Discussion
The results from the present study demonstrate that wounds inflicted on well-differentiated respiratory epithelium, grown from primary NECs in ALI culture, heal at different rates depending on the atopic and asthmatic status of the individual donors. Healing is slowest in atopic asthmatics. However, atopic status appears to have more influence than asthmatic status. In addition, our data strongly suggest that the regular use of inhaled corticosteroids prior to nasal cells being harvested improves wound healing in our ex vivo model. Finally, wounded epithelium takes longer to heal when the wound occurs at the peak of an RSV infection, regardless of health status, and that prior regular use of inhaled corticosteroids improves wound healing under these circumstances as well.
Before discussing the implications of our findings, some technical issues need to be acknowledged. Wound healing involves three processes: spreading, migration and proliferation 4,6. Our model only addresses spreading and migration as our end-point is when the wound is physically closed. Thus we are not able to comment on proliferative events. Our ex vivo model of wound closure uses a fully-differentiated epithelium grown from primary NECs in ALI culture. Many previous studies have wounded monolayer cultures of tracheal epithelial cells 15-18 . A major advantage of ALI cultures is they contain the full variety of epithelial cells6,19; this is not the case with monolayer cultures, which consist of basal cells only. Thus the results from the present study may differ from monolayer wounding studies. However, the general impairment of wound repair we report following RSV infection in ALI culture is similar to previous reports of delayed wound repair following rhinovirus infection of monolayer cultures 16. Finally, ALI cultures are not well suited to measuring cytokine secretion into the surface lining fluid. The surface lining fluid layerin vitro is thin and the maximum volume that can be removed is around 1-2µL without the addition of a wash solution and even this stimulates the cells 20 . Thus lack of cytokine responses in the present study should be treated with caution and is unlikely to reflect in vivo conditions.
Previous monolayer culture studies, reported incomplete wound healing in asthmatic children 15,17,18 . In the present study, delayed wound repair was related more close to atopy than asthma; cultures from non-atopic asthmatics healed at similar rates to those from healthy subjects and delayed healing was seen in atopic non-asthmatics. Atopy is associated with increased levels of the T-helper(Th)-2 cytokine, IL-13 12. IL-13 induces epithelial proliferation by inducing TGF α, which in turn binds to the EGF receptor (EGFR) on epithelial cells 12. In the present study, we confirm that signalling through EGFR is necessary for epithelial wound repair; repair rate was markedly reduced by blocking EGFR. However, adding additional EGF did not hasten wound repair. The role of IL-13 is more difficult to understand. Exogenous IL-13 did reduce wound healing, however, the amount added was at least six orders of magnitude (ng/ml) greater than endogenous production by epithelial cells (pg/ml). Blocking the effects of endogenous IL-13 had no effect on the rate of wound healing. Taken together, these data suggest that excessive production of IL-13 could influence wound healing but not in concentrations made by epithelial cells. One caveat to that premise is that epithelial cells grown in monolayer culture have been shown to secrete high enough levels of IL-13 to facilitate wound healing21. However, that study used 1HAEo cells, a SV-40 transformed cell line. How these results relate to normal epithelial cells is uncertain. Monocyte derived macrophages secrete IL-13 in ng/ml quantities, especially when polarized to the alternatively activated phenotype 22 . IL-13 is also secreted by Th-2 T cells. As such, IL-13 secreted into the airway in vivo could be involved in an exaggerated delay in wound healing in atopic non-asthmatics as well as in atopic asthmatics and warrants further investigation.
The data from the present study confirm previous reports, using different respiratory viruses 16,23, that infection of the respiratory epithelium impairs wound repair in all subjects, but especially in asthmatics. In the context of the present study, these results serve to demonstrate the utility of the model rather than advancing knowledge about epithelial response to external stressors.
Perhaps the most surprising result from the present study was the improved wound healing in subjects taking regular inhaled corticosteroids prior to cell harvesting. There is an implied, if not written, “belief” that harvested cells are unlikely to be influenced by in vivo exposures after several culture passages in vitro . Our data definitely challenge this premise. In our case, these cells can only have been exposed via the systemic circulation, either by the extremely low levels of corticosteroid absorbed into the circulation or by some downstream effect of corticosteroids in the lung. Despite this, the effects of prior use of inhaled corticosteroids in improving the rate of wound healing, including after RSV infection, are clear and indisputable (Table 2). The real challenge is in understanding what these findings mean! By definition, innate immune responses, such as those from epithelial cells, do not have a response memory. However, this concept has recently been challenged by elegant studies showing that skin wounds in mice heal more quickly when the mouse had previously been wounded in the same area 24-26. Whether similar mechanisms operate in human respiratory epithelium remains to be demonstrated.
In summary, data from the present study have demonstrated that atopic subjects per se are more likely to have delayed healing of a wounded respiratory epithelium, however the mechanisms involved remain unclear. Exposure to inhaled corticosteroids prior to harvest improve wound healing in vitro , including following RSV infection.