adjacent to the wound edge. Our data demonstrate that TIMP-1 interacts with matrix metalloproteinases and regulates matrilysin activity during airway epithelial repair. Furthermore, we speculate that TIMP-1 overexpression restricts airway re-epithelialization by inhibiting matrilysin activity, contributing to a stereotypic injury response that promotes airway fibrosis via bronchiole airway epithelial damage and obliteration.
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Células Epiteliales/fisiología , Metaloproteinasa 7 de la Matriz/fisiología , Regeneración , Mucosa Respiratoria/patología , Inhibidor Tisular de Metaloproteinasa-1/fisiología , Animales , Bronquiolitis Obliterante/inducido químicamente , Bronquiolitis Obliterante/enzimología , Bronquiolitis Obliterante/patología , Línea Celular , Movimiento Celular , Células Cultivadas , Activación Enzimática , Células Epiteliales/enzimología , Humanos , Pulmón/enzimología , Masculino , Ratones , Ratones Noqueados , Naftalenos , Unión Proteica , Mucosa Respiratoria/enzimología , Inhibidor Tisular de Metaloproteinasa-1/genéticaRESUMEN
Obliterative bronchiolitis (OB) is thought to be a form of chronic allograft rejection. However, immunosuppressive therapy is not effective once fibrosis has developed. We hypothesize that disordered tissue remodeling is a mechanism for the pathogenesis of OB. We examined allograft airway fibrosis in an intrapulmonary tracheal transplant model of OB. Allograft airways were completely obliterated at day 21 by fibrotic tissue; however, tissue remodeling continued thereafter, as demonstrated by the change of collagen deposition density, shift from type I to type III collagen, shift from fibroblasts to myofibroblasts and shift of expression profiles and activities of matrix metalloproteinases (MMPs). We then used a broad-spectrum MMP inhibitor, SC080, to attempt to manipulate tissue remodeling. Administration of the MMP inhibitor from day 0 to day 28 reduced airway obliteration, without inhibiting T-cell activation. MMP inhibition from day 14 to day 28 showed similar effects on airway obliteration. MMP inhibition from day 21 to day 35 did not reverse the airway obliteration, but significantly reduced the collagen deposition, type III collagen and myofibroblasts in the lumen. We conclude that tissue remodeling plays a critical role in the development and maintenance of fibrosis after transplantation.
Asunto(s)
Bronquiolitis Obliterante/patología , Rechazo de Injerto/patología , Pulmón/patología , Metaloproteinasas de la Matriz/metabolismo , Tráquea/patología , Tráquea/trasplante , Animales , Bronquiolitis Obliterante/enzimología , Bronquiolitis Obliterante/prevención & control , Fibrosis , Rechazo de Injerto/enzimología , Rechazo de Injerto/prevención & control , Pulmón/enzimología , Activación de Linfocitos , Masculino , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Ratas , Ratas Endogámicas , Linfocitos T/inmunología , Tráquea/enzimología , Trasplante HomólogoRESUMEN
Parenchymal disease in the allograft lung is associated with interstitial remodeling believed to be mediated by matrix metalloproteinases (MMPs). Recent studies suggest high levels of MMP-9 are associated with bronchiolitis obliterans syndrome (BOS) in lung transplant recipients. Since BOS occurs late in the posttransplant period and may be preceded by episodes of acute rejection or infection, which are associated with interstitial remodeling, we examined MMP profiles in allograft bronchoalveolar lavage (BAL) fluid in the early posttransplant period (preceding BOS). Gelatin zymography, protein array analysis and specific ELISA on BAL fluids from transplanted lungs indicated that MMP-8, MMP-9 and TIMP-1 were strongly expressed in allograft BAL fluid from stable patients, or those with infection or rejection compared to BAL fluid from normal volunteers. Elevated expression of MMP-8, MMP-9 and TIMP-1 occurred early, and was sustained for the 3.2 years covered in this study. Elevations of MMP-8, MMP-9 and TIMP-1 in the first 2 years posttransplant appear to be associated with lung transplantation itself, and not infection or rejection. These data suggest that ongoing and clinically silent MMP activity could perpetuate progressive disease in the allograft lung.
