Rose F. Vrtis

Decreased Expression of Membrane IL-5 Receptor α on Human Eosinophils: I. Loss of Membrane IL-5 Receptor α on Airway Eosinophils and Increased Soluble IL-5 Receptor α in the Airway After Allergen Challenge

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-5Rα on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Rα 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-5Rα on airway eosinophils compared with circulating cells. Furthermore, sIL-5Rα concentrations were elevated in BAL fluid, but steady state levels of sIL-5Rα 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-5Rα on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Rα, the presence of sIL-5Rα, 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.

Detection of pathogenic bacteria during rhinovirus infection is associated with increased respiratory symptoms and asthma exacerbations

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.

Decreased Expression of Membrane IL-5 Receptor α on Human Eosinophils: II. IL-5 Down-Modulates Its Receptor Via a Proteinase-Mediated Process

In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Rα expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Rα is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Rα 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-5Rα, which in turn contributes to the presence of sIL-5Rα. 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-5Rα was accompanied by an increase in sIL-5Rα in the supernatant. IL-5 had no ligand-specific effect on mIL-5Rα or sIL-5Rα 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-5Rα, suggesting that sIL-5Rα may be produced by proteolytic cleavage of mIL-5Rα. IL-5 transiently reduced surface expression of β-chain, but had no effect on the expression of GM-CSFRα. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Rα 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-5Rα and release sIL-5Rα in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.

Stimulus-dependent differences in superoxide anion generation by normal human eosinophils and neutrophils

The role of eosinophils in allergic and hypersensitivity diseases has yet to be fully established and remains limited by techniques to isolate the eosinophil in high purity. Consequently, most studies that evaluate and characterize eosinophil function are conducted with isolates from patients with hypereosinophilia. There is, however, evidence to suggest that isolates from such patients do not represent normal function. Now, with new techniques to isolate and purify eosinophils from normal subjects without eosinophilia, metabolic function of the normal eosinophil can be assessed. To accomplish this, granulocytes from healthy volunteers were separated by continuous density Percoll gradients into populations of purified eosinophils (90.3 +/- 1.9%) and neutrophils (98.2 +/- 0.4%). Superoxide (O2-) generation was measured with a microassay of superoxide dismutase-inhibitable cytochrome c reduction in response to several soluble and particulate agonists. Normal eosinophils generated significantly more O2- in response to either phorbol myristate acetate or calcium ionophore A23187 than their matched neutrophil fractions. In contrast, differences in granulocyte response to zymosan and chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine, were dependent on the presence of cytochalasin B (CB) in the reaction. N-formyl-methionyl-leucyl-phenylalanine-stimulated eosinophils generated less O2- in the absence of CB but similar amounts in the presence of CB, compared to neutrophils. Activation by zymosan in the presence of 10% autologous serum generated similar amounts of O2- in all the cell populations when CB was present; however, in the absence of CB, neutrophils produced less O2- when they were compared to eosinophils. Therefore, normal eosinophils respond differently to some activators, compared to neutrophils, and these differences may prove significant as the contribution of eosinophils to inflammation becomes established.

Lower Airway Rhinovirus Burden and the Seasonal Risk of Asthma Exacerbation

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.

The Relationship of Rhinovirus-Associated Asthma Hospitalizations with Inhaled Corticosteroids and Smoking

Abstract BackgroundAlthough rhinovirus (RV) respiratory infections trigger asthma exacerbations, the etiologic association between this virus and severe exacerbations, as well as the clinical characteristics of adults at risk for RV-associated asthma that necessitates hospitalization, have not been established MethodsDuring 1999–2003, we conducted a cohort study of 101 adults prospectively enrolled at hospital admission for an asthma exacerbation. Patient characteristics and frequencies of RV in nasal specimens were analyzed, by reverse-transcription polymerase chain reaction (RT-PCR), at asthma-related hospital admission and at a 3-month convalescent follow-up visit ResultsRV was detected by RT-PCR in 21% of hospitalized patients over a 4-year period and in 1.3% of patients who returned for a 3-month follow-up visit. RV detection was strongly associated with hospitalization for asthma (adjusted odds ratio [OR], 15.1 [95% confidence interval {CI}, 1.88–121.4]). After adjustment for baseline asthma severity, RV-positive patients were more likely than RV-negative patients to be current smokers and nonusers of inhaled corticosteroids (ICSs) (adjusted OR, 11.18 [95% CI, 2.37–52.81]; P=.002) ConclusionsRV respiratory infection is an etiologic agent in severe asthma exacerbations necessitating hospitalization in adults. Compared with hospitalized patients with asthma who were RV negative, RV-positive patients were significantly more likely to be smokers and nonusers of ICSs

Similar colds in subjects with allergic asthma and nonatopic subjects after inoculation with rhinovirus-16.

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.

Increased H1N1 Infection Rate in Children with Asthma

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.

The presence of hypodense eosinophils and diminished chemiluminescence response in asthma

Although peripheral blood eosinophilia is a prominent feature of asthma, the contribution of eosinophils to asthma has yet to be fully comprehended. Furthermore, study of isolated eosinophil function in asthma has been complicated by difficult purification methods and, now, the presence of hypodense eosinophils. In our study, eosinophils were isolated from normal subjects and patients with asthma. Two principal evaluations were performed: (1) a comparison of the density-gradient profiles on peripheral blood leukocytes from normal subjects and patients with asthma and (2) a comparison of the chemiluminescence (CL) response with normal dense eosinophils from these two study groups. Granulocyte preparations were initially isolated from Ficoll-Hypaque gradients and were then applied to a continuous Percoll density gradient. In asthma, 40.8 +/- 5.8% of the peripheral blood eosinophils were hypodense (defined as a density less than 1.081 gm/ml), whereas normal subjects had only 9.1 +/- 1.9% of this subpopulation (p less than 0.01). Functional assessment of purified (greater than 90%) normal dense eosinophils was made by measurement of CL to opsonized zymosan particles and the soluble stimulus phorbol myristate acetate. In asthma, eosinophil CL to zymosan, but not phorbol myristate acetate, was significantly less. Differences in eosinophil CL between normal subjects and subjects with asthma did not correlate with the severity of airway obstruction or the peripheral blood eosinophil count. The reasons for the appearance of hypodense eosinophils and diminished metabolic activity in asthma are not established but raise the possibility that their presence represents previous eosinophil activation.

Rhinovirus-Specific T Cells Recognize both Shared and Serotype-Restricted Viral Epitopes

To characterize rhinovirus (RV)-specific T cells, RV16- and RV49-specific CD4 T cells were cloned from peripheral blood, and cytokine secretion and serotype specificity were defined. Each RV-specific clone secreted high levels of interferon-gamma, and several also produced interleukin-4 and -5. To test serotype specificity, each clone was incubated separately with five different RV serotypes. Although 2 of 31 clones proliferated only in response to the virus used in cloning, the rest had significant proliferation in response to 2-5 different serotypes. Thus, RV-specific T cells can be activated by either serotype-specific or shared viral epitopes, raising the possibility that repeated activation of T cells by shared viral determinants in vivo could induce potent recall T cell responses. It is likely that enhanced T cell responses to shared viral epitopes contribute to antiviral activity, airway inflammation, or both.