Airway inflammation and AHR are hallmarks of allergic asthma. There are many reports in humans and in animal models linking airway inflammation and altered airway function to increases in Th2 cytokine and/or chemokine levels produced by immune cells (27
); the common theme is that these factors produce AHR indirectly through the recruitment of inflammatory cells. However, there remains significant controversy as to which cytokine(s) or chemokine(s) are essential to one or both processes. Despite their importance, only limited data are available with respect to the kinetics of development and maintenance or persistence of airway immune/inflammatory responses and AHR after repetitive allergen exposure of sensitized hosts. The primary purpose of the present study was to examine the kinetics of the development and resolution of AHR and airway inflammation after repetitive allergen exposure and relate these changes, when possible, to cytokine and chemokine responses.
A characteristic and consistent feature of the murine response to sensitization and short-term airway allergen challenge is the development of AHR in response to inhaled MCh. Changes in lung function, measured in a variety of ways, have been demonstrated in both BALB/c and C57BