RESUMO
We describe a mechanical method for delivery of adenoviral vector to the adventitial surface of arteries and to other tissues. Our goal was to characterize, principally in intact carotid artery, the morphological, biochemical, and functional effects of mechanical delivery of a recombinant beta-galactosidase-expressing adenoviral vector following its direct application using a small paintbrush. Our ex vivo and in vivo data demonstrate efficient, accurate, and rapid transduction of arteries without compromise of their morphological, biochemical, and functional integrity. We also demonstrate the general applicability of this technique in vivo via transduction of skeletal muscle, fibrotendinous tissue, peritoneum, serosal surface of bowel, and wounded skin. We conclude that direct mechanical delivery of an adenoviral vector to tissues using a suitable paintbrush represents an intuitive, accurate, and effective means of augmenting gene transfer efficiency, and may be a useful adjunct to other delivery methods.
Assuntos
Adenoviridae/genética , Artérias Carótidas , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Transdução Genética/métodos , Administração Tópica , Animais , Artérias Carótidas/enzimologia , Cães , Modelos Animais , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
BACKGROUND: In patients with quiescent asthma, macrophages are the most prevalent cells recovered by bronchoalveolar lavage (BAL). Through activation via their FcepsilonRII receptors or by acting as antigen-presenting cells, macrophages could, in theory, promote the late airway response to allergen. OBJECTIVE: In order to investigate the importance of macrophages and other airway luminal cells in inducing the late airway response, a novel washout experiment was designed. METHODS: Five patients with ragweed-allergic asthma underwent bronchoscopy and segmental bronchial challenge with either normal saline or short ragweed extract in two segments of one lung. In a third segment of the opposite lung, 12 successive BALs (25 mL each) were performed, followed by challenge with an identical dose of short ragweed (washed-challenged segment). After 24 h, all three challenged segments underwent BAL. RESULTS: Initially, in the washed-challenged segment, over 80% (mean 80.4%, range 68-88%) of the recoverable airway dwelling cells were removed. Unexpectedly, 24 h later these same washed-challenged segments contained more eosinophils in the BAL than the challenged segments from the opposite lung (P = 0.033). CONCLUSIONS: Removing the majority of airway luminal cells followed by allergen bronchoprovocation increased the number of eosinophils recovered 24 h after challenge. Our results suggest that in quiescent allergic asthma, the airway luminal cells are protective and attenuate the late eosinophilic response to allergen challenge.
Assuntos
Antígenos/imunologia , Asma/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Eosinófilos/imunologia , Adulto , Asma/patologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Divisão Celular , Eosinófilos/patologia , Feminino , Humanos , Contagem de Leucócitos , Pulmão/imunologia , Macrófagos/fisiologia , Masculino , Pessoa de Meia-Idade , Proteínas de Plantas/imunologia , Pólen/imunologia , Fatores de TempoRESUMO
Human eosinophil major basic protein (MBP) is strongly implicated as a mediator of disease, especially bronchial asthma. We recently isolated a highly divergent human homologue of MBP (MBPH). Given human MBP's importance in disease and the restricted expression of it and human MBPH, we isolated the 4.6-kb human MBPH gene (HGMW-approved symbol PRG3). Comparisons among the human MBP (PRG2), human MBPH, and murine MBP-1 (mMBP-1; Prg2) genes suggest that the human MBP and mMBP-1 genes are more closely related than either is to the human MBPH gene. Proximal promoters of these three genes show conservation of potential binding sites for IK2 and STAT and of a known GATA site. However, a known C/EBP site is altered in the human MBPH gene's proximal promoter. The human MBP and MBPH genes localized to chromosome 11 in the centromere to 11q12 region. Thus, the human MBP and MBPH genes have diverged considerably, probably following a gene duplication event. Furthermore, the identified conserved and distinct proximal promoter elements likely contribute to the eosinophil-restricted and relatively reduced transcription of the human MBPH gene.
Assuntos
Proteínas Sanguíneas/genética , Proteína Básica Maior de Eosinófilos , Regiões Promotoras Genéticas , Ribonucleases , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Sanguíneas/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Centrômero/ultraestrutura , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Sequência Conservada , DNA Complementar/metabolismo , Proteínas Granulares de Eosinófilos , Evolução Molecular , Éxons , Biblioteca Gênica , Humanos , Hibridização in Situ Fluorescente , Íntrons , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Gene therapy is being investigated as a putative treatment option for cardiovascular diseases, including cerebral vasospasm. Because there is presently no information regarding gene transfer to human cerebral arteries, the principal objective of this study was to characterize adenovirus-mediated expression and function of recombinant endothelial nitric oxide synthase (eNOS) gene in human pial arteries. Pial arteries (outer diameter 500 to 1,000 microm) were isolated from 30 patients undergoing temporal lobectomy for intractable seizures and were studied using histologic staining, histochemistry, electron microscopy, and isometric force recording. Gene transfer experiments were performed ex vivo using adenoviral vectors encoding genes for bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVLacZ). In transduced arteries, studied 24 hours after exposure to vectors, expression of recombinant beta-galactosidase and eNOS was detected by histochemistry, localizing mainly to the adventitia (n = 4). Immunoelectron microscopy localized recombinant eNOS in adventitial fibroblasts. During contractions to U46619, bradykinin-induced relaxations were significantly augmented in AdCMVeNOS-transduced rings compared with control and AdCMVLacZ-transduced rings (P < 0.01; n = 6). The NOS inhibitor L-nitroarginine methylester (L-NAME) caused significantly greater contraction in AdCMVeNOS-transduced rings (P < 0.001; n = 4) and inhibited bradykinin-induced relaxations in control and transduced rings (P < 0.001; n = 6). The current findings suggest that in AdCMVeNOS-transduced human pial arteries, expression of recombinant eNOS occurs mainly in adventitial fibroblasts where it augments relaxations to NO-dependent agonists such as bradykinin. Findings from the current study might be beneficial in future clinical applications of gene therapy for the treatment or prevention of cerebral vasospasm.
Assuntos
Artérias Cerebrais/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Óxido Nítrico Sintase/genética , Adenoviridae , Adolescente , Adulto , Idoso , Animais , Bovinos , Criança , Feminino , Vetores Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III , Proteínas Recombinantes/genéticaRESUMO
Human eosinophils have been reported to express both the mRNA and protein for the high affinity IgE receptor (FcepsilonRI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the FcepsilonRI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low affinity IgG receptor (FcgammaRII, CD32), but no expression of FcepsilonRI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-FcepsilonRI mAb, suggesting that eosinophils do express minimal amounts of FcepsilonRI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express approximately 0.5% of the FcepsilonRI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE.
Assuntos
Sítios de Ligação de Anticorpos , Eosinófilos/metabolismo , Hipersensibilidade/sangue , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Adolescente , Adulto , Animais , Antígenos CD/biossíntese , Antígenos CD/sangue , Basófilos/imunologia , Biomarcadores/sangue , Biotinilação , Degranulação Celular/imunologia , Eosinófilos/imunologia , Humanos , Imunoglobulina E/genética , Leucotrieno C4/biossíntese , Leucotrieno C4/sangue , Ativação Linfocitária , Camundongos , Pessoa de Meia-Idade , Receptores de IgE/biossíntese , Receptores de IgE/sangue , Receptores de IgE/fisiologia , Receptores de IgG/biossíntese , Receptores de IgG/sangue , Receptores de IgG/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Rinite Alérgica Sazonal/sangue , Rinite Alérgica Sazonal/imunologia , Superóxidos/sangueRESUMO
Eosinophils are important effector cells in defense against helminth infection and in allergic diseases. To identify novel eosinophil proteins, large scale sequencing of a cDNA library prepared from interleukin-5-stimulated umbilical cord precursor cells was performed, and the major genes expressed by maturing eosinophils were determined. This resulted in the identification of a cDNA with 64% identity to human prepro-major basic protein (hprepro-MBP). This cDNA was designated hprepro-MBP homolog (hprepro-MBPH). Interestingly, the calculated pI values for hMBPH and hMBP differed by >100-fold, with pI values of 8.7 and 11.4, respectively. Given this pronounced basicity difference, the homolog transcript's abundance (1.1%), and MBP's critical role in eosinophil biological activity, we further characterized the homolog. Reverse transcription-polymerase chain reaction detected transcription of hprepro-MBPH in bone marrow only, and this result was confirmed by analysis of a large cDNA data base (electronic Northern). hMBPH was isolated from human eosinophil granule lysates, and its identity was verified by amino acid sequencing and by mass spectrometry. Analyses of the biological activities showed that hMBPH had effects similar to hMBP in cell killing and neutrophil (superoxide anion production and interleukin-8 release) and basophil (histamine and leukotriene C4 release) stimulation assays, but usually with reduced potency. Overall, this novel homolog's unique physical properties indicated that the high net positive charge of hMBP is important but not essential for biological activity.
Assuntos
Proteínas Sanguíneas/química , Eosinófilos/química , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Ribonucleases , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/química , Proteínas Granulares de Eosinófilos , Eosinófilos/efeitos dos fármacos , Humanos , Interleucina-5/farmacologia , Dados de Sequência Molecular , Precursores de Proteínas/química , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil-specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112+/-4 ng/10(6) cells, and ECP, 163+/-2 ng/10(6) cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold-labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription-polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross-reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflammation in human disease.
Assuntos
Proteínas Sanguíneas/metabolismo , Neutrófilos/metabolismo , Proteínas/metabolismo , Ribonucleases , Biópsia , Grânulos Citoplasmáticos/metabolismo , Proteínas Granulares de Eosinófilos , Neurotoxina Derivada de Eosinófilo , Eosinófilos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Microscopia Imunoeletrônica , Reação em Cadeia da Polimerase , RNA Mensageiro/sangue , Pele/metabolismo , Transcrição GênicaRESUMO
Chronic eosinophilic pneumonitis (CEP) is characterized by longstanding respiratory symptoms accompanied by a massive pulmonary eosinophil infiltration. We hypothesized that cytokine(s) produced in the disease sites are implicated in the pathophysiology of CEP. We studied peripheral blood and bronchoalveolar lavage fluids (BALF) obtained from two lung segments of a patient with CEP. Seventy times more eosinophils were found in the BALF from an involved lung segment (showing patchy opacification on a chest roentgenogram) than from an uninvolved segment. The eosinophil-active cytokines interleukin-5 (IL-5), IL-6, and IL-10 were strikingly elevated in the BALF from the involved lung segment, whereas no or minimal levels of these cytokines were detectable in the BALF from the uninvolved segment or serum, respectively. Leukocytes in the involved lung segment, but not those in peripheral blood, expressed messenger ribonucleic acid (mRNA) for IL-5, IL-6, and IL-10. In contrast, IL-2, IL-3, IL-4, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha) were not detected in any sample. These findings suggest that increased production of several cytokines, such as IL-5, IL-6, and IL-10, in the involved lung segment, but not in the uninvolved lung segment or peripheral blood, is a critical pathophysiologic feature of CEP.
Assuntos
Citocinas/biossíntese , Eosinofilia Pulmonar/fisiopatologia , Adulto , Sequência de Bases , Líquido da Lavagem Broncoalveolar/química , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Interleucina-5/análise , Interleucina-5/biossíntese , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Eosinofilia Pulmonar/metabolismoRESUMO
To test whether eosinophil recruitment after pulmonary allergen challenge is associated with interleukin (IL)-5 in patients with asthma, we performed segmental bronchoprovocation (SBP) with saline, and with low and high dosages of ragweed extract in six patients with allergic asthma. Bronchoalveolar lavage (BAL) of the challenged segments was performed 5 min after challenge (immediate BAL fluid) and repeated 24 h later (late BAL fluid). Allergen challenge resulted in recruitment of eosinophils, and increased levels of eosinophil-active cytokines. A bioassay showed the predominant eosinophil-active cytokine in the late BAL fluids to be IL-5. Analysis of the late BAL fluids revealed that IL-5 levels correlated with the numbers of eosinophils and lymphocytes. This study provides evidence that IL-5 is a critical cytokine associated with eosinophil and lymphocyte recruitment into the airways of patients with asthma following exposure to allergen.