Asunto(s)
Bronquiolitis Obliterante/enzimología , Trasplante de Pulmón/fisiología , Metaloproteasas/metabolismo , Complicaciones Posoperatorias/enzimología , Biomarcadores/metabolismo , Bronquiolitis Obliterante/diagnóstico , Lavado Broncoalveolar , Ensayo de Inmunoadsorción Enzimática , Humanos , Trasplante de Pulmón/efectos adversos , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Periodo Posoperatorio , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Trasplante HomólogoRESUMEN
Obliterative bronchiolitis (OB) develops insidiously in nearly half of all lung transplant recipients. Although typically preceded by a CD8(+) T cell-rich lymphocytic bronchitis, it remains unresponsive to conventional immunosuppression. Using an airflow permissive model to study the role of gases flowing over the transplanted airway, it is shown that prolonged inhalation of sublethal doses of carbon monoxide (CO), but not nitric oxide (NO), obliterate the appearance of the obstructive airway lesion. Induction of the enzyme responsible for the synthesis of CO, heme oxygenase (Hmox) 1, increased carboxyhemoglobin levels and suppressed lymphocytic bronchitis and airway luminal occlusion after transplantation. In contrast, zinc protoporphyrin IX, a competitive inhibitor of Hmox, increased airway luminal occlusion. Compared with wild-type allografts, expression of inducible NO synthase (iNOS), which promotes the influx of cytoeffector leukocytes and airway graft rejection, was strikingly reduced by either enhanced expression of Hmox-1 or exogenous CO. Hmox-1/CO decreased nuclear factor (NF)-kappaB binding activity to the iNOS promoter region and iNOS expression. Inhibition of soluble guanylate cyclase did not interfere with the ability of CO to suppress OB, implicating a cyclic guanosine 3',5'-monophosphate-independent mechanism through which CO suppresses NF-kappaB, iNOS transcription, and OB. Prolonged CO inhalation represents a new immunosuppresive strategy to prevent OB.
Asunto(s)
Bronquiolitis Obliterante/enzimología , Monóxido de Carbono/administración & dosificación , Hemo Oxigenasa (Desciclizante)/metabolismo , Inmunosupresores/administración & dosificación , Trasplante de Pulmón , Óxido Nítrico Sintasa/biosíntesis , Animales , Bronquiolitis Obliterante/tratamiento farmacológico , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/patología , Inhibidores Enzimáticos/administración & dosificación , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/metabolismo , Rechazo de Injerto/patología , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Hemo Oxigenasa (Desciclizante)/genética , Hemo-Oxigenasa 1 , Leucocitos/enzimología , Leucocitos/patología , Trasplante de Pulmón/patología , Proteínas de la Membrana , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II , Protoporfirinas/administración & dosificación , Trasplante HomólogoRESUMEN
Bronchiolitis obliterans (BO) is a survival-limiting factor in lung transplantation. There are no common BO markers in use. Since BO is associated with extracellular matrix remodeling, we asked whether matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs) could serve as BO markers. In 72 lung transplant patients (34 BO syndrome (BOS) 0, 15 BOS 0-p, and 23 BOS 1) serum and broncho-alveolar lavage (BAL) MMP and TIMP levels were examined by ELISA. The BAL cell counts were additionally analyzed. The serum MMP-2, MMP-8, MMP-9 and TIMP-2 levels were not different in all groups. In contrast, the BAL MMP-8, -9 and TIMP-1 levels were significantly elevated in BOS 0-p (p = 0.003; p = 0.007; p = 0.0003, respectively) and BOS 1 (p = 0.003; p = 0.001; p = 0.0004, respectively) as compared to BOS 0 patients. The BAL MMP-8, -9 and TIMP-1 levels were significant predictors of BOS 0-p (p = 0.01; p = 0.01; p = 0.01, respectively) and BOS-1 (p = 0.007; p = 0.01; p = 0.006, respectively) in receiver operating characteristic analysis. Except for BAL macrophages that were significantly decreased in BOS 0-p versus BOS 0 patients; other cell counts were not different between the groups. BAL MMP-8, -9 and TIMP-1 might be useful markers to detect BO in lung transplant patients.
Asunto(s)
Biomarcadores/metabolismo , Bronquiolitis Obliterante/enzimología , Bronquiolitis Obliterante/etiología , Líquido del Lavado Bronquioalveolar/química , Enfermedades Pulmonares/enzimología , Trasplante de Pulmón/efectos adversos , Metaloproteinasas de la Matriz/metabolismo , Adulto , Anciano , Bronquiolitis Obliterante/diagnóstico , Estudios Transversales , Ensayo de Inmunoadsorción Enzimática , Humanos , Enfermedades Pulmonares/sangre , Enfermedades Pulmonares/terapia , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Sensibilidad y Especificidad , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
BACKGROUND: Epithelial cell injury, inflammation, fibrosis, and airway obliteration are associated in post-transplant obliterative bronchiolitis. Fibrosis is a consequence of fibroblastic activity and of collagen deposition after disturbances in the balance of protein formation and degradation. Proteolytic enzymes such as the matrix metalloproteinases mediate degradation. To assess matrix metalloproteinases during obliterative bronchiolitis development, we studied porcine, heterotopic bronchial allografts. METHODS: A total of 119 allografts or autografts were harvested serially at 3 to 60 days after transplantation and processed for histology and in situ hybridization for matrix metalloproteinases 2 and 9. Immunocytochemistry for vimentin and alpha-smooth-muscle-cell actin was performed with specific antibodies. RESULTS: Implants had initial ischemic injury to airway epithelium and to the bronchial wall. Recovery was rapid in autografts and in immunosuppressed allografts. In matrix metalloproteinase-2 mRNA activity in fibroblasts, correlation with endothelial expression and expression in macrophages occurred during intense fibroproliferation. We observed intense matrix metalloproteinase-9 positivity during onset of inflammation and fibroproliferation in endothelial cells (p < 0.01), fibroblasts (p < 0.05), macrophages (p < 0.05), and lymphocytes (p < 0.05). Matrix metalloproteinase-9 mRNA activity in fibroblasts correlated with that in endothelial and inflammatory cells and also proved predictive of early obliteration. CONCLUSIONS: Matrix metalloproteinase-2, and especially matrix metalloproteinase-9, gene activity was associated with onset of inflammation and fibroblastic proliferation in allografts, predicting early obliteration. Although this may be the case in the model described, its role in human-allograft post-transplant obliterative bronchiolitis requires further supportive data.
