RESUMEN
SETTING: Aerosolized interferon-gamma (IFN-gamma) leads to transient conversion of sputum smears in multidrug-resistant pulmonary tuberculosis (MDR-TB). OBJECTIVE: To test long-term conversion of sputum smears using the new Jena protocol. DESIGN: Four MDR-TB patients were treated with aerosolized recombinant IFN-gamma (rIFN-gamma) twice weekly for 8 weeks and anti-tuberculosis drugs. Patients were monitored clinically and T-cell subpopulations were analyzed. RESULTS: The treatment was well tolerated. All sputum smears cleared within 6-8 weeks, and radiological signs of recovery lasted in all patients for 73-106 months (the entire follow-up period). Before treatment, a patient with a 20+ year history of TB showed no gammadelta T-cells; these cells appeared during treatment. The proportion of natural killer (NK) cells was enhanced during treatment and remained elevated. The proportion of CD4+/CD25+ T-cells in the blood rose after treatment and remained elevated at 2 and 10 months afterwards. No significant change in T-cell levels appeared in patients with a shorter history of TB, except for a tendency toward a slight increase in gammadelta T-cells during treatment. CONCLUSION: We invite further confirmation, but aerosolized rIFN-gamma plus anti-microbial treatment cured MDR-TB in this case study. The optimal dosing schedule needs to be determined.
Asunto(s)
Antituberculosos/administración & dosificación , Interferón gamma/administración & dosificación , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Pulmonar/tratamiento farmacológico , Adolescente , Adulto , Aerosoles , Protocolos Clínicos , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Radiografía , Esputo/microbiología , Subgrupos de Linfocitos T , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico por imagenRESUMEN
Flow cytometry has evolved from single- and two-color analysis to the current use of 11-16 colors. The relatively bright excitation spectra of most fluorochromes have made color compensation a challenge especially when performed manually. We describe how by choosing filters with narrower bandwidths results in the color compensation values between FITC, PE, PE-TxR (ECD), PE-Cy5, and PE-Cy7 that range from 0 % to 50% depending on the combination of fluorochromes. Peripheral blood mononuclear cells were stained with alpha-CD4-FITC, alpha-CD27-PE, alpha-CD62L-ECD, alpha-CD45RA-PE-Cy5 and alpha-CD3-PE-Cy7. The samples were acquired on a MO Flo. The initial (first) and second filter sets for our experiments consisted of 530/30 or 519/20 for FITC, 580/30 or 575/20for PE, 630/30 or 630/22 for PE-TxR (ECD), 670/30 or 675/20 for PE-Cy5 and 740LP or 780/40 for PE-Cy7. Nonstained cells were used to adjust the threshold values of detection for each photo multiplier tube (PMT) for each filter set. The mean fluorescent intensity (MFI) of each fluorochrome was not reduced to any great extent by either filter set. However, the compensation value between PE and PE-TxR (ECD) with the first filter selection ranged from 84% to 89% and with the second set of filters it was 25-36%. In addition, the compensation between PE-TxR (ECD) and PE-Cy5 were reduced to 30.2% from 44.2% with the second filter set. The reduction of filter bandwidths that results in minimizing spectral overlaps without lost of signal provides a method by which discrimination of signals between PE containing fluorochromes can be achieved.
Asunto(s)
Citometría de Flujo/métodos , Recuento de Leucocitos/métodos , Citometría de Flujo/instrumentación , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Rayos Láser , Recuento de Leucocitos/instrumentación , Espectrometría de Fluorescencia/métodosRESUMEN
Pulmonary fibrosis (PF) may develop following successful chemotherapy for malignancy, even if such therapy is not combined with radiotherapy. Bleomycin, which is known to induce acute pneumonitis and lung fibrosis, is especially associated with chemotherapy-induced PF, and bleomycin-induced pulmonary fibrosis can occur more than five years after such therapy. Additionally, supplemental oxygen therapy can trigger the onset of pneumonitis and lethal PF in patients who have previously received bleomycin therapy. Careful assessment of lung function via spiroergometry and arterial blood gas analysis during exercise are required if the administration of supplemental oxygen is considered. Two case reports reveal the potential lethal risk of oxygen for patients who have been treated with bleomycin: (1) a patient with successfully resected and treated basal tongue carcinoma and (2) a patient in remission after being treated for non-Hodgkin lymphoma. Single and double lung transplantation is the only therapeutic option for patients with severe, oxygen-induced PF and should be included as an indication for lung transplantation. Early recognition of pulmonary diffusion abnormalities and establishing a risk profile, as well as consequent monitoring of pulmonary function, may help to avoid or at least reduce the risk of PF induced by oxygen therapy when administered to patients who have previously been given bleomycin.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Enfermedad de Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/tratamiento farmacológico , Fibrosis Pulmonar/inducido químicamente , Insuficiencia Respiratoria/inducido químicamente , Adulto , Bleomicina/efectos adversos , Análisis de los Gases de la Sangre , Femenino , Humanos , Trasplante de Pulmón , Masculino , Persona de Mediana Edad , Fibrosis Pulmonar/prevención & control , Pruebas de Función Respiratoria , Insuficiencia Respiratoria/prevención & controlRESUMEN
OBJECTIVE: To determine the diversity of Salmonella serotypes isolated from a large population of cull (market) dairy cows at slaughter. DESIGN: Cross-sectional study. SAMPLE POPULATION: Salmonella organisms isolated from the cecal-colon contents of 5,087 market dairy cows. PROCEDURE: During winter and summer 1996, cecal-colon contents of cull dairy cows at slaughter were obtained from 5 US slaughter establishments. Specimens were subjected to microbiologic culturing for Salmonella spp at 1 laboratory. Identified isolates were compared with Salmonella isolation lists published by the Centers for Disease Control and Prevention (CDC) and the National Veterinary Services Laboratory (NVSL) for approximately the same period. The Simpson diversity index was used to calculate the likelihood that Salmonella isolates selected randomly by establishment were different. RESULTS: Of 58 Salmonella serotypes identified, Salmonella ser. Montevideo was the most prevalent. Two of the top 10 CDC serotypes identified from in 1996, Salmonella ser. Typhimurium and S Montevideo, appeared on our top 10 list; 8 of the top 10 were found on NVSL listings. Thirty-one of 59 S. Typhimurium isolates were identified as DT104 and found at a west slaughter establishment, 30 during the winter and 1 during the summer. The greatest diversity of serotypes was at a southeast establishment during the summer; the least diversity was at a central establishment in the winter. CONCLUSIONS AND CLINICAL RELEVANCE: 58 Salmonella serotypes were isolated from market dairy cows at slaughter and could pose a threat for food-borne illness. Salmonella Montevideo was the most frequently isolated serotype and may contribute substantially to salmonellosis in dairy cattle.
Asunto(s)
Enfermedades de los Bovinos/microbiología , Salmonelosis Animal/microbiología , Salmonella/clasificación , Animales , Bovinos , Ciego/microbiología , Colon/microbiología , Estudios Transversales , Femenino , Microbiología de Alimentos , Salud Pública/estadística & datos numéricos , Salmonella/genética , Salmonella/aislamiento & purificación , Estaciones del Año , Serotipificación/veterinaria , Estados UnidosRESUMEN
OBJECTIVE: To determine the prevalence of Salmonella spp in the cecal-colon contents of cull (market) dairy cows at slaughter because of potential public health ramifications. DESIGN: Survey study. SAMPLE POPULATION: Cecal-colon contents collected from 5,087 cull (market) dairy cows at slaughter at 5 slaughter establishments across the United States. PROCEDURE: During 2 periods of the year, winter (January and February) and summer (July through September), 5 cull (market) cow slaughter establishments in the United States--west (WE), southeast (SEE), central (CE), north central (NCE), and south central (SCE)--establishments were visited, and cecal-colon contents of cull dairy cows were obtained at the time of slaughter. Samples were examined by microbiologic culture at a single laboratory for Salmonella spp. RESULTS: Salmonella spp were detected in 23.1% of cecal-colon content samples from cull dairy cows across the 5 slaughter establishments. The highest site prevalence (54.5%) was detected at the WE during the summer period, whereas the lowest was found at the CE during the summer (4.3%) and at the NCE during the winter (4.5%). Considerable variation in the daily prevalence of Salmonella spp was found, particularly at the WE and the SCE. Salmonella spp were isolated from 93% of cecal-colon contents collected on a summer day at the WE. CONCLUSIONS AND CLINICAL RELEVANCE: Results strongly suggest that there is a high prevalence of Salmonella spp in cull dairy cows at slaughter, which could burden Hazard Analysis Critical Control Point programs implemented in slaughter establishments. Procedures to reduce Salmonella load at the dairy farm and during transport to slaughter could reduce the risk of spread during the slaughter process.