Assuntos
Alérgenos/efeitos adversos , Asma/imunologia , Quimiotaxia de Leucócito/imunologia , Eosinófilos/imunologia , Interleucina-5/imunologia , Linfócitos/imunologia , Ribonucleases , Adolescente , Adulto , Proteínas Sanguíneas/análise , Testes de Provocação Brônquica , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Broncoscopia , Proteínas Granulares de Eosinófilos , Humanos , Ensaio Imunorradiométrico , Mediadores da Inflamação/análise , Masculino , Elastase Pancreática/análise , RadioimunoensaioRESUMO
BACKGROUND: The eosinophilia-myalgia syndrome, caused by a contaminant or contaminants in epidemiologically implicated L-tryptophan products, is characterized by eosinophilia and eosinophil degranulation. We hypothesized that immune cells are stimulated by implicated L-tryptophan and produce eosinophil-active cytokines. OBJECTIVES: This study was designed to identify substances in L-tryptophan causing the eosinophilia-myalgia syndrome. METHODS: Peripheral blood mononuclear cells were cultured with L-tryptophan products, and supernatants were tested for their ability to enhance eosinophil degranulation and survival in vitro and for their cytokine content. Subsequently, 46 different L-tryptophan lots were analyzed for their in vitro biologic activities. RESULTS: After peripheral blood mononuclear cells were cultured with implicated L-tryptophan, their supernatants enhanced eosinophil degranulation and survival. These activities were blocked by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody; immunoreactive GM-CSF was measurable in the supernatants. Monocytes, but not T lymphocytes, were the responding cells. However, no correlation was observed between the in vitro biologic activity and lots of epidemiologically implicated L-tryptophan products. This biologic activity in the L-tryptophan products was characterized as endotoxin. CONCLUSION: Although L-tryptophan products stimulate peripheral blood mononuclear cells to produce GM-CSF, this response is caused by endotoxin contamination of the L-tryptophan products and not by a specific L-tryptophan contaminant. Endotoxin contamination must be considered as a possible cause of eosinophil-active cytokine production by peripheral blood mononuclear cells.
Assuntos
Citocinas/análise , Síndrome de Eosinofilia-Mialgia/sangue , Eosinófilos/imunologia , Leucócitos Mononucleares/imunologia , Triptofano/imunologia , Sobrevivência Celular , Células Cultivadas , Endotoxinas/química , Síndrome de Eosinofilia-Mialgia/imunologia , Eosinófilos/patologia , Humanos , Triptofano/química , Triptofano/farmacologiaRESUMO
Segmental bronchoprovocation (SBP) with allergen was used in an attempt to study eosinophils recruited to the airway 24 h after challenge. Unexpectedly, in the first four patients, neutrophils (rather than eosinophils) were recruited in the bronchoalveolar lavage (BAL) fluids, and we hypothesized that the allergen extracts were contaminated with endotoxin. The extracts used for challenge in the first four patients tested positive for bacterial endotoxin in a limulus amebocyte lysate assay. Rechallenge of one patient from the first group with a comparable dose of an endotoxin-free extract and SBP with endotoxin-free extract in five additional patients resulted in preferential recruitment of eosinophils rather than neutrophils. The number of neutrophils recovered from the challenged segments in the patients challenged with endotoxin-free extract was significantly less than that observed in the first four patients. Taken together, these observations suggest that neutrophil recruitment in the 24-h BAL fluids from the first four patients was probably due to endotoxin contamination of the allergen extract. We caution investigators that endotoxin contamination of allergen extract may alter the cellular inflammation during the late airway response following allergen challenge.