Asunto(s)
Bronquiolitis Obliterante/enzimología , Trasplante de Pulmón/efectos adversos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Animales , Biomarcadores/metabolismo , Bronquios/enzimología , Bronquios/patología , Bronquios/trasplante , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/patología , Proliferación Celular , Modelos Animales de Enfermedad , Fibroblastos/enzimología , Fibroblastos/patología , Inmunohistoquímica , Hibridación in Situ , Trasplante de Pulmón/patología , Linfocitos/enzimología , Linfocitos/patología , Macrófagos/enzimología , Macrófagos/patología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mucosa Respiratoria/enzimología , Mucosa Respiratoria/patología , PorcinosRESUMEN
This study was designed to define the roles of matrix metalloproteinase (MMP)-2 and MMP-9 in obliterative airway disease (OAD) in heterotopic murine tracheal allografts, considered a suitable animal model for chronic lung allograft rejection. BALB/c tracheal allografts were transplanted into MMP-2-deficient (-/-) and MMP-9-/- mice. Also, wild-type recipients were treated with doxycycline, a nonspecific MMP inhibitor. After 10, 20 and 30 days, allografts were analyzed for OAD development, intragraft levels of MMP-2 and MMP-9 and the frequency and cytokine/chemokine production profile of alloreactive T cells. Allografts transplanted into wild-type mice developed OAD lesions within 30 days. These allografts revealed significant upregulation of both MMP-2 and MMP-9. Allografts transplanted into MMP-9-/- and doxycycline-treated recipients did not develop OAD. In contrast, allografts transplanted into MMP-2-/- mice developed OAD lesions with normal kinetics. Interestingly, MMP-9-/- recipients showed an enhanced T cell alloreactivity associated with an abnormal profile of cytokine/chemokine production. The enhanced T cell alloreactivity in MMP-9-/- mice was mediated by enhanced dendritic cell stimulatory capacity as well as enhanced T cell responsive capacity. These results suggest that MMP-9 plays an important role in the pathogenesis of OAD and may represent a target for the therapeutic intervention of chronic lung allograft rejection.
Asunto(s)
Obstrucción de las Vías Aéreas/prevención & control , Bronquiolitis Obliterante/prevención & control , Metaloproteinasa 9 de la Matriz/deficiencia , Tráquea/trasplante , Obstrucción de las Vías Aéreas/enzimología , Obstrucción de las Vías Aéreas/inmunología , Obstrucción de las Vías Aéreas/metabolismo , Animales , Bronquiolitis Obliterante/enzimología , Bronquiolitis Obliterante/inmunología , Bronquiolitis Obliterante/metabolismo , Doxiciclina/farmacología , Inmunosupresores/farmacología , Metaloproteinasa 2 de la Matriz/deficiencia , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factores de Tiempo , Tráquea/enzimología , Tráquea/inmunología , Trasplante HomólogoRESUMEN
Bronchiolitis obliterans syndrome (BOS) is a severe complication after lung transplantation (LTX). In a retrospective cohort study 12 stable healthy recipients (non-BOS) and eight patients with BOS were enrolled after LTX and matrix metalloproteinases (MMP)-9, TIMP-1 and cell characteristics in bronchoalveolar lavage (BAL) samples (n = 145) were analysed. BALs from patients with BOS were further divided according to whether they were obtained before (pre-BOS) or after manifestation of BOS (BOS group). The MMP-9/TIMP-1 ratio was significantly increased in the BOS group compared with non-BOS or pre-BOS; furthermore, the ratio was negatively correlated with forced expiratory volume in one second. In zymography, the active form of MMP-9 was detected predominantly in the BOS group. In addition, zymography showed the banding pattern of neutrophil-derived MMP-9, indicating that polymorphonuclear neutrophils (PMNs) were the main source of MMP-9. According to that, MMP-9 was significantly correlated with the number of PMN. In immunocytochemistry, MMP-9 was also associated predominantly with PMN. This is the first study to evaluate the expression of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinases-1 over time during manifestation of a fibroproliferative lung disease in patients. It demonstrates development of bronchiolitis obliterans syndrome after lung transplantation is associated with an imbalance of matrix metalloproteinases-9/tissue inhibitors of metalloproteinase-1 ratio.