Asunto(s)
Enfermedades de los Bovinos/epidemiología , Salmonelosis Animal/epidemiología , Salmonella/aislamiento & purificación , Mataderos , Animales , Bovinos , Ciego/microbiología , Colon/microbiología , Femenino , Encuestas Epidemiológicas , Prevalencia , Salud Pública , Estaciones del Año , Estados Unidos/epidemiologíaAsunto(s)
Membrana Mucosa/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta , Subgrupos de Linfocitos T/inmunología , Animales , Colorantes , Enfermedades Transmisibles/inmunología , Enfermedades Transmisibles/patología , Células Epiteliales/inmunología , Humanos , Inmunidad Activa , Inmunidad Celular , Inflamación/inmunología , Inflamación/fisiopatología , Membrana Mucosa/patologíaRESUMEN
Infiltration of eosinophils into the airways plays a central role in the pathophysiology of asthma. Human blood eosinophils express apoptosis-inducing receptors (e.g. CD95R and CD69R) regulating both viability and survival and, thus, the extent of eosinophil infiltration into the airways. Signal transduction processes induced by occupation of the CD69 receptor expressed by eosinophils are insufficiently known. Purified human peripheral blood eosinophils (MACS, purity > 99%) were pre-incubated with a GM-CSF for 18 h and stimulated with alpha-CD69mAb (clon TP1/55), alpha-CD95mAb (clon CH-11), and as a control alpha-CD11bmAb (clon Bear-1). The specificity of receptor ligation was assessed using a blocking mAb (Klon ZB4). Phenotype, viability, apoptosis and bcl-2-expression were measured employing flow cytometry. alpha-CD95mAb (1 microgram/ml) induced apoptosis both in control and GM-CSF (10 ng/ml) treated eosinophils. Similarly, alpha-CD69mAb (10 micrograms/ml) induced apoptosis of GM-CSF-stimulated CD69+ cells after an incubation period of 114 h which was not affected by a CD95 blocking mAb. Naive eosinophils showed a basale, bcl-2-expression, which decreased to 30% after 66 h. In the presence of GM-CSF, intracellular bcl-2-concentration remained unchanged. Following stimulation with alpha-CD69mAb or alpha-CD95mAb, a dose-dependent decline of the bcl-2-expression was detected, whereas alpha-CD11bmAb (10 micrograms/ml) had no effect. The data suggest that both CD95R- and CD69R-induced apoptosis of human eosinophils involves a bcl-2-dependent signal transduction mechanism.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Apoptosis/fisiología , Eosinófilos/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Transducción de Señal/fisiología , Receptor fas/fisiología , Anticuerpos Monoclonales/farmacología , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos T/genética , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Técnicas In Vitro , Lectinas Tipo C , Transducción de Señal/efectos de los fármacos , Receptor fas/genéticaRESUMEN
Substance P (SP), a neurotransmitter of the central and peripheral nervous system, has been implicated as a mediator of the pulmonary inflammatory response through its stimulatory effects on neutrophils. We investigated the role of SP in priming the production of reactive oxygen species by human neutrophils with the cytochrome c reduction assay and by flow cytometry using the intracellular oxidizable probe dichlorofluorescein. We also investigated SP-induced formation of nitrite and nitrate as an index of nitric oxide (NO) production. Our results indicate that SP primes two distinct pathways with respect to the induction of reactive oxygen species in the human neutrophil: the production of superoxide anion and hydrogen peroxide by the calmodulin-dependent NADPH oxidase, and the generation of NO by a constitutive NO synthase. Preincubation of neutrophils with inhibitors of calmodulin and NO synthase diminished the oxidative response in an additive fashion. These results give insight into distinct signal transduction pathways in the SP-primed neutrophil with respect to the formation of superoxide anion, hydrogen peroxide, and NO.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Neutrófilos/metabolismo , Óxido Nítrico/biosíntesis , Sustancia P/farmacología , Adulto , Fluoresceínas/metabolismo , Humanos , Inflamación/inducido químicamente , Neurotransmisores/farmacología , Neutrófilos/fisiología , Oxidantes/metabolismo , Oxidación-Reducción , Superóxidos/sangreRESUMEN
