RESUMEN
Moraxella catarrhalis is found almost exclusively within the human respiratory tract. This pathobiont is associated with ear infections and the development of respiratory illnesses, including allergies and asthma. Given the limited ecological distribution of M. catarrhalis, we hypothesized that we could leverage the nasal microbiomes of healthy children without M. catarrhalis to identify bacteria that may represent potential sources of therapeutics. Rothia was more abundant in the noses of healthy children compared to children with cold symptoms and M. catarrhalis. We cultured Rothia from nasal samples and determined that most isolates of Rothia dentocariosa and "Rothia similmucilaginosa" were able to fully inhibit the growth of M. catarrhalis in vitro, whereas isolates of Rothia aeria varied in their ability to inhibit M. catarrhalis. Using comparative genomics and proteomics, we identified a putative peptidoglycan hydrolase called secreted antigen A (SagA). This protein was present at higher relative abundance in the secreted proteomes of R. dentocariosa and R. similmucilaginosa than in those from non-inhibitory R. aeria, suggesting that it may be involved in M. catarrhalis inhibition. We produced SagA from R. similmucilaginosa in Escherichia coli and confirmed its ability to degrade M. catarrhalis peptidoglycan and inhibit its growth. We then demonstrated that R. aeria and R. similmucilaginosa reduced M. catarrhalis levels in an air-liquid interface culture model of the respiratory epithelium. Together, our results suggest that Rothia restricts M. catarrhalis colonization of the human respiratory tract in vivo. IMPORTANCE Moraxella catarrhalis is a pathobiont of the respiratory tract, responsible for ear infections in children and wheezing illnesses in children and adults with chronic respiratory diseases. Detection of M. catarrhalis during wheezing episodes in early life is associated with the development of persistent asthma. There are currently no effective vaccines for M. catarrhalis, and most clinical isolates are resistant to the commonly prescribed antibiotics amoxicillin and penicillin. Given the limited niche of M. catarrhalis, we hypothesized that other nasal bacteria have evolved mechanisms to compete against M. catarrhalis. We found that Rothia are associated with the nasal microbiomes of healthy children without Moraxella. Next, we demonstrated that Rothia inhibit M. catarrhalis in vitro and on airway cells. We identified an enzyme produced by Rothia called SagA that degrades M. catarrhalis peptidoglycan and inhibits its growth. We suggest that Rothia or SagA could be developed as highly specific therapeutics against M. catarrhalis.
Asunto(s)
Asma , Moraxella catarrhalis , Niño , Adulto , Humanos , Peptidoglicano/metabolismo , Ruidos RespiratoriosRESUMEN
BACKGROUND: Detection of either viral or bacterial pathogens is associated with wheezing in children; however, the influence of both bacteria and viruses on illness symptoms has not been described. OBJECTIVE: We evaluated bacterial detection during the peak rhinovirus season in children with and without asthma to determine whether an association exists between bacterial infection and the severity of rhinovirus-induced illnesses. METHODS: Three hundred eight children (166 with asthma and 142 without asthma) aged 4 to 12 years provided 5 consecutive weekly nasal samples during September and scored cold and asthma symptoms daily. Viral diagnostics and quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were performed on all nasal samples. RESULTS: Detection rates were 53%, 17%, and 11% for H influenzae, S pneumoniae, and M catarrhalis, respectively, with detection of rhinovirus increasing the risk of detecting bacteria within the same sample (odds ratio [OR], 2.0; 95% CI, 1.4-2.7; P < .0001) or the following week (OR, 1.6; 95% CI, 1.1-2.4; P = .02). In the absence of rhinovirus, S pneumoniae was associated with increased cold symptoms (mean, 2.7 [95% CI, 2.0-3.5] vs 1.8 [95% CI, 1.5-2.2]; P = .006) and moderate asthma exacerbations (18% [95% CI, 12% to 27%] vs 9.2% [95% CI, 6.7% to 12%]; P = .006). In the presence of rhinovirus, S pneumoniae was associated with increased moderate asthma exacerbations (22% [95% CI, 16% to 29%] vs 15% [95% CI, 11% to 20%]; P = .01). Furthermore, M catarrhalis detected alongside rhinovirus increased the likelihood of experiencing cold symptoms, asthma symptoms, or both compared with isolated detection of rhinovirus (OR, 2.0 [95% CI, 1.0-4.1]; P = .04). Regardless of rhinovirus status, H influenzae was not associated with respiratory symptoms. CONCLUSION: Rhinovirus infection enhances detection of specific bacterial pathogens in children with and without asthma. Furthermore, these findings suggest that M catarrhalis and S pneumoniae contribute to the severity of respiratory tract illnesses, including asthma exacerbations.
