RESUMEN

The role of innate immunity in the pathogenesis of asthma is unclear. Although increased presence of neutrophils is associated with persistent asthma and asthma exacerbations, how neutrophils participate in the pathogenesis of asthma remains controversial. In this study, we show that the absence of dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease found in neutrophils, dampens the acute inflammatory response and the subsequent mucous cell metaplasia that accompanies the asthma phenotype induced by Sendai virus infection. This attenuated phenotype is accompanied by a significant decrease in the accumulation of neutrophils and the local production of CXCL2, TNF, IL-1beta, and IL-6 in the lung of infected DPPI-/- mice. Adoptive transfer of DPPI-sufficient neutrophils into DPPI-/- mice restored the levels of CXCL2 and enhanced cytokine production on day 4 postinfection and subsequent mucous cell metaplasia on day 21 postinfection. These results indicate that DPPI and neutrophils play a critical role in Sendai virus-induced asthma phenotype as a result of a DPPI-dependent neutrophil recruitment and cytokine response.

Asunto(s)

Catepsina C/fisiología , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Neutrófilos/patología , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/patología , Virus Sendai/inmunología , Animales , Asma/enzimología , Asma/inmunología , Asma/patología , Catepsina C/deficiencia , Catepsina C/genética , Inflamación/enzimología , Inflamación/inmunología , Inflamación/virología , Masculino , Metaplasia/enzimología , Metaplasia/inmunología , Metaplasia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/enzimología , Mucosa Respiratoria/enzimología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Infecciones por Respirovirus/enzimología , Linfocitos T Citotóxicos/enzimología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología

RESUMEN

RATIONALE: Different sensitivities to profibrotic compounds such as bleomycin are observed among mouse strains. OBJECTIVES: To identify genetic factors contributing to the outcome of lung injury. METHODS: Physiological comparison of C57BL/6 (sensitive) and BALB/c (resistant) mice challenged by intratracheal bleomycin instillation revealed several early differences: global gene expression profiles were thus established from lungs derived from the two strains, in the absence of any bleomycin administration. MEASUREMENTS AND MAIN RESULTS: Expression of 25 genes differed between the two strains. Among them, two molecules, not previously associated with pulmonary fibrosis, were identified. The first corresponded to dipeptidyl-peptidase I (DPPI), a cysteine peptidase (also known as cathepsin C) essential for the activation of serine proteinases produced by immune/inflammatory cells. The second corresponded to tissue inhibitor of matrix metalloproteinase-3, which also inhibits members of the ADAM (a disintegrin and metalloproteinase) family, such as the tumor necrosis factor-converting enzyme. In functional studies performed in the bleomycin-induced lung fibrosis model, the level of expression of these two genes was closely correlated with specific early events associated with lung fibrosis, namely activation of polymorphonuclear neutrophil-derived serine proteases and tumor necrosis factor-alpha-dependent inflammatory syndrome. Surprisingly, genetic deletion of DPPI in the context of a C57BL/6 genetic background did not protect against bleomycin-mediated fibrosis, suggesting additional function(s) for this key enzyme. CONCLUSIONS: This study highlights the importance of the early inflammatory events that follow bleomycin instillation in the development of lung fibrosis, and describes for the first time the roles that DPPI and tissue inhibitor of matrix metalloproteinase-3 may play in this process.

Asunto(s)

Bleomicina , Catepsina C/metabolismo , Neumonía/inducido químicamente , Neumonía/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animales , Apoptosis , Bleomicina/administración & dosificación , Líquido del Lavado Bronquioalveolar/química , Catepsina C/deficiencia , Eosinofilia/inducido químicamente , Eosinofilia/etiología , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Instilación de Medicamentos , Interleucina-5/metabolismo , Pulmón/metabolismo , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C/genética , Ratones Endogámicos BALB C/metabolismo , Ratones Endogámicos C57BL/genética , Ratones Endogámicos C57BL/metabolismo , Ratones Noqueados , Neutrófilos/enzimología , Análisis de Secuencia por Matrices de Oligonucleótidos , Neumonía/fisiopatología , Serina Endopeptidasas/metabolismo , Especificidad de la Especie , Tráquea , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

BACKGROUND & AIMS: We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. METHODS: We injected TxA into ileal loops in PAR(2) or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR(2) and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. RESULTS: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR(2) deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%-28% and tissue and fluid myeloperoxidase by 31%-71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. CONCLUSIONS: PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.

Asunto(s)

Toxinas Bacterianas , Catepsina C/metabolismo , Enteritis/inducido químicamente , Enterotoxinas , Péptido Hidrolasas/metabolismo , Receptor PAR-2/metabolismo , Animales , Toxinas Bacterianas/farmacología , Catepsina C/deficiencia , Células Cultivadas , Colon/citología , Colon/metabolismo , Enteritis/etiología , Enteritis/metabolismo , Enteritis/patología , Enterotoxinas/farmacología , Granulocitos/patología , Ileítis/etiología , Íleon/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/patología , Ratones , Ratones Noqueados , Sistema Nervioso/metabolismo , Peroxidasa/metabolismo , Receptor PAR-2/deficiencia , Receptores de Neuroquinina-1/metabolismo , Tripsina/metabolismo , Inhibidores de Tripsina/farmacología , Triptasas/farmacología , Regulación hacia Arriba

RESUMEN

Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease critical for the activation of granule-associated serine proteases, including neutrophil elastase, cathepsin G, and proteinase 3. DPPI and granule-associated serine proteases have been shown to play a key role in regulating neutrophil recruitment at sites of inflammation. It has recently been suggested that neutrophils and neutrophil-associated proteases may also be important in the development and progression of abdominal aortic aneurysms (AAAs), a common vascular disease associated with chronic inflammation and destructive remodeling of aortic wall connective tissue. Here we show that mice with a loss-of-function mutation in DPPI are resistant to the development of elastase-induced experimental AAAs. This is in part because of diminished recruitment of neutrophils to the elastase-injured aortic wall and impaired local production of CXC-chemokine ligand (CXCL) 2. Furthermore, adoptive transfer of wild-type neutrophils is sufficient to restore susceptibility to AAAs in DPPI-deficient mice, as well as aortic wall expression of CXCL2. In addition, in vivo blockade of CXCL2 by using neutralizing antibodies directed against its cognate receptor leads to a significant reduction in aortic dilatation. These findings suggest that DPPI and/or granule-associated serine proteases are necessary for neutrophil recruitment into the diseased aorta and that these proteases act to amplify vascular wall inflammation that leads to AAAs.

Asunto(s)

Aneurisma de la Aorta Abdominal/enzimología , Catepsina C/metabolismo , Infiltración Neutrófila/inmunología , Traslado Adoptivo , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/patología , Catepsina C/deficiencia , Quimiocinas CXC/biosíntesis , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/citología , Neutrófilos/enzimología , Elastasa Pancreática , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

Papillon-Lefevre syndrome is characterized by increased susceptibility to early-onset periodontitis and is caused by mutations in the cathepsin C gene. How deficiency of the enzyme relates to an increased periodontal infection risk is still not entirely clear. One possibility is that the deficiency leads to changes in the structure of the periodontal tissues as a result of which its barrier function to pathogens is compromised. We studied the structure of the periodontium in 9-month-old cathepsin C-deficient mice (cathepsin C(-/-)) and compared this with age-matched wild-type mice. Our observations showed that the overall structure of the gingiva, periodontal ligament, alveolar process, and cementum layer are normal in cathepsin C(-/-) mice, with one exception, namely that epithelial rests of Malassez in the periodontal ligament of the cathepsin C(-/-) mice are slightly enlarged. In both experimental and control animals, we noted cyst formation in rests of Malassez. No signs of periodontal infection were observed. It is concluded that cathepsin C deficiency does not lead to major changes in the structure of the periodontium.

Asunto(s)

Catepsina C/deficiencia , Periodoncio/patología , Proceso Alveolar/patología , Animales , Quistes/patología , Cemento Dental/patología , Epitelio/patología , Encía/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ligamento Periodontal/patología

RESUMEN

A variety of neutral serine proteases are important for the effector functions of immune cells. The neutrophil-derived serine proteases cathepsin G and neutrophil elastase are implicated in the host defense against invading bacterial and fungal pathogens. Likewise, the cytotoxic lymphocyte and NK cell granule-associated granzymes A and B are important for the elimination of virus-infected cells. The activation of many of these serine proteases depends on the N-terminal processing activity of the lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI). Although mice deficient in DPPI have defects in serine protease activation in multiple cellular compartments, the role of DPPI for human serine protease activation is largely undefined. Papillon-Lefevre syndrome (PLS) is a rare autosomal recessive disease associated with loss-of-function mutations in the DPPI gene locus. In this study, we established that the loss of DPPI activity is associated with severe reduction in the activity and stability of neutrophil-derived serine proteases. Surprisingly, patients with PLS retain significant granzyme activities in a cytotoxic lymphocyte compartment (lymphokine-activated killer) and have normal lymphokine-activated killer-mediated cytotoxicity against K562 cells. Neutrophils from patients with PLS do not uniformly have a defect in their ability to kill Staphylococcus aureus and Escherichia coli, suggesting that serine proteases do not represent the major mechanism used by human neutrophils for killing common bacteria. Therefore, this study defines the consequences of DPPI deficiency for the activation of several immune cell serine proteases in humans, and provides a molecular explanation for the lack of a generalized T cell immunodeficiency phenotype in patients with PLS.

Asunto(s)

Catepsina C/deficiencia , Catepsina C/genética , Enfermedad de Papillon-Lefevre/enzimología , Enfermedad de Papillon-Lefevre/genética , Adolescente , Adulto , Actividad Bactericida de la Sangre , Catepsina C/sangre , Catepsina G , Catepsinas/sangre , Catepsinas/deficiencia , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Activación Enzimática/genética , Femenino , Tamización de Portadores Genéticos , Granzimas , Humanos , Células Asesinas Activadas por Linfocinas/inmunología , Elastasa de Leucocito/sangre , Elastasa de Leucocito/deficiencia , Masculino , Mutación Missense , Mieloblastina , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/microbiología , Enfermedad de Papillon-Lefevre/inmunología , Serina Endopeptidasas/sangre , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/metabolismo

RESUMEN

Recent evidence suggests that protease release by neutrophils in the bone marrow may contribute to hematopoietic progenitor cell (HPC) mobilization. Matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE), and cathepsin G (CG) accumulate in the bone marrow during granulocyte colony-stimulating factor (G-CSF) treatment, where they are thought to degrade key substrates including vascular cell adhesion molecule-1 (VCAM-1) and CXCL12. To test this hypothesis, HPC mobilization was characterized in transgenic mice deficient in one or more hematopoietic proteases. Surprisingly, HPC mobilization by G-CSF was normal in MMP-9-deficient mice, NE x CG-deficient mice, or mice lacking dipeptidyl peptidase I, an enzyme required for the functional activation of many hematopoietic serine proteases. Moreover, combined inhibition of neutrophil serine proteases and metalloproteinases had no significant effect on HPC mobilization. VCAM-1 expression on bone marrow stromal cells decreased during G-CSF treatment of wild-type mice but not NE x CG-deficient mice, indicating that VCAM-1 cleavage is not required for efficient HPC mobilization. G-CSF induced a significant decrease in CXCL12 alpha protein expression in the bone marrow of Ne x CG-deficient mice, indicating that these proteases are not required to down-regulate CXCL12 expression. Collectively, these data suggest a complex model in which both protease-dependent and -independent pathways may contribute to HPC mobilization.

Asunto(s)

Endopeptidasas/deficiencia , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/citología , Animales , Médula Ósea/metabolismo , Médula Ósea/ultraestructura , Catepsina C/deficiencia , Catepsina C/genética , Catepsina G , Catepsinas/deficiencia , Catepsinas/genética , Quimiocina CXCL12 , Quimiocinas CXC/biosíntesis , Ensayo de Unidades Formadoras de Colonias , Endopeptidasas/genética , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Humanos , Interleucina-8/genética , Interleucina-8/farmacología , Metaloproteinasa 9 de la Matriz/sangre , Metaloproteinasa 9 de la Matriz/deficiencia , Metaloproteinasa 9 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Neutrófilos/fisiología , Inhibidores de Proteasas/farmacología , Receptores de Quimiocina/biosíntesis , Proteínas Recombinantes/farmacología , Serina Endopeptidasas , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

