RESUMO
The integrin alpha9 subunit forms a single heterodimer, alpha9beta1. The alpha9 subunit is most closely related to the alpha4 subunit, and like alpha4 integrins, alpha9beta1 plays an important role in leukocyte migration. The alpha4 cytoplasmic domain preferentially enhances cell migration and inhibits cell spreading, effects that depend on interaction with the adaptor protein, paxillin. To determine whether the alpha9 cytoplasmic domain has similar effects, a series of chimeric and deleted alpha9 constructs were expressed in Chinese hamster ovary cells and tested for their effects on migration and spreading on an alpha9beta1-specific ligand. Like alpha4, the alpha9 cytoplasmic domain enhanced cell migration and inhibited cell spreading. Paxillin also specifically bound the alpha9 cytoplasmic domain and to a similar level as alpha4. In paxillin(-/-) cells, alpha9 failed to inhibit cell spreading as expected but surprisingly still enhanced cell migration. Further, mutations that abolished the alpha9-paxillin interaction prevented alpha9 from inhibiting cell spreading but had no effect on alpha9-dependent cell migration. These findings suggest that the mechanisms by which the cytoplasmic domains of integrin alpha subunits enhance migration and inhibit cell spreading are distinct and that the alpha9 and alpha4 cytoplasmic domains, despite sequence and functional similarities, enhance cell migration by different intracellular signaling pathways.
Assuntos
Movimento Celular/fisiologia , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Cadeias alfa de Integrinas , Integrinas/metabolismo , Fosfoproteínas/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Células CHO , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Cricetinae , Proteínas do Citoesqueleto/farmacologia , Adesões Focais/fisiologia , Integrinas/genética , Paxilina , Fosfoproteínas/farmacologia , Mutação Puntual/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The integrin alpha9beta1 is expressed on epithelial cells, smooth muscle cells, skeletal muscle, and neutrophils and recognizes at least three distinct ligands: vascular cell adhesion molecule 1 (VCAM-1), tenascin-C, and osteopontin. The alpha9 subunit is structurally similar to the integrin alpha4 subunit, and alpha9beta1 and alpha4beta1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of alpha4 integrins to ligands, would also be a functional inhibitor of alpha9beta1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of alpha9beta1-mediated adhesion to VCAM-1 and of neutrophil migration across tumor necrosis factor-activated endothelial cells. A peptide containing a novel MLDG motif shared by both of these disintegrins also inhibited alpha9beta1- and alpha4beta1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of alpha9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other alpha9beta1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SVVYGLR, which we have previously shown inhibit binding of alpha9beta1-expressing cells to tenascin-C and osteopontin, respectively, had no effect on adhesion to VCAM-1. These data suggest that there are structurally distinct requirements for interactions of the alpha9beta1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.
Assuntos
Desintegrinas/farmacologia , Integrinas/metabolismo , Sialoglicoproteínas/metabolismo , Tenascina/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Venenos de Víboras/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Dimerização , Desintegrinas/química , Desintegrinas/isolamento & purificação , Desintegrinas/metabolismo , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Humanos , Integrinas/antagonistas & inibidores , Integrinas/genética , Dados de Sequência Molecular , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Osteopontina , Fragmentos de Peptídeos/farmacologia , Alinhamento de Sequência , Análise de Sequência de Proteína , Especificidade por Substrato , Venenos de Víboras/química , Venenos de Víboras/isolamento & purificação , Venenos de Víboras/metabolismoRESUMO
Oxidant/antioxidant imbalance is thought to be involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Therefore, antioxidants, such as superoxide dismutase (SOD), are expected to have an inhibitory potential against IPF. To elucidate whether a lecithinized SOD (phosphatidylcholine [PC]-SOD) has the potential to be a new therapeutic agent for IPF, we investigated the inhibitory effects of PC-SOD at doses of 1 mg/kg/d (low dose) and 10 mg/kg/d (high dose) and of methylprednisolone (mPSL) on bleomycin (BLM)-induced pulmonary fibrosis in mice. Histopathologic evaluation and lung hydroxyproline content revealed that the severity of fibrosis was attenuated in mice treated with low-dose PC-SOD, whereas no significant effect was observed in other mice. In bronchoalveolar lavage fluid on Day 1 after treatment with BLM, BLM-induced increases in total cell number, populations of lymphocytes and neutrophils, and expression of messenger RNA for interleukin-1beta and platelet-derived growth factor (PDGF)-A were significantly suppressed in PC-SOD-treated mice. The suppression of PDGF-A expression was significantly greater in mice treated with low-dose PC-SOD than in mice treated with high-dose PC-SOD or mPSL. In summary, this study demonstrated the inhibitory effects of low-dose PC-SOD on the development of pulmonary fibrosis, which indicates the potential usefulness of PC-SOD as a new treatment agent for IPF or at least for BLM-induced pulmonary fibrosis in humans.
Assuntos
Bleomicina , Fosfatidilcolinas/farmacologia , Fibrose Pulmonar/prevenção & controle , Superóxido Dismutase/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Hidroxiprolina/análise , Interleucina-1/metabolismo , Pulmão/metabolismo , Masculino , Metilprednisolona/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fibrose Pulmonar/induzido quimicamenteRESUMO
OBJECTIVE: We previously observed increased expression of interleukin-1beta, platelet-derived growth factor-A, and insulin-like growth factor-I in bronchoalveolar lavage (BAL) cells during the development of pulmonary fibrosis after an intraperitoneal administration of bleomycin in mice. The purpose of this study was to investigate the roles of tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 in this model. METHODOLOGY: We investigated the mRNA expression levels of TNF-alpha and TGF-beta1 in BAL cells of Institute for Cancer Research mice after 10 days of the intraperitoneal administration of bleomycin with or without treatment with a specific neutrophil elastase inhibitor, ONO-5046 x Na. RESULTS: On day 1 but not on days 15 and 29, the relative amount of TGF-beta1 mRNA in the bleomycin-treated mice was significantly decreased compared with control mice. In the mice treated with both bleomycin and ONO-5046 x Na intermediate values for TGF-beta1 were obtained. No significant differences in TNF-alpha expression were observed in any of the treatment groups. CONCLUSIONS: These results suggest that a reduced expression of TGF-beta1 in BAL cells in the early phase may be important during the development of murine pulmonary fibrosis induced by an intraperitoneal administration of bleomycin.
Assuntos
Líquido da Lavagem Broncoalveolar/química , Fibrose Pulmonar/metabolismo , Fator de Crescimento Transformador beta/análise , Fator de Necrose Tumoral alfa/análise , Animais , Biomarcadores/análise , Bleomicina , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Injeções Intraperitoneais , Camundongos , Probabilidade , Fibrose Pulmonar/induzido quimicamente , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Estatísticas não ParamétricasRESUMO
The integrin alpha(9)beta(1) mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH(2)-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for alpha(9)beta(1). In this report, we used site-directed mutagenesis and synthetic peptides to identify the alpha(9)beta(1) recognition sequence in osteopontin. alpha(9)-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH(2)-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-alpha(9) monoclonal antibody Y9A2, indicating the presence of a non-RGD alpha(9)beta(1) recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on alpha(9)beta(1)-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr(165), blocked alpha(9)beta(1)-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin beta(1) subunit on alpha(9)-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to alpha(9)beta(1) and is responsible for alpha(9)beta(1)-mediated cell adhesion to the NH(2)-terminal fragment of osteopontin.
Assuntos
Integrinas/metabolismo , Sialoglicoproteínas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Células CHO , Cricetinae , Humanos , Integrinas/química , Integrinas/genética , Dados de Sequência Molecular , Mutação , Osteopontina , Ligação Proteica , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Trombina/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.