We reported previously that treatment with antibody to transforming growth factor-beta (TGF-beta) caused a marked attenuation of bleomycin (BL)-induced lung fibrosis (LF) in mice. Decorin (DC), a proteoglycan, binds TGF-beta and thereby down-regulates all of its biological activities. In the present study, we evaluated the antifibrotic potential of DC in a three-dose BL-hamster model of lung fibrosis. Hamsters were placed in the following groups: (1) saline (SA) + phosphate-buffered saline (PBS) (SA + PBS); (2) SA + DC; (3) BL + PBS; and (4) BL + DC. Under pentobarbital anesthesia, SA (4 mL/kg) or BL was instilled intratracheally in three consecutive doses (2.5, 2.0, 1.5 units/kg/4 mL) at weekly intervals. DC (1 mg/mL) or PBS was instilled intratracheally in 0.4 mL/hamster on days 3 and 5 following instillation of each dose of SA or BL. In week 4, hamsters received three doses of either DC or PBS every other day. The hamsters were killed at 30 days following the first instillation, and their lungs were appropriately processed. Lung hydroxyproline levels in SA + PBS, SA + DC, BL + PBS, and BL + DC groups were 965, 829, 1854, and 1387 microg/lung, respectively. Prolyl hydroxylase activities were 103, 289, and 193% of SA + PBS control in SA + DC, BL + PBS, and BL + DC groups, respectively. The myeloperoxidase activities in the corresponding groups were 222, 890, and 274% of control (0.525 units/lung). Intratracheal instillation of BL caused significant increases in these biochemical markers, and instillation of DC diminished these increases in the BL + DC group. DC treatment also caused a significant reduction in the infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) of hamsters in the BL + DC group. However, DC treatment had little effect on BL-induced increases in lung superoxide dismutase activity and lipid peroxidation and leakage of plasma proteins in the BALF of the BL + DC group. Hamsters in the BL + PBS group showed severe multifocal fibrosis and accumulation of mononuclear inflammatory cells and granulocytes. In contrast, hamsters in the BL + DC group showed mild multifocal septal thickening with aggregations of mononuclear inflammatory cells. Hamsters in both control groups (SA + PBS and SA + DC) showed normal lung structure. Frozen lung sections following immunohistochemical staining revealed an intense staining for EDA-fibronectin and collagen type I in the BL + PBS group as compared with all other groups. It was concluded that DC potentially offers a novel pharmacological intervention that may be useful in treating pulmonary fibrosis.
Asunto(s)
Proteoglicanos/uso terapéutico , Fibrosis Pulmonar/tratamiento farmacológico , Animales , Bleomicina/toxicidad , Cricetinae , Decorina , Proteínas de la Matriz Extracelular , Hidroxiprolina/análisis , Inmunohistoquímica , Pulmón/química , Pulmón/patología , Masculino , Mesocricetus , Peroxidasa/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Superóxido Dismutasa/metabolismoRESUMEN
T lymphocytes expressing the alpha E beta 7 integrin are localized and selectively retained in mucosal tissues. To investigate a potential relationship between alpha E beta 7 expression and pulmonary inflammation, the distribution of alpha E beta 7-bearing CD4+ and CD8+ T cells in peripheral blood and bronchoalveolar lavage (BAL) fluids obtained from patients with allergic asthma, sarcoidosis, hypersensitivity pneumonitis, and idiopathic pulmonary fibrosis (IPF) was determined. In contrast to the distribution in peripheral blood, BAL fluid from these patients contained high number of cells expressing alpha E beta 7 with markedly different expression patterns on CD4 or CD8 cells as well as among the various diseases. Despite similar numbers of activated CD4 cells, alpha E beta 7+CD4+ T cells ranged from 15% in asthmatics to 70% in IPF. In contrast, even in normal individuals, 60% to 90% of BAL fluid CD8+ T cells express alpha E beta 7, suggesting differential induction mechanisms on CD4 and CD8 cells. In vitro experiments revealed that a substantial proportion of peripheral blood CD+ T cells express alpha E beta 7 after stimulation with anti-CD3 antibodies, and up to 80% positive cells were found after the addition of TGF-beta. In contrast, less than 10% of CD4 cells express this particular integrin after in vitro stimulation, and the presence of TGF-beta only increased the number to 30%. Supernatants from in vitro-activated BAL cells as well as concentrated BAL fluid from patients with high alpha E beta 7 expression had no further enhancing effect. However, crosslinking of alpha 4 beta 1-, but not beta 2-integrins, significantly increased the number of alpha E beta 7 expressing CD4+ and CD8+ T cells, even in the absence of TGF-beta. These data indicate that in addition to TGF-beta, the interaction of particular T-cell subsets with specific endothelial cell and extracellular matrix proteins may upregulate alpha E beta 7 integrin expression and thereby contribute to the selective accumulation of these cells in inflammatory lung diseases.
