Infection of airway epithelium by rhinovirus is the most common cause of asthma exacerbations. Even in mild asthma, airway epithelium shows mucous metaplasia, and the degree of this metaplasia increases with increasing severity of the disease. We previously showed that squamous cultures of human airway epithelium show levels of viral infection ~ 100-fold greater than those of mucociliary cultures derived from cells from the same trachea (9
). Here, we further show that the change from the mucociliary to the mucous metaplasia phenotype is also associated with increased susceptibility to rhinoviral infection.
We induced mucous metaplasia with IL-13. Several lines of evidence suggest that this cytokine plays a major role in the induction of mucous metaplasia seen in asthma (26
). IL-13 has been shown to increase in bronchial tissues from patients with asthma (30
). The IL-13 treatment of primary cultures from human airway epithelium increases the ratio of secretory cells to ciliated cells, induces goblet-cell metaplasia, and decreases ciliary beat frequency, and these effects can be reversed by blocking the receptor to IL-13 (25
). IL-13 is produced by most resident airway cells, with differential expression between cell types (33
). IL-13 has been shown to play a direct role in propagating and perpetuating airway inflammation, initiating subepithelial fibrosis, regulating mucous hypersecretion, and causing alterations to epithelial cell function (34
). Polymorphisms in the IL-13 gene have been linked to an increased incidence of, and susceptibility to asthma (35
We found that treatment with 10 ng/ml IL-13 for 7 days increased the release of mucin, as measured by ELISA. Moreover, observable increases occurred in the number of cells that stained for either antibody B6E8 ( and ) or FPAS (), or that appeared to be mucous cells by light microscopy (). The increase in the number of mucous cells was accompanied by a reduction in the degree of ciliation of cultures, with significantly decreased Rte. These results suggest that IL-13–treated cultures represent a good model for the epithelial phenotype of mucous hyperplasia and metaplasia associated with asthma.
The effects of IL-13 on susceptibility to rhinoviral infection appeared entirely attributable to the induction of mucous metaplasia. Thus, the increase in numbers of infected cells equaled the change in numbers of infected goblet cells. Furthermore, the odds of a given mucous cell being infected were increased by IL-13 treatment.
Further evidence that the effects of IL-13 on levels of viral infection were attributable to the induction of mucous metaplasia in mucociliary cultures was obtained by using squamous cultures. Pretreatment of these cultures with IL-13 caused no structural changes whatsoever. In particular, mucous cells did not appear, and levels of rhinoviral infection did not increase.
We investigated the hypothesis that the increased levels of rhinoviral infection seen with mucous metaplasia were attributable to increased levels of ICAM-1 on the apical membrane. Immunocytochemical staining for ICAM-1 was most intense on the apical membrane, with no overt differences in staining pattern or intensity between mucociliary and mucous cultures. Furthermore, total levels of ICAM-1, as determined by Western blotting, were identical in the two cell types (although squamous cells contained about four times more ICAM-1 than either). However, we acknowledge the need for caution in the interpretation of Western blots. Doubtless, ICAM-1 is densest on apical membranes, but the area of a basolateral membrane is greater, and ICAM-1 from this latter membrane (or from the cytoplasm) may make a greater contribution to total levels than anticipated from the immunocytochemical data. Interestingly, total levels of ICAM-1 in squamous cultures were ~ 4 times greater than in mucociliary cultures (), but the levels of virus produced were at least 100 times greater (9
). This finding emphasizes the importance of factors other than binding sites in the total amount of virus produced by any epithelial phenotype.
Next, we tested the hypothesis that rhinovirus preferentially infected cycling or dividing cells. Many examples exist of both DNA and RNA viruses (36
) that require host factors for translation and replication, and that replicate better in dividing than nondividing cells. However, we never observed a colocalization of Ki-67 and rhinovirus. We interpret this result to mean that dividing cells do not have apical membranes, and cannot be infected by rhinovirus. This interpretation is further validated by the finding that cycling cells were only found at the bottom layers of cell sheets in both treatment conditions.
A highly ciliated epithelium, as seen in healthy airways, may sterically block rhinovirus from accessing its receptor ICAM-1. Thus, we speculate that the ability of rhinovirus to bind to ICAM-1 and to be endocytosed is increased in cells with flatter apical surfaces. This would explain the very high levels of infection seen in squamous cultures (9
). Consistent with this hypothesis, we found large apical membranes with reduced ciliation in IL-13–treated sheets. To test this idea further, we compared levels of infection in control cultures of different ages. Cultures after ~10 days showed no ciliation and no goblet cells, and possessed comparatively undifferentiated apical membranes (13
). However, the levels of infection were at least four times greater than in cultures after > 21 days, at which time mucociliary differentiation is well advanced (13
Ezrin is a key component in the clathrin-mediated internalization of rhinovirus (41
). After a 12-day treatment of cultured spheroids from human tracheal epithelium with IL-13, levels of ezrin declined by 30% (25
), although no change was evident after 7 days (the treatment duration used in the present studies). Furthermore, with the transition from a mucociliary to a mucous phenotype, the predominant ezrin staining shifted from the microvilli of ciliated cells to the cytoplasm, although the authors did not indicate whether ciliated or secretory cells were involved (25
). Clearly, such changes in the levels and distribution of ezrin will affect the internalization of rhinovirus, and may be responsible for the increased uptake of virus seen with mucous metaplasia, or alternatively, may alter the effects of potentiating factors that potentiate viral replication.