Asunto(s)
Asma , Bacterias , Infecciones Bacterianas/microbiología , ADN Bacteriano/genética , Infecciones por Picornaviridae , Rhinovirus , Asma/complicaciones , Asma/genética , Asma/microbiología , Asma/virología , Bacterias/genética , Bacterias/aislamiento & purificación , Infecciones Bacterianas/genética , Niño , Preescolar , Femenino , Humanos , Masculino , Infecciones por Picornaviridae/genética , Infecciones por Picornaviridae/microbiología , Reacción en Cadena de la PolimerasaRESUMEN
RATIONALE: Most virus-induced attacks of asthma are caused by rhinoviruses (RVs). OBJECTIVES: To determine whether people with asthma are susceptible to an increased viral load during RV infection. METHODS: Seventy-four children (4-18 yr old) were enrolled; 28 with wheezing, 32 with acute rhinitis, and 14 without respiratory tract symptoms. Nasal washes were evaluated using quantitative polymerase chain reaction for RV to judge viral load along with gene sequencing to identify strains of RV. Soluble intercellular adhesion molecule-1, IFN-λ1, and eosinophil cationic protein in nasal washes, along with blood eosinophil counts and total and allergen-specific IgE in sera, were also evaluated. Similar assessments were done in 24 young adults (16 with asthma, 8 without) who participated in an experimental challenge with RV (serotype 16). MEASUREMENTS AND MAIN RESULTS: Fifty-seven percent of wheezing children and 56% with acute rhinitis had nasal washes testing positive for RV. The geometric mean of viral loads by quantitative polymerase chain reaction in washes from wheezing children was 2.8-fold lower, but did not differ significantly from children with rhinitis (7,718 and 21,612 copies of viral RNA per microliter nasal wash, respectively; P = 0.48). The odds for wheezing were increased if children who tested positive for RV were sensitized to one or more allergens (odds ratio, 3.9; P = 0.02). Similarly, neither peak nor cumulative viral loads differed significantly in washes from adults with asthma compared with those without asthma during the experimental RV challenge. CONCLUSIONS: During acute symptoms, children infected with RV enrolled for wheezing or acute rhinitis had similar viral loads in their nasal washes, as did adults with and without asthma infected with RV-16 experimentally.
Asunto(s)
Asma/virología , Infecciones por Picornaviridae/virología , Ruidos Respiratorios/etiología , Rinitis/virología , Rhinovirus/aislamiento & purificación , Carga Viral , Enfermedad Aguda , Adolescente , Asma/sangre , Asma/complicaciones , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Preescolar , Estudios Transversales , Progresión de la Enfermedad , Proteína Catiónica del Eosinófilo/sangre , Eosinófilos/metabolismo , Femenino , Humanos , Inmunoglobulina E/sangre , Molécula 1 de Adhesión Intercelular/sangre , Interferones , Interleucinas/sangre , Recuento de Leucocitos , Modelos Logísticos , Masculino , Líquido del Lavado Nasal/virología , Oportunidad Relativa , Infecciones por Picornaviridae/sangre , Infecciones por Picornaviridae/complicaciones , Infecciones por Picornaviridae/diagnóstico , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Ruidos Respiratorios/fisiología , Rinitis/sangre , Rhinovirus/genética , Adulto JovenRESUMEN
RATIONALE: The 2009 H1N1 flu appeared to cause more severe cold symptoms during the 2009-2010 flu season. OBJECTIVES: We evaluated H1N1 infections during peak viral season in children with and without asthma to determine whether the H1N1 infectivity rate and illness severity were greater in subjects with asthma. METHODS: One hundred and eighty children, 4-12 years of age, provided eight consecutive weekly nasal mucus samples from September 5 through October 24, 2009, and scored cold and asthma symptoms daily. Viral diagnostics were performed for all nasal samples. MEASUREMENTS AND MAIN RESULTS: One hundred and sixty-one children (95 with asthma, 66 without asthma) completed at least 6 of the 8 nasal samples. The incidence of H1N1 infection was significantly higher in children with asthma (41%) than in children without asthma (24%; odds ratio, 4; 95% confidence interval, 1.8-9; P < 0.001), but rates of human rhinovirus infection (90% each) and other viral infections (47 vs. 41%) were similar. In children with asthma, there was a nonsignificant trend for increased loss of asthma control during H1N1 infections compared with human rhinovirus infections (38 vs. 21%; odds ratio, 2.6; 95% confidence interval, 0.9-7.2; P = 0.07). CONCLUSIONS: During peak 2009 H1N1 flu season, children with asthma were infected almost twice as often with H1N1 compared with other respiratory viruses. H1N1 infection also caused increased severity of cold symptoms compared with other viral infections. Given the increased susceptibility of children with asthma to infection, these findings reinforce the need for yearly influenza vaccination to prevent infection, and raise new questions about the mechanism for enhanced susceptibility to influenza infection in asthma.
Asunto(s)
Asma/epidemiología , Susceptibilidad a Enfermedades/epidemiología , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/epidemiología , Distribución por Edad , Asma/diagnóstico , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Niño , Preescolar , Comorbilidad , Femenino , Humanos , Incidencia , Gripe Humana/diagnóstico , Modelos Logísticos , Masculino , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/epidemiología , Estudios Retrospectivos , Rhinovirus/aislamiento & purificación , Medición de Riesgo , Índice de Severidad de la Enfermedad , Distribución por Sexo , Estadísticas no Paramétricas , Estados Unidos/epidemiologíaRESUMEN
RATIONALE: Most asthma exacerbations are initiated by viral upper respiratory illnesses. It is unclear whether human rhinovirus (HRV)induced exacerbations are associated with greater viral replication and neutrophilic inflammation compared with HRV colds. OBJECTIVES: To evaluate viral strain and load in a prospective asthma cohort during a natural cold. METHODS: Adults were enrolled at the first sign of a cold, with daily monitoring of symptoms, medication use, and peak expiratory flow rate until resolution. Serial nasal lavage and induced sputum samples were assessed for viral copy number and inflammatory cell counts. MEASUREMENTS AND MAIN RESULTS: A total of 52 persons with asthma and 14 control subjects without atopy or asthma were studied for over 10 weeks per subject on average; 25 participants developed an asthma exacerbation. Detection of HRVs in the preceding 5 days was the most common attributable exposure related to exacerbation. Compared with other infections, those by a minor group A HRV were 4.4- fold more likely to cause exacerbation (P = 0.038). Overall, sputum neutrophils and the burden of rhinovirus in the lower airway were similar in control subjects without atopy and the asthma group. However, among HRV-infected participants with asthma, exacerbations were associated with greater sputum neutrophil counts (P = 0.005). CONCLUSIONS: HRV infection is a frequent cause of exacerbations in adults with asthma and a cold, and there may be group-specific differences in severity of these events. The absence of large differences in viral burden among groups suggests differential lower airway sensitization to the effects of neutrophilic inflammation in the patients having exacerbations.
Asunto(s)
Asma/virología , Infecciones por Picornaviridae/complicaciones , Rhinovirus/aislamiento & purificación , Estaciones del Año , Esputo/virología , Enfermedad Aguda , Adolescente , Adulto , Asma/inmunología , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Volumen Espiratorio Forzado , Humanos , Inflamación/complicaciones , Recuento de Leucocitos , Masculino , Lavado Nasal (Proceso) , Neutrófilos , Ápice del Flujo Espiratorio , Estudios Prospectivos , Recurrencia , Sistema Respiratorio/virología , Índice de Severidad de la Enfermedad , Esputo/citología , Carga ViralRESUMEN
A fundamental aspect of climate change is the potential shifts in flowering phenology and pollen initiation associated with milder winters and warmer seasonal air temperature. Earlier floral anthesis has been suggested, in turn, to have a role in human disease by increasing time of exposure to pollen that causes allergic rhinitis and related asthma. However, earlier floral initiation does not necessarily alter the temporal duration of the pollen season, and, to date, no consistent continental trend in pollen season length has been demonstrated. Here we report that duration of the ragweed (Ambrosia spp.) pollen season has been increasing in recent decades as a function of latitude in North America. Latitudinal effects on increasing season length were associated primarily with a delay in first frost of the fall season and lengthening of the frost free period. Overall, these data indicate a significant increase in the length of the ragweed pollen season by as much as 13-27 d at latitudes above ~44°N since 1995. This is consistent with recent Intergovernmental Panel on Climate Change projections regarding enhanced warming as a function of latitude. If similar warming trends accompany long-term climate change, greater exposure times to seasonal allergens may occur with subsequent effects on public health.
Asunto(s)
Ambrosia , Polen , Estaciones del Año , Temperatura , Asma/etiología , Clima , Humanos , América del Norte , Rinitis Alérgica Estacional/etiologíaRESUMEN
BACKGROUND: Leukotrienes are induced by viral infections. OBJECTIVES: To determine whether treatment with montelukast would improve asthma disease control in patients with mild allergic asthma during an experimentally induced rhinovirus infection. METHODS: Patients with mild allergic asthma were randomized to receive treatment with either montelukast or placebo, and 7 days later both groups were inoculated with human rhinovirus 16. Patients were evaluated at baseline, during the acute infection phase, and during the recovery phase for asthma and cold symptoms by questionnaire. Sputum, nasal lavage fluid, and blood were analyzed for viral shedding and cellular inflammation, and peak expiratory flow was measured daily. RESULTS: A total of 19 patients (11 in the placebo group and 8 in the active group) completed the study. No significant differences were found in asthma control and cold symptom scores between the control and treatment groups. The change in peak expiratory flow from the randomization to acute illness phase was greater in the placebo group than the treatment group (mean, -22 vs 0 L/min; P = .05). During the recovery phase, the percentage of sputum eosinophils increased in the placebo group and remained at baseline levels in the montelukast group (median, 2.7% vs 0.2%; P = .05 between groups). CONCLUSIONS: In this pilot study, montelukast did not improve asthma control or cold symptom scores caused by experimental rhinovirus infection. Analysis of secondary outcomes suggests that montelukast may prevent reductions in lung function and increases in sputum eosinophils caused by common cold infections. Further studies are needed to determine whether these effects are associated with clinically significant improvements in health outcomes during natural colds. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00359073.
Asunto(s)
Acetatos/uso terapéutico , Asma/tratamiento farmacológico , Asma/virología , Resfriado Común/inmunología , Antagonistas de Leucotrieno/uso terapéutico , Quinolinas/uso terapéutico , Rhinovirus/inmunología , Asma/inmunología , Resfriado Común/tratamiento farmacológico , Resfriado Común/virología , Ciclopropanos , Método Doble Ciego , Femenino , Humanos , Masculino , Proyectos Piloto , ARN Viral/química , ARN Viral/genética , Pruebas de Función Respiratoria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rhinovirus/genética , Sulfuros , Adulto JovenRESUMEN
BACKGROUND: Rhinovirus infections are frequent causes of asthma exacerbations. OBJECTIVE: This study was conducted to test whether subjects with and without allergic asthma have different responses to infection and to identify baseline patient risk factors that predict cold outcomes. METHODS: Twenty subjects with mild persistent allergic asthma and 18 healthy subjects were experimentally inoculated with rhinovirus-16. Subjects were evaluated at baseline, during the acute infection, and during recovery for asthma and cold symptoms by using a validated questionnaire. Sputum and nasal lavage fluid were evaluated for viral shedding, cytokines, and cellular inflammation. RESULTS: There were no group-specific significant differences in peak cold symptom scores (10.0 +/- 5.8 vs 11.1 +/- 6.2, asthmatic vs healthy subjects), peak nasal viral titers (log(10) 4.3 +/- 0.8 vs 3.7 +/- 1.4 50% tissue culture infective dose/mL, respectively), or changes in peak flow during the study (10% +/- 10% vs 8% +/- 6%, respectively). Rhinovirus-16 infection increased peak asthma index values in the asthmatic group (median, 6 --> 13; P = .003) but only marginally in the healthy group (median, 4 --> 7; P = .09). More asthmatic subjects had detectable eosinophils in nasal lavage and sputum samples at baseline and during infection, but otherwise, cellular and cytokine responses were similar. Baseline sputum eosinophilia and CXCL8 (IL-8) levels were positively associated with cold symptoms, whereas CCL2 (monocyte chemotactic protein 1) levels were inversely associated with nasal viral shedding. CONCLUSIONS: These findings suggest that subjects with mild allergic asthma and healthy subjects have similar cold symptoms and inflammatory and antiviral responses. In addition, eosinophilia and other selective baseline measures of airway inflammation in subjects with or without asthma might predict respiratory outcomes with rhinovirus infection.
Asunto(s)
Asma/inmunología , Asma/virología , Resfriado Común/complicaciones , Infecciones por Picornaviridae/complicaciones , Rhinovirus , Adulto , Citocinas/biosíntesis , Citocinas/inmunología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Eosinófilos/virología , Femenino , Humanos , Inmunoglobulina E/sangre , Masculino , Líquido del Lavado Nasal/virología , ARN Viral/análisis , Esputo/metabolismo , Esputo/virologíaRESUMEN
BACKGROUND: There is evidence that CD4(+)CD25(high) T-regulatory cells are important for establishing tolerance to allergens, but information in children is limited. OBJECTIVE: To test the hypothesis that greater numbers and function of CD4(+)CD25(high) T cells are associated with a reduced risk of childhood allergies and wheezing. METHODS: A cohort of 151 six-year-old children from atopic families was analyzed for peripheral blood CD4(+)CD25(high) and CD4(+)CD25(int) T cells by flow cytometry and for clinical and immunologic correlates of atopy. The associations between these variables were assessed by regression analysis. RESULTS: Factors positively associated with % CD4(+)CD25(high)/CD4 T cells were male sex, number of positive allergen-specific IgE tests, total IgE, season, and 1-month average total pollen count preceding blood draw. The percentage of CD4(+)CD25(high)/total CD4 T cells did not correlate with induced cytokine production, and correlated negatively with suppressive capacity of CD4(+)CD25(+) T cells (r = -0.45; P = .034). The percentage of CD4(+)CD25(int)/CD4 T cells was 54% higher in pollen-sensitized children compared with nonsensitized children in spring (P = .023 for interaction), and correlated positively with IL-5, IL-10, and IL-13 (P < or = .001 for all). CONCLUSION: Our findings suggest that blood CD4(+)CD25(high) cells are a mixture of activated and regulatory T cells, and that these cells could be seasonally regulated by environmental factors such as pollen exposure. CLINICAL IMPLICATIONS: Seasonal increases in CD4CD25(high) expression in children with allergy may represent systemic immune activation caused by pollen exposures.