Asunto(s)
Integrinas/análisis , Integrinas/inmunología , Enfermedades Pulmonares Intersticiales/inmunología , Subgrupos Linfocitarios/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/farmacología , Adulto , Alveolitis Alérgica Extrínseca/inmunología , Anticuerpos , Asma/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Células Cultivadas , Reactivos de Enlaces Cruzados , Eosinófilos/inmunología , Femenino , Antígenos HLA-DR/análisis , Humanos , Integrina alfa4beta1 , Integrinas/sangre , Antígenos Comunes de Leucocito/análisis , Activación de Linfocitos , Macrófagos Alveolares/inmunología , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Fibrosis Pulmonar/inmunología , Receptores de Interleucina-2/análisis , Sarcoidosis/inmunologíaRESUMEN
CD4, CD8, and gamma delta T-cells located in the epithelium express the integrin alpha E beta 7 that binds to E-cadherin on the epithelium. Gamma delta T-cells mediate specific cellular immune functions and can recognize damaged cells directly. It was, therefore, of interest to analyse the presence of gamma delta T-cells and the expression of alpha E beta 7 on gamma delta T-cells in the bleomycin (BLM) model of pulmonary fibrosis. Lung fibrosis was induced by a single intratracheal instillation of BLM (0.125 U.mouse-1), and bronchoalveolar lavage (BAL) T-cell subpopulations were examined at various time-points for the expression of the integrin alpha E beta 7 by flow cytometry. CD4+ T-cells accounted for about 40% of the lymphocytes, compared to about 10% of CD8+ T-cells and 10-14% gamma delta T-cells. Within the CD4+ T-cell population the proportion of alpha E beta 7+ cells decreased between Days 2 and 22 from 36 to 11%. The percentage of alpha E beta 7+ CD8+ T-cells increased at the same time from 4 to 68%. However, more than 80% of the gamma delta T-cells in BAL fluid expressed alpha E beta 7 at all time-points. The surface-expression of this integrin on gamma delta T-cells was 2-3 times higher than on CD4+ or CD8+ T-cells. This predominant expression of alpha E beta 7 on gamma delta T-cells suggests a role for these cells in the pathogenesis of bleomycin-induced lung fibrosis.
Asunto(s)
Bleomicina/efectos adversos , Líquido del Lavado Bronquioalveolar/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Integrinas/biosíntesis , Fibrosis Pulmonar/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Cadherinas/fisiología , Epitelio/química , Citometría de Flujo , Hidroxiprolina/análisis , Recuento de Linfocitos , Masculino , Ratones , Fibrosis Pulmonar/inducido químicamente , Receptores de Antígenos de Linfocitos T gamma-delta/inmunologíaRESUMEN
Neutrophil adhesion to microvascular endothelium at sites of acute inflammation is regulated by both chemotactic peptides and lipid-derived mediators. Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory peptide that up-regulates endothelial expression of intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (E-selectin), while platelet-activating factor (PAF) is a potent lipid mediator that induces vascular changes via an unknown mechanism. Both have been shown to increase leucocyte-endothelial adhesion in various in vitro models of acute inflammation; however, the combined effects of recombinant TNF-alpha (rTNF-alpha) and PAF on neutrophil-endothelium adhesion have not been well described. In this study, we found rTNF-alpha at 0.5 ng/ml and PAF at 10 microM acted synergistically to increase neutrophil adherence to cultured umbilical vein endothelial cells 4 hr after stimulation. This increased neutrophil-endothelial adhesion was, in part, dependent on up-regulated expression of ICAM-1 and E-selectin since application of anti-ICAM1 and anti-E-selectin F(ab')2 fragments markedly diminished adhesion. Cultures stimulated with rTNF-alpha (0.5 ng/ml) or PAF (10 microM) alone did not show a significant increase in neutrophil adhesion, and neither ICAM-1 nor E-selectin expression was up-regulated as determined by flow cytometric analysis of endothelial cells. These results indicate that rTNF-alpha and PAF act synergistically to increase neutrophil-endothelial adhesion by stimulating endothelial expression of ICAM-1 and E-selectin and, thus, may play important roles in the onset and severity of acute inflammatory reactions.
Asunto(s)
Selectina E/fisiología , Endotelio Vascular/fisiología , Molécula 1 de Adhesión Intercelular/fisiología , Neutrófilos/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Sinergismo Farmacológico , Humanos , Neutrófilos/fisiología , Proteínas Recombinantes/farmacología , Estimulación Química , Regulación hacia ArribaRESUMEN
Increasing evidence suggests an important role for cytokines in the regulation of eosinophilic inflammation. In the present study we investigated the distribution of leukocytes, lymphocyte subsets, their activation state, and the cytokine profile present in BAL fluid from patients with various lung diseases associated with eosinophilia. For this purpose, we analyzed the levels of IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, GM-CSF, TNF-alpha, and IFN-gamma, as well as soluble IL-2 and TNF receptors, in concentrated bronchoalveolar lavage (BAL) fluid obtained from clearly defined patients with allergic and nonallergic asthma, eosinophilic pneumonia, allergic bronchopulmonary aspergillosis (ABPA), hypersensitivity pneumonitis, and idiopathic pulmonary fibrosis. BAL fluid from normal individuals and sarcoidosis patients was analyzed as noneosinophilic controls. BAL cytokine levels were compared with the cellular infiltrate and the activation state of CD4+ and CD8+ T cells as measured by the expression of IL-2 receptors (CD25), HLA-DR, and the very late activation antigen VLA-1. Beside the characteristic leukocyte infiltrate in the various lung diseases, all patients demonstrated significantly increased numbers of activated CD4 and CD8 T cells compared with normal individuals. The analysis of the cytokine profile present in BAL fluid revealed a T helper type 2 (Th2) cell cytokine pattern, with elevated IL-4 and IL-5 but normal levels of IL-2 or IFN-gamma in allergic asthma. ABPA patients demonstrated significantly increased levels of IL-4 and IL-5, with low but significantly elevated concentrations of IL-2 and IFN-gamma. In contrast, the analysis of the cytokine profile in sarcoidosis patients revealed a Th1 cell cytokine pattern characterized by increased concentrations of IL-2 and IFN-gamma but normal levels of IL-4 or IL-5. All other patient groups showed a cytokine pattern incompatible with a pure Th1 or Th2 cell response, because IL-5, IL-2, and IFN-gamma were found to be significantly increased. The BAL fluid analysis of the other, mainly non-T cell-derived cytokines and soluble receptors showed increased levels in all patients compared with normal individuals and may represent the ongoing inflammatory responses. In conclusion, whereas increased IL-4 levels were found only in diseases characterized by increased IgE production, IL-5 was elevated in all patients with increased numbers of eosinophils. The close correlation between IL-5 levels, number of eosinophils, and activated T cells further supports a role for IL-5 in causing tissue eosinophilia.
Asunto(s)
Líquido del Lavado Bronquioalveolar/inmunología , Citocinas/análisis , Activación de Linfocitos/inmunología , Eosinofilia Pulmonar/inmunología , Linfocitos T/inmunología , Adulto , Alveolitis Alérgica Extrínseca/epidemiología , Alveolitis Alérgica Extrínseca/etiología , Alveolitis Alérgica Extrínseca/inmunología , Aspergilosis Broncopulmonar Alérgica/epidemiología , Aspergilosis Broncopulmonar Alérgica/etiología , Aspergilosis Broncopulmonar Alérgica/inmunología , Aspergillus fumigatus , Asma/epidemiología , Asma/etiología , Asma/inmunología , Líquido del Lavado Bronquioalveolar/citología , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Eosinofilia Pulmonar/epidemiología , Eosinofilia Pulmonar/etiología , Fibrosis Pulmonar/epidemiología , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/inmunología , Sarcoidosis Pulmonar/epidemiología , Sarcoidosis Pulmonar/etiología , Sarcoidosis Pulmonar/inmunología , Estadísticas no ParamétricasRESUMEN
Eosinophils from sputum, nasal polyps, and bronchoalveolar lavages of asthmatics demonstrated a considerably increased CD11b expression, compared with blood eosinophils. Furthermore, the tissue eosinophils expressed ICAM-1 (CD54) and HLA-DR, whereas peripheral blood eosinophils did not. In vitro migration of peripheral blood eosinophils across IL-1-activated human umbilical vein endothelial cell monolayers caused a considerable up-regulation of CD11b and CD35 expression, no induction of ICAM-1 or HLA-DR, and a small but significant decrease in CD11a, CD29, and CD32 expression. These changes were only partially inducible with supernatants from nonactivated or IL-1-activated endothelial cells, platelet-activating factor, or a variety of recombinant cytokines. Thus, cell-cell interactions mediated by receptor-ligand binding or endothelial cell membrane-bound mediators, rather than soluble factors, are responsible for the altered eosinophil surface marker expression. Indeed, preparations of membrane fragments from IL-1-stimulated endothelial cells were able to induce up-regulation of CD11b, which was not inhibitable with the platelet-activating factor antagonist WEB 2086 or antibodies against ELAM-1, VCAM-1, or ICAM-1. Investigation of the functional significance of the increased CD11b expression on eosinophils revealed only minimal changes in the adherence or transmigration capacity. Nevertheless, increased CD11b expression was related to an increased capacity to generate superoxide after stimulation with opsonized zymosan. Thus, cell-cell interactions between eosinophils and endothelial cells induce a considerable up-regulation of CD11b and CD35 on eosinophils and an increased capacity to generate an oxidative burst.
