This is the first study to demonstrate impairment of AM phagocytosis in children with poorly controlled asthma. In subjects with moderate and severe asthma, phagocytosis was decreased by more than 50% compared with that seen in adult and pediatric control subjects. AM apoptosis was also greater in asthmatic subjects and increased further with LPS stimulation. These data suggest that the airway innate immune response might be impaired in children with poorly controlled asthma, a finding that might account for the aberrant response to respiratory tract infection commonly observed in this population.14,15
AMs internalize foreign airway particles through a variety of mechanisms, including pinocytosis, receptor-mediated endocytosis, and phagocytosis. Whereas pinocytosis refers to the nonspecific uptake of fluid and solutes, receptor-mediated endocytosis is a specific process in which small particles (typically <0.5 μm) enter cells. In the presence of larger particles (>0.5 μm), phagocytosis occurs either through opsonization of the antigen or unopsonized nonspecific uptake.16
In this study phagocytosis was assessed by adding inactivated S aureus
(particle size, 0.8–1.1 μm) to AMs in culture media containing 2% FBS. Therefore our results do not permit conclusion as to the specific type of phagocytosis observed. Our findings might reflect impairment in both opsonized and unopsonized phagocytosis in children with poorly controlled asthma, although further studies are needed to define the specific factors responsible for our observations.
AM phagocytosis is a complex process triggered by a variety of activation pathways.17
Here we focused on phagocytosis resulting from innate activation of the AM after a bacterial microbial stimulus (S aureus)
. With innate activation, microbes are recognized by pattern-recognition receptors, which induce proinflammatory cytokine production and promote phagocytosis.17
Although it is true that the majority of asthma exacerbations in young children and older school-age children are triggered by respiratory tract viruses and not by acute bacterial infections,2,18
innate AM activation is also important for respiratory viral clearance. Huber et al19
recently observed phagocytosis of influenza virus resulting from direct binding of the opsonized virus to Fc scavenger receptors on the AM surface. Furthermore, adenovirus and respiratory syncytial virus infection result in increased AM Fcγ scavenger receptor expression, suggesting that opsonization and phagocytic engulfment are important for viral clearance.20,21
Direct binding of rhinovirus to the AM cell surface has also been observed,22
although the associated receptors and mechanisms of engulfment remain unclear.22,23
Recently, a 3-fold increase in AM expression of the pattern-recognition Toll-like receptor 4 was observed in rhinovirus-infected children,24
which might contribute to AM engulfment of the virus. Although these studies highlight the importance of innate AM activation in respiratory tract virus clearance, the precise mechanisms involved with this process are far from understood and can vary between viral strains. Further studies of AM phagocytosis as it relates to viral respiratory tract infection in asthmatic children are needed.
Our findings of decreased AM phagocytosis in children with poorly controlled asthma are similar to those previously observed in other chronic airway disorders. In patients with cystic fibrosis25
and chronic obstructive pulmonary disease,26
phagocytosis of bacteria and apoptotic cells is reduced to half of the levels of healthy control subjects. In patients with chronic obstructive pulmonary disease, ex vivo
treatment with broad anti-inflammatory agents, such as lovastatin and azithromycin, improves phagocytic activity by 50% or more.27,28
Although the precise mechanisms responsible for AM dysfunction can vary between disease states, these findings suggest that underlying inflammation might have an important effect on AM function in the human airway.
There are a limited number of studies on AM phagocytosis in patients with asthma, and few have targeted patients with poor asthma control. In one study phagocytosis did not differ between control subjects and subjects with mild intermittent asthma with good symptom control, although the total number of opsonized particles was lower in asthmatic subjects with airway eosinophilia compared with those without.29
In a similar sample the number of opsonized particles in airway macrophages was reduced by approximately 50% six hours after endotoxin (LPS) inhalation.30
These data suggest that even despite good symptom control, asthmatic subjects might be more susceptible to a secondary airway insult. This effect is more pronounced in those with severe disease. Recently, Huynh et al7
observed no phagocytic differences between adults with mild-to-moderate asthma and control subjects, but phagocytosis was impaired by nearly 50% in subjects with severe asthma. LPS stimulation further decreased phagocytosis in subjects with severe asthma, a finding that was reversed with ex vivo
These data suggest that asthmatic AMs might be functionally modulated by airway inflammation, thus rendering the asthmatic patient more susceptible to a secondary airway insult. However, the focus of that study was to determine whether asthma compromised phagocytosis of apoptotic cells, a process critical for the clearance of neutrophils or eosin-ophils recruited to the airspace. In contrast, the goal of the present study was to assess phagocytosis in response to innate immune activation of the AM and clearance of infectious particles.