Asunto(s)
Hipersensibilidad Inmediata/inmunología , Linfocitos T Reguladores/inmunología , Niño , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Hipersensibilidad Inmediata/diagnóstico , Subunidad alfa del Receptor de Interleucina-2/análisis , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Masculino , Estaciones del AñoRESUMEN
Eosinophils migrate from the vascular circulation to the inflamed airways during asthma exacerbations. While the mechanism(s) of this process is not known, the expression of urokinase-type plasminogen activator receptor (uPAR) has been found to modulate neutrophil adhesion and migration to inflammatory sites. We hypothesized that increased expression of uPAR and its ligand, uPA, enhance eosinophil adhesion in patients with asthma. Patients with allergic asthma underwent segmental bronchoprovocation with allergen; 48 h later, peripheral blood and airway (from bronchoalveolar lavage fluid) eosinophils were isolated. uPA and uPAR protein expression were measured by flow cytometry and Western blot; mRNA was quantified by real-time PCR. Eosinophil adhesion to intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was assessed by eosinophil peroxidase activity. Airway eosinophils expressed significantly more uPA and uPAR protein and uPAR mRNA than peripheral blood eosinophils. Removal of cell-bound uPA and/or addition of exogenous uPA had no effect on blood eosinophil adhesion to ICAM-1 or VCAM-1. In contrast, exogenous uPA stimulated ICAM and VCAM adhesion of airway eosinophils. N-formyl-methionyl-leucyl-phenylalanine-activated airway eosinophil adherence to VCAM-1 and ICAM-1 (VCAM-1, 52.8 +/- 4.7%; ICAM-1, 49.2 +/- 5.3%) was increased over blood eosinophil adhesion (VCAM-1, 38.4 +/- 3.6%; ICAM-1, 27.7 +/- 4.9%; P < 0.05). Removal of cell-bound uPA from airway eosinophils decreased adhesion to blood cell levels; reintroduction of exogenous uPA completely restored adhesion levels. These data suggest that constitutive uPA primes, and exogenous uPA can activate, airway eosinophil adhesion following segmental allergen challenge and that increased uPA expression may be a mechanism of increased eosinophil infiltration and function in asthma.
Asunto(s)
Asma/metabolismo , Bronquios/metabolismo , Adhesión Celular , Eosinófilos/fisiología , ARN Mensajero/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Adolescente , Adulto , Bronquios/citología , Líquido del Lavado Bronquioalveolar/química , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Solubilidad , Regulación hacia Arriba , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Capacidad VitalRESUMEN
BACKGROUND: One mechanism of the eosinophil's contribution to airway inflammation in asthma is through release of cationic granule proteins to cause airway injury. Differences in either the intracellular concentration of granule proteins or the extent of activated degranulation between eosinophils from healthy patients and those with allergy and asthma could, therefore, relate to fundamental differences in this cell's function. OBJECTIVE: To identify phenotypic differences in eosinophil-derived neurotoxin (EDN) content and release in eosinophils from healthy patients, those with allergy, and those with allergy and asthma. METHODS: Peripheral blood eosinophils were isolated by negative anti-CD16 selection. Total intracellular and cytokine-activated release of EDN protein was measured by radioimmunoassay. EDN mRNA was assessed by real-time PCR. RESULTS: Eosinophils from patients with asthma contained significantly more EDN per cell than comparable cells from healthy patients, those with allergy but without asthma, or those with asthma treated with inhaled corticosteroids, but they had concentrations similar to airway eosinophils isolated from bronchoalveolar lavage fluid 48 hours after segmental bronchoprovocation with allergen. Furthermore, this increased granule protein was reflected in more EDN degranulation by IL-5- or GM-CSF-activated eosinophils when calculated as nanograms of protein secreted but not when calculated as a percentage of total EDN release. Levels of EDN mRNA were similar in all subject groups. CONCLUSIONS: These data suggest that peripheral blood eosinophils from subjects with untreated asthma have increased inflammatory capacity, as reflected by greater intracellular concentrations of EDN.