Asunto(s)
Asma/inmunología , Endotelio Vascular/fisiología , Eosinófilos/fisiología , Antígeno de Macrófago-1/análisis , Antígenos CD/análisis , Adhesión Celular , Moléculas de Adhesión Celular/análisis , Movimiento Celular , Células Cultivadas , Citocinas/farmacología , Eosinófilos/inmunología , Antígenos HLA-DR/análisis , Humanos , Molécula 1 de Adhesión Intercelular , Factor de Activación Plaquetaria/farmacología , Receptores de Antígeno muy Tardío/fisiologíaRESUMEN
We present evidence that human blood eosinophils produce interleukin (IL)-8 when stimulated with calcium ionophore. Following in vitro culture of 99% pure eosinophils with calcium ionophore, released IL-8 was detectable by enzyme-linked immunosorbent assay in supernatants. Eosinophil IL-8 production was considerably greater than that of IL-3 or granulocyte macrophage colony-stimulating factor. Furthermore, eosinophil production of IL-8 in the presence of calcium ionophore could be inhibited with the immunomodulating agent cyclosporin A and the protein synthesis inhibitor cycloheximide. In addition, following stimulation of highly purified blood eosinophils with calcium ionophore, IL-8 mRNA was detectable after polymerase chain reaction amplification. In comparison with other cells on stimulation with calcium ionophore, eosinophils produce about half as much IL-8 as neutrophils but significantly more than purified T cells. In contrast to monocytes and neutrophils, IL-8 production was not inducible with IL-1 or tumor necrosis factor. Finally, following calcium ionophore stimulation blood eosinophils were shown to contain cytoplasmic IL-8 by employing a monoclonal antibody against IL-8 in conjunction with immunohistochemistry. These observations demonstrate that eosinophils are capable of IL-8 production and release, which may contribute to defense against parasites and to the pathophysiology of allergic and asthmatic disease.
Asunto(s)
Calcio/fisiología , Eosinófilos/metabolismo , Interleucina-8/biosíntesis , Calcimicina/farmacología , Células Cultivadas , Eosinófilos/efectos de los fármacos , Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Granulocitos/metabolismo , Humanos , Técnicas In Vitro , Interleucina-3/biosíntesis , Interleucina-8/metabolismo , Leucocitos Mononucleares/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Linfocitos T/metabolismoRESUMEN
Cytokines act on eosinophils to regulate eosinophil function, with IL-5 recognised to be especially important in control of eosinopoiesis, eosinophil survival and eosinophil priming. In addition, eosinophils have the capacity to produce cytokines involved in acute and chronic inflammatory and repair processes, as well as to produce cytokines that stimulate eosinophils within an autocrine loop. This paper describes (A) an immunomagnetic selection technique for the purification of human blood eosinophils, and (B) a method that employs immunofluorescence with flow cytometry for measurement of blood and sputum eosinophil surface markers. Having demonstrated that sputum eosinophils express HLA-DR, highly purified blood eosinophils were used to analyse (C) the induction and function of eosinophil HLA-DR. Cytokines have the capacity to induce eosinophil HLA-DR, and are produced by eosinophils as an accessory function during antigen presentation. Finally, preliminary data on (D) eosinophil production of IL-8 is presented. Hence, eosinophils have the capacity to act as immunomodulatory cells within cells networks in allergic and asthmatic inflammation.