The increase in the number of goblet cells in IL-13–treated sheets was accompanied by a decrease in Rte
. We therefore considered the possibility that the increased levels of infection in mucous cultures compared with mucociliary cultures may be attributable to reduced barrier function. However, we reject this hypothesis for a number of reasons. First, we show here that in control cells and in cells treated with IL-13, there is no dependence of levels of infection on Rte
. Second, in other work (Sachs and Widdicombe, unpublished findings), we exposed cells to Ca–Mg-free medium. This exposure opened tight junctions, as revealed by a decline in Rte
to zero, but had no effect on the numbers of cells infected with virus. Third, the addition of virus to the basolateral side of the cultures produced levels of infection considerably lower than with additions to the apical surface (Sachs and Widdicombe, unpublished findings). This is consistent with our immunocytochemical finding that ICAM-1 has a predominantly apical localization (). Fourth, we find little reason to believe that pores the size of rhinoviruses exist in airway epithelium in general or in our cultures in particular. Multiple images from both scanning electron microscopy and confocal fluorescence microscopy showed that our cultures, regardless of resistance, were all completely confluent. No gaps were visible in the cell sheet, and ZO-1 staining revealed that every cell was surrounded by an intact tight junction. Hence virus would have to penetrate through tight junctions, and the effective pore size of even the leakiest of these is ~4 Å (42
) Tricellular junctions, i.e., the points where tight junctions branch, constitute an exception to this statement. But even here, the effective pore diameter was estimated as ~ 10 nm at most (44
), although functional studies indicate that it is usually much less (45
Our viral preparation was a crude cell lysate, and thus the increased level of viral infection seen in mucous cells may be attributable to a selective action of these contaminants on this cell type. Unfortunately, such an effect can never be ruled out completely, because eliminating contaminants entirely is impossible. Thus, concentrating a virus with centrifugal filters or similar devices will merely enhance the concentration of virus relative to contaminants. Key contaminants may still be present in the suspension at concentrations supramaximal for the observed effect. However, we think a selective action of contaminants is unlikely for three reasons. First, exposure times to virus were brief (1 hour), thereby minimizing the time available for any contaminants to induce significant changes in the cellular apparatus for the binding and internalization of virus. Second, viral suspension was added to the mucosal surface of confluent cell sheets, and epithelial receptors for cytokines and other bioactive agents are generally on the basolateral membrane. Third, we used virus at a concentration of 105 TCID50/ml, a 63-fold dilution of the original suspension (106.8 TCID50/ml).
We show that mucous metaplasia in vitro
is associated with increased levels of viral infection. Even patients with mild asthma have more goblet cells in their airway epithelium. Moreover, consistent with higher levels of viral infection, patients with asthma contract abnormally severe lower respiratory tract symptoms during colds (46
). Therefore, during a cold, patients with asthma should demonstrate more shedding of virus from the epithelium. However, studies involving experimental inoculations of rhinovirus failed to show an effect of asthma on viral titers in nasal lavages or induced sputum (48
). Several reasons for this discrepancy between in vivo
and in vitro
studies are possible. First, most patients in studies of experimental inoculations in vivo
have comparatively mild asthma, and therefore presumably less mucous metaplasia than with in vitro
systems. Second, comparisons of viral release from patients with asthma and normal subjects in vivo
are made difficult by the extreme variability in levels of virus recovered. For instance, in a study by Message and colleagues (47
), viral titers in nasal lavage or induced sputum varied over at least five orders of magnitude, and although virus retrieved in lavages or sputum from patients with asthma averaged from 0.5–2 logs greater than normal, these differences were not statistically significant. Finally, however good the culture model, the possibility always remains that results obtained in vitro
do not apply to the more complex situation in vivo
In conclusion, our study shows that mucous metaplasia increases the susceptibility of the airway epithelium to rhinovirus infection. Furthermore, our data suggest that the increased susceptibility is attributable to a loss of apical membrane complexity. We induced mucous metaplasia with IL-13, and this agent may also be largely responsible for the mucous metaplasia of airway epithelium in asthma. Our results therefore have important implications concerning IL-13 as a target for asthma therapy. Most of the costs, and most of the morbidity, of asthma are attributable to asthma exacerbations, and most exacerbations are triggered by viral respiratory infections (49
), and especially by rhinovirus. A recent study described the association of increased levels of serum IL-13 with rhinovirus-induced asthmatic symptoms (47
). Allergen ovalbumin (OVA)-challenged mice infected with rhinovirus also have increased levels of serum IL-13 (51
). Therefore, blockade of IL-13 may inhibit the mucous metaplasia often associated with asthma (52
), and by so doing may reduce the frequency and severity of asthma exacerbations.