Assuntos
Adesão Celular/fisiologia , Quimiotaxia de Leucócito , Endotélio Vascular/fisiologia , Cadeias alfa de Integrinas , Integrinas/fisiologia , Neutrófilos/fisiologia , Molécula 1 de Adesão de Célula Vascular/fisiologia , Animais , Antígenos CD/genética , Antígenos CD/fisiologia , Células CHO , Células Cultivadas , Cricetinae , Humanos , Técnicas In Vitro , Integrina alfa4 , Integrinas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Transfecção , Veias Umbilicais , Molécula 1 de Adesão de Célula Vascular/farmacologiaRESUMO
Telomerase maintains telomere length and is considered to be necessary for the indefinite proliferation of human cells. Telomerase activity is detected not only in germline and immortal cancer cells, but also in stem/progenitor cells of renewal tissues and activated lymphocytes. While it is generally agreed that telomerase is a useful tumor marker, the utility of telomerase activity in non-cancerous cells should also be considered. In the present study, we quantitatively examined telomerase activity in 56 cytology samples and 106 bronchoalveolar lavage samples obtained from patients with various respiratory diseases. Fourteen of 34 samples obtained from lung cancer patients showed detectable telomerase activity, while only 7 of 128 samples obtained from patients without lung cancer did (p<0.001). Moreover, 12 of 14 telomerase-positive samples with lung cancer showed strong signals, while none without lung cancer did. Among 106 non-cancerous bronchoalveolar lavage samples, 4 telomerase positive samples had increased number of lymphocytes and increased disease progression. These findings indicate that evaluation of telomerase activity may not only be a useful diagnostic test for lung cancer, but may also be a marker of disease aggressiveness for immune-associated lung diseases.
Assuntos
Pneumopatias/enzimologia , Neoplasias Pulmonares/enzimologia , Telomerase/metabolismo , Biomarcadores/análise , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Pneumopatias/imunologia , Neoplasias Pulmonares/diagnósticoRESUMO
Neutrophils play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). To elucidate the possible involvement of neutrophil elastase (NE) in pulmonary fibrosis, we investigated the efficacy of a new specific NE inhibitor (ONO-5046 Na) in a murine model of human IPF, bleomycin-induced pulmonary fibrosis. Bronchoalveolar lavage (BAL) and histopathological analysis were performed on bleomycin-treated mice (group A), bleomycin and ONO-5046 Na-treated mice (group B), and saline control groups at 1, 15, and 29 d after the end of bleomycin treatment. At 29 d, multifocal fibrosis was observed in group A, whereas no fibrotic regions were observed in group B. Interleukin-1 beta and macrophage inflammatory protein-2 mRNA levels in BAL cells on day 1, and platelet-derived growth factor-A and insulin-like growth factor-1 mRNA levels on days 1 and 15, were significantly lower in group B than in group A. Thus, we demonstrated an inhibitory effect of ONO-5046. Na on pulmonary fibrosis in mice, indicating the involvement of NE in the pathogenesis of pulmonary fibrosis. We propose that this effect might be related to suppressed expression of particular cytokines in alveolar macrophages and that this specific NE inhibitor could be a novel therapeutic agent for IPF.
Assuntos
Glicina/análogos & derivados , Elastase de Leucócito/antagonistas & inibidores , Neutrófilos/enzimologia , Fibrose Pulmonar/fisiopatologia , Inibidores de Serina Proteinase/farmacologia , Sulfonamidas/farmacologia , Animais , Bleomicina , Glicina/farmacologia , Masculino , Camundongos , Fibrose Pulmonar/induzido quimicamenteRESUMO
A 36-year old woman was admitted because of painful dysesthesia of her extremities, suggesting the presence of mononeuritis multiplex. Laboratory data was almost within normal limits, with the exception of lupus anticoagulant positivity and increase of IgM level. We considered the possibility of connective tissue diseases and examined the patient accordingly. Keratoconjunctivitis sicca without dry eye symptoms, identified by rose-bengal and fluorescence testing, was the only recognizable abnormality. Oral sicca symptoms were not revealed although lip biopsy showed infiltration by a moderate number of plasma cells and lymphocytes. Under the diagnosis of subclinical Sjögren's syndrome, the following examination was carried out. Sural nerve biopsy specimens revealed wallerian degeneration and perivascular mononuclear cell infiltration of the vasa nervorum. We therefore concluded that the peripheral neuropathy was caused by subclinical Sjögren's syndrome. Magnetic resonance imaging (MRI) of the brain demonstrated multiple small lesions with increased spin echo images (T2 weighted) in the white matter. So, this patient was suffered from not only peripheral but also central nervous system complications. The mechanism of nervous system involvement was considered to be mononuclear cell-dependent ischemic damage caused by infiltration of the vasa nervorum. Both steroid pulse therapy and oral corticosteroid administration were ineffective in treatment of the peripheral neuropathy. Alternative use of cyclophosphamide (75 mg per day) was dramatically effective in relieving peripheral nervous system disorders. This was evident in the remarkable improvement of painful dysesthesia, grip strength and motor nerve conduction velocities. This case could be considered valuable for understanding the pathophysiology of Sjögren's syndrome and associated nervous system complications.
Assuntos
Doenças do Sistema Nervoso Central/etiologia , Ciclofosfamida/uso terapêutico , Doenças do Sistema Nervoso Periférico/etiologia , Síndrome de Sjogren/tratamento farmacológico , Adulto , Feminino , Humanos , Síndrome de Sjogren/complicaçõesRESUMO
A 26-year old woman, who was diagnosed as having systemic lupus erythematosus at the age of 23 year old, presented diarrhea and headache. She showed severe hypoproteinemia (serum total protein 3.7 g/dl, serum albumin 1.4 g/dl) and hyperlipidemia. She revealed to have protein-losing enteropathy with the result of alpha-1-antitrypsin clearance test using stool. Increase of prednisolone improved the loss of albumin into the bowel and abnormal laboratory findings. She also showed watershed infarction in the area of middle cerebral artery and posterior cerebral artery. Protein-losing enteropathy is a rare complication of SLE, only 18 cases are available on literature. No case is found to have cerebral infarction in patients with protein-losing enteropathy associated with SLE. It is known that blood levels of anticoagulation factors decrease in protein-losing enteropathy due to the leakage of plasma protein into intestinal lumen. Serum antithrombin III was decreased in this case. Hyperlipidemia found in this case seems to be caused by same mechanism in nephrotic syndrome. Lupus anticoagulant was also positive in this patient. These factors seems to be related to the occurrence of cerebral infarction. This case suggests the possibility of cerebral infarction in patients with protein-losing enteropathy in SLE.
Assuntos
Infarto Cerebral/etiologia , Lúpus Eritematoso Sistêmico/complicações , Enteropatias Perdedoras de Proteínas/etiologia , Adulto , Feminino , HumanosRESUMO
In 1985, McCarty et al reported 10 patients with a symmetrical synovitis affecting predominately the wrists and flexor digitorum tendon sheaths associated with marked pitting edema of the dorsum of both hands and both feet. It was insisted on the clinical entity as remitting seronegative symmetrical synovitis with pitting edema (RS3PE syndrome). These patients were mostly elderly men whose sera revealed negative rheumatoid factor and had a benign clinical course. Patients with RS3PE syndrome remitted completely within 3-36 months and the remission was maintained even after all medications were discontinued. We experienced 2 interesting cases which were similar to RS3PE syndrome. One case with SLE-like conditions evolved into RA-like conditions. On the contrary, the other which had been effectively treated as RA developed into SLE-like conditions. Both cases were seronegative and had the characteristic pitting edema of both hands and feet demonstrating the symmetrical synovitis without bony erosions. They went into complete remission by corticosteroid therapy, although it did not continue for a long time. We should consider that such cases are similar to RS3PE syndrome and must be distinct from it.
Assuntos
Artrite Reumatoide/diagnóstico , Edema/diagnóstico , Lúpus Eritematoso Sistêmico/diagnóstico , Sinovite/diagnóstico , Idoso , Artrite Reumatoide/complicações , Artrite Reumatoide/tratamento farmacológico , Diagnóstico Diferencial , Feminino , Humanos , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Pessoa de Meia-Idade , Prednisolona/uso terapêutico , SíndromeRESUMO
A 40-year old man with Behçet's disease was admitted for severe decrease of visual acuity. Since 1987, he had suffered from oral aphtha, retinitis, erythema nodosum, genital ulcer and epididymitis. He was diagnosed as complete Behçet's disease and has been administered cyclosporin A (CYA) and colchicine (Col). Because of repeated ocular attacks and reduced visual acuity, CYA was increased from 3.49 mg/kg/day (220 mg/day) to 6.35 mg/kg/day (400 mg/day) and Col, 0.5 mg/day to 1.0 mg/day. 2 weeks later, he revealed fever, generalized myalgia, muscle weakness and general fatigue, accompanying marked elevation of creatine kinase (4962 IU/l). CYA was discontinued and Col was diminished to 0.5 mg/day. The myalgia disappeared in 4 days and general conditions including creatine kinase were normalized within 2 weeks. We concluded that CYA was highly suspected of the cause of myopathy considering his clinical course.