Assuntos
Alérgenos , Asma/diagnóstico , Asma/imunologia , Testes de Provocação Brônquica/efeitos adversos , Líquido da Lavagem Broncoalveolar/citologia , Contaminação de Medicamentos , Endotoxinas/efeitos adversos , Eosinófilos , Hipersensibilidade/complicações , Neutrófilos , Adolescente , Adulto , Asma/etiologia , Líquido da Lavagem Broncoalveolar/química , Feminino , Humanos , Hipersensibilidade/diagnóstico , Ensaio Imunorradiométrico , Inflamação , Contagem de Leucócitos , Elastase de Leucócito , Teste do Limulus , Masculino , Elastase Pancreática/análise , Testes Cutâneos , Fatores de TempoRESUMO
In many diseases, retrospective analysis for determining the presence of mast cells has been difficult because of their loss of metachromatic staining properties once tissue has undergone formalin fixation. We quantified mast cells in peribronchiolar tissue of idiopathic pulmonary fibrosis (IPF) and in normal human lung by using rabbit antiserum to human mast cell tryptase. In lung biopsy specimens from 15 patients with IPF, the mean number of mast cells per high-power field in connective tissue directly adjacent to the lumen of small airways (0.5 to 2 mm in diameter) and other fibrotic foci was 29.9 +/- 10.8 in comparison with 13.7 +/- 3.5 in 16 normal controls (P < 0.001). In addition, mast cells in cases of IPF had an altered appearance--irregularity of the plasma membrane and release of extracellular tryptase. We conclude that the number of mast cells is increased in IPF and that the altered appearance of the mast cells suggests that they are activated and undergoing degranulation.
Assuntos
Pulmão/imunologia , Mastócitos/enzimologia , Fibrose Pulmonar/imunologia , Serina Endopeptidases/metabolismo , Biópsia , Estudos de Casos e Controles , Contagem de Células , Linhagem Celular , Quimases , Imunofluorescência , Humanos , Pulmão/citologia , Pulmão/patologia , Mastócitos/patologia , Fibrose Pulmonar/patologia , TriptasesRESUMO
Eosinophils isolated from normal individuals were cultured in the presence of human rIL-5 (hrIL-5) for up to 14 days, and the effects of this exposure were determined. First, the hrIL-5-cultured eosinophils were activated and degranulated more readily than freshly isolated eosinophils. For example, eosinophils cultured for 7 days with hrIL-5 released 30 and 10% of granule eosinophil-derived neurotoxin (EDN) when exposed to Sepharose 4B beads coupled to secretory IgA and IgG, respectively, whereas freshly isolated eosinophils released only 19 and 4%, respectively, of their EDN in response to the same stimuli. Degranulation of hrIL-5-cultured eosinophils was not augmented by further exposure to hrIL-5, whereas degranulation of freshly isolated cells to secretory IgA and IgG beads was increased by exposure to hrIL-5. Second, eosinophils cultured with hrIL-5 had prolonged viability in vitro. For example, after four days of culture with 50 U/ml of hrIL-5, 86% of eosinophils were viable compared to 12% in medium alone. Third, hrIL-5-cultured eosinophils became hypodense, and electron microscopy showed that they contained granules with core and matrix lucency and with evidence of granule fusion. Fourth, hrIL-5-cultured eosinophils spontaneously lost 30 to 60% of their EDN, eosinophil cationic protein, and eosinophil peroxidase and about 50% of their eosinophil granule major basic protein content compared to freshly isolated eosinophils, and all four of the granule proteins were released into the culture medium. Fifth, detailed studies of eosinophils cultured in hrIL-5 showed that 89 +/- 10% of the starting quantity of EDN could be recovered at 7 days. Whereas 99 +/- 1% of the EDN at day 0 was cell associated, by 7 days 60 +/- 9% was in the cell supernatants. Thus, hrIL-5 activates eosinophils, increases their viability, decreases their density, and their content of granule proteins and causes release of the granule proteins into culture fluids. The striking loss of granule proteins during culture with hrIL-5 may be an important mechanism for deposition of these cationic toxins in various diseases where IL-5 plays a role.
Assuntos
Proteínas Sanguíneas/metabolismo , Eosinófilos/efeitos dos fármacos , Interleucina-5/farmacologia , Ribonucleases , Proteínas Sanguíneas/análise , Degranulação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Granulares de Eosinófilos , Neurotoxina Derivada de Eosinófilo , Eosinófilos/metabolismo , Humanos , Imunoglobulinas/fisiologia , Neurotoxinas/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Umbilical cord mononuclear cells, HL-60 cells, HL-60 clones selected for eosinophil differentiation, and the eosinophil leukemia cell line EoL were tested for their ability to produce eosinophil peroxidase. HL-60 clones selected for eosinophil differentiation produced eosinophil peroxidase, as judged by staining of cells for cyanide-resistant peroxidase activity; however, these cells lost their ability to produce eosinophil peroxidase in long-term culture. In contrast, eosinophil precursors from human umbilical cord blood mononuclear cells stimulated with murine EL-4 conditioned medium (EL-4 CM) were regularly induced to eosinophil protein synthesis, including eosinophil peroxidase, major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin, as assessed by cyanide-resistant peroxidase and immunofluorescence staining. This induction by EL-4 CM is either at the level of gene transcription or mRNA stabilization, as shown by the increase of total mRNA for eosinophil peroxidase, major basic protein, and eosinophil-derived neurotoxin by Northern blot analyses. Purified peripheral blood eosinophils incubated for 4 days with EL-4 CM had increased survival over control eosinophils. Moreover, this enhanced survival was specifically blocked by antiserum to interleukin 5. Our results suggest that the effects of EL-4 CM on human umbilical cord mononuclear cells and mature eosinophils are due to the presence of interleukin 5.
Assuntos
Eosinófilos/citologia , Sangue Fetal/citologia , Ribonucleases , Animais , Proteínas Sanguíneas/metabolismo , Diferenciação Celular , Sobrevivência Celular , Meios de Cultura , Proteínas Granulares de Eosinófilos , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Humanos , Interleucina-5/farmacologia , Leucócitos Mononucleares/citologia , Peroxidases/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas/imunologiaRESUMO
The neutral protease tryptase has been isolated from a human mast cell line, HMC-1. The HMC-1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC-1-derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin-agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS-PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl-L-arginine methyl ester by purified tryptase was inhibited by dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone 2 HCl, HgCl2, tosyl-L-lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl-L-phenyl-alanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary-derived tryptase.
Assuntos
Compostos de Dansil , Mastócitos/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Clorometilcetonas de Aminoácidos/farmacologia , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Glicerol/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Leucemia de Mastócitos/enzimologia , Leucemia de Mastócitos/patologia , Leucemia de Mastócitos/fisiopatologia , Mastócitos/efeitos dos fármacos , Mastócitos/patologia , Mastocitose/enzimologia , Mastocitose/patologia , Mastocitose/fisiopatologia , Cloreto de Mercúrio/farmacologia , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Peptídeo Hidrolases/análise , Pele/citologiaRESUMO
Cells from the human immature mast cell line, HMC-1, were tested for their sensitivity to 11 chemotherapeutic agents by changes in viability and incorporation of tritiated thymidine ([3H]-thymidine) after 72 h of in vitro drug exposure. Doxorubicin hydrochloride and cytosine arabinoside were the most active agents against HMC-1 cells at the concentrations tested. Doxorubicin hydrochloride inhibited the incorporation of [3H]-thymidine and decreased the viability of HMC-1 cells by greater than 90% at a concentration of 0.06 microgram/ml. Cytosine arabinoside inhibited the incorporation of [3H]-thymidine and decreased the viability by greater than 95% at a concentration of 0.1 microgram/ml. A clone of HMC-1 cells, A4, with an enhanced percentage (70-80%) of metachromatically staining cells was equally sensitive to these two agents; however, A4 cells were more sensitive to vinblastine sulfate and less sensitive to methylprednisolone than was the parent cell line. Both HMC-1 cells and A4 cells showed approximately equal sensitivity to etoposide and to mitomycin. These results show that human mast cells are susceptible in vitro to a number of commonly used chemotherapeutic drugs.