Asunto(s)
Asma/fisiopatología , Hipersensibilidad Inmediata/fisiopatología , Ribonucleasas/sangre , Adolescente , Adulto , Asma/inmunología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Degranulación de la Célula , Neurotoxina Derivada del Eosinófilo , Eosinófilos/inmunología , Eosinófilos/metabolismo , Femenino , Humanos , Hipersensibilidad Inmediata/inmunología , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasas/genéticaRESUMEN
BACKGROUND: Intercellular adhesion molecule 3 (ICAM-3) has recently been identified on the surface of eosinophils. OBJECTIVE: The purpose of this study was to characterize ICAM-3 expression on eosinophils in response to cytokines and to determine whether ligand binding of ICAM-3 modulates inflammatory responses of eosinophils, as it does in other leukocytes. METHODS: To determine effects of ICAM-3 on eosinophil function, we isolated human eosinophils and used a monoclonal antibody directed against the epitope of ICAM-3 that binds to leukocyte-function antigen-1 to mimic binding of ICAM-3 and this natural ligand. We measured granulocyte-macrophage colony stimulating factor (GM-CSF) production by unstimulated eosinophils and eosinophils stimulated with ionomycin (1 micromol/L), both in the presence and absence of this anti-ICAM-3 antibody. RESULTS: We found that 99% of eosinophils expressed ICAM-3, regardless of whether allergic symptoms were present or absent. Expression of ICAM-3 was not enhanced by proinflammatory cytokines. Expression of ICAM-3 was reduced in apoptotic cells and in cells incubated with the combination of GM-CSF and tumor necrosis factor-alpha (n = 3). Antibody binding of ICAM-3, which mimics leukocyte-function antigen-1 binding, had no effect on baseline GM-CSF production but reduced by 80% the production of GM-CSF stimulated by ionomycin (control 1969 pg/mL +/- 1259 SD versus anti-ICAM-3 396 pg/mL +/- 207 SD, n = 8) and reduced GM-CSF mRNA content. CONCLUSIONS: ICAM-3 is highly expressed on the surface of human eosinophils, and downregulation of GM-CSF production by anti-ICAM-3 mAb suggests that ICAM-3 ligation may inhibit eosinophil inflammatory responses and survival.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Eosinófilos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Adulto , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/genética , Citocinas/farmacología , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Inflamación/prevención & control , Molécula 1 de Adhesión Intercelular/fisiología , Ionomicina/farmacología , Persona de Mediana Edad , ARN Mensajero/análisisRESUMEN
IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.
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Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Eosinófilos/enzimología , Eosinófilos/inmunología , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Adulto , Alérgenos/administración & dosificación , Pruebas de Provocación Bronquial , Degranulación de la Célula/efectos de los fármacos , Membrana Celular/inmunología , Ensayo de Inmunoadsorción Enzimática , Eosinófilos/efectos de los fármacos , Femenino , Citometría de Flujo , Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Hipersensibilidad Inmediata/genética , Hipersensibilidad Inmediata/inmunología , Hipersensibilidad Inmediata/patología , Técnicas In Vitro , Interleucina-5/metabolismo , Interleucina-5/farmacología , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina-5 , Proteínas Recombinantes , SolubilidadRESUMEN
In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Ralpha expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Ralpha is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Ralpha mRNA, and BAL eosinophils become refractory to IL-5 for ex vivo degranulation. We hypothesized that IL-5 regulates its receptor through proteolytic release of mIL-5Ralpha, which in turn contributes to the presence of sIL-5Ralpha. Purified human peripheral blood eosinophils were incubated with IL-5 under various conditions and in the presence of different pharmacological agents. A dose-dependent decrease in mIL-5Ralpha was accompanied by an increase in sIL-5Ralpha in the supernatant. IL-5 had no ligand-specific effect on mIL-5Ralpha or sIL-5Ralpha mRNA levels. The matrix metalloproteinase-specific inhibitors BB-94 and GM6001 and tissue inhibitor of metalloproteinase-3 partially inhibited IL-5-mediated loss of mIL-5Ralpha, suggesting that sIL-5Ralpha may be produced by proteolytic cleavage of mIL-5Ralpha. IL-5 transiently reduced surface expression of beta-chain, but had no effect on the expression of GM-CSFRalpha. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Ralpha rendered these cells unable to degranulate in response to further IL-5 stimulation, but they were fully responsive to GM-CSF. These findings suggest that IL-5-activated eosinophils may lose mIL-5Ralpha and release sIL-5Ralpha in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.