Leukocyte recruitment in inflammation is critical for host defense, but excessive accumulation of inflammatory cells can lead to tissue damage. Neutrophil-derived serine proteases (cathepsin G [CG], neutrophil elastase [NE], and proteinase 3 [PR3]) are expressed specifically in mature neutrophils and are thought to play an important role in inflammation. To investigate the role of these proteases in inflammation, we generated a mouse deficient in dipeptidyl peptidase I (DPPI) and established that DPPI is required for the full activation of CG, NE, and PR3. Although DPPI(-/-) mice have normal in vitro neutrophil chemotaxis and in vivo neutrophil accumulation during sterile peritonitis, they are protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen. Specifically, there is no accumulation of neutrophils in the joints of DPPI(-/-) mice. This protective effect correlates with the inactivation of neutrophil-derived serine proteases, since NE(-/-) x CG(-/-) mice are equally resistant to arthritis induction by anti-collagen antibodies. In addition, protease-deficient mice have decreased response to zymosan- and immune complex-mediated inflammation in the subcutaneous air pouch. This defect is accompanied by a decrease in local production of TNF-alpha and IL-1 beta. These results implicate DPPI and polymorphonuclear neutrophil-derived serine proteases in the regulation of cytokine production at sites of inflammation.

Asunto(s)

Artritis Experimental/enzimología , Artritis Experimental/etiología , Catepsina C/metabolismo , Neutrófilos/enzimología , Serina Endopeptidasas/metabolismo , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Catepsina C/deficiencia , Catepsina C/genética , Catepsina G , Catepsinas/deficiencia , Catepsinas/genética , Catepsinas/metabolismo , Citocinas/biosíntesis , Activación Enzimática , Hematopoyesis , Interleucina-1/biosíntesis , Elastasa de Leucocito/deficiencia , Elastasa de Leucocito/genética , Elastasa de Leucocito/metabolismo , Ratones , Ratones Noqueados , Mieloblastina , Neutrófilos/patología , Factor de Necrosis Tumoral alfa/biosíntesis

Asunto(s)

Catepsina C/deficiencia , Enfermedad de Papillon-Lefevre/enzimología , Periodontitis/enzimología , Periodontitis/etiología , Catepsina C/genética , Humanos , Enfermedad de Papillon-Lefevre/genética

Asunto(s)

Catepsina C/deficiencia , Mutación/genética , Enfermedades Periodontales/genética , Catepsina C/genética , Cromosomas Humanos Par 11/genética , Humanos

RESUMEN

Papillon-Lefèvre syndrome, or keratosis palmoplantaris with periodontopathia (PLS, MIM 245000), is an autosomal recessive disorder that is mainly ascertained by dentists because of the severe periodontitis that afflicts patients. Both the deciduous and permanent dentitions are affected, resulting in premature tooth loss. Palmoplantar keratosis, varying from mild psoriasiform scaly skin to overt hyperkeratosis, typically develops within the first three years of life. Keratosis also affects other sites such as elbows and knees. Most PLS patients display both periodontitis and hyperkeratosis. Some patients have only palmoplantar keratosis or periodontitis, and in rare individuals the periodontitis is mild and of late onset. The PLS locus has been mapped to chromosome 11q14-q21 (refs 7, 8, 9). Using homozygosity mapping in eight small consanguineous families, we have narrowed the candidate region to a 1.2-cM interval between D11S4082 and D11S931. The gene (CTSC) encoding the lysosomal protease cathepsin C (or dipeptidyl aminopeptidase I) lies within this interval. We defined the genomic structure of CTSC and found mutations in all eight families. In two of these families we used a functional assay to demonstrate an almost total loss of cathepsin C activity in PLS patients and reduced activity in obligate carriers.

Asunto(s)

Periodontitis Agresiva/enzimología , Periodontitis Agresiva/genética , Catepsina C/deficiencia , Catepsina C/genética , Enfermedad de Papillon-Lefevre/enzimología , Enfermedad de Papillon-Lefevre/genética , Mutación Puntual , Periodontitis Agresiva/patología , Secuencia de Bases , Cromosomas Humanos Par 11/genética , Cartilla de ADN/genética , ADN Complementario/genética , Exones , Femenino , Genes Recesivos , Ligamiento Genético , Humanos , Intrones , Masculino , Repeticiones de Microsatélite , Enfermedad de Papillon-Lefevre/patología , Linaje