Although few studies have examined the capacity of asthmatic AMs to phagocytose foreign particles, previous studies have observed increased activation of AMs from asthmatic patients. Compared with control subjects, asthmatic subjects have increased basal spontaneous generation of superoxide anion,31
and regulators of inflammatory gene expression, such as histone acetyltransferase.33
These alterations are further increased with antigen stimulation34
and are accompanied by decreased production of anti-inflammatory cytokines, such as IL-10.35
Taken together, these findings provide evidence that the respiratory burst of asthmatic AMs might be impaired, thus inhibiting microbe killing. However, it is important to note that the AM respiratory burst and phagocytosis are regulated by different cellular mechanisms and might not necessarily occur in parallel.35
Further study of the dynamic relationships between the respiratory burst and phagocytosis is warranted in asthmatic subjects to better define the mechanisms associated with respiratory infection in this population.
This study has a number of limitations. Because bronchoscopy cannot be performed on otherwise healthy children solely for research purposes, our pediatric control group was limited to children with symptomatic respiratory tract illnesses undergoing bronchoscopy for diagnostic purposes. Although this sample was sufficient to detect differences in AM phagocytosis and apoptosis between groups, the AMs isolated from these children might be phenotypically different from those of true pediatric control subjects. The fact that our healthy adult control subjects were significantly older than our pediatric sample also raises the question as to whether age is a determinant of AM function across the lifespan. Because AM function was similar between our adult and pediatric control subjects, it is unlikely that age contributed to our findings, particularly because age was treated as a potential confounder in multivariate analyses. However, additional studies are needed to more adequately address the effect of age on AM function in children.
Because pediatric bronchoscopies were not performed for research purposes, BAL samples from children were pooled before analysis. This practice is common at our institution and provides an increased sample yield for clinical laboratory analysis. Because pooling of the BAL fluid intermixes the bronchial and alveolar airway constituents, it is possible that the macrophages obtained from children for this study were not purely alveolar (or bronchial) in origin. It is also possible that bronchial macrophages and AMs have distinct functional abilities. The BAL samples from adult control subjects were similarly pooled to minimize this potential effect on our results. However, given the differences between adults and children with regard to airway structure and the lavage volumes used, we cannot exclude the possibility that the samples from healthy adults contained more alveolar cells. Further studies are needed to characterize the differences between the bronchial and alveolar cellular constituents, particularly in asthmatic subjects.
It is also possible the differences in AM function that we observed could be attributed to the confounding effects of asthma treatment or other unmeasured clinical variables. Although the effects of unmeasured clinical variables remain unknown, we do not believe that corticosteroid use sufficiently explains the discrepancies in AM function between asthmatic children and control subjects. Because our pediatric control subjects were symptomatic, all children in this group were treated empirically with ICSs for a minimum of 16 weeks, yet AM phagocytosis in this group was similar to that of healthy adult control subjects. A subgroup analysis of children with severe asthma receiving oral corticosteroids further revealed no differences in AM function compared with subjects with severe asthma treated with ICSs alone. Finally, statistical control of ICS dose and other potential confounding variables revealed that asthma severity alone, as defined by this study, was the most significant predictor of AM function. Although this evidence suggests a limited association between corticosteroid use and AM function, additional studies are needed to thoroughly examine this relationship in children with poorly controlled asthma.
Our findings of impaired AM phagocytosis and increased AM apoptosis warrant further study. Although apoptosis correlated with phagocytosis, the association was modest and did not sufficiently account for the differences in phagocytosis observed between groups. Furthermore, despite increased apoptosis in children with severe asthma, the apoptotic cells isolated from these children were not less likely to undergo phagocytosis compared with the other groups. These data suggest that other factors aside from apoptosis are related to the impairment in AM function that we observed.
In conclusion, the mechanisms associated with respiratory tact infection in asthmatic children are not well understood. These data support the hypothesis that AMs are functionally impaired in children with poorly controlled asthma and are characterized by increased apoptosis and decreased phagocytosis of pathogenic bacteria. Although there are limited studies on the epidemiology of bacterial infection in asthma, a recent study found that asthma was a risk factor for invasive pneumococcal disease.36
These findings, when taken into account with those demonstrating an increased prevalence of atypical mycobacteria in subjects with acute asthma,3
suggest that bacterial infection might play a role in asthma morbidity. However, the relationship between bacterial and respiratory tract viral infection in children with asthma is not clear. Further studies are needed to define how impairments in AM phagocytosis relate to bacterial phagocytosis and viral clearance in patients with poorly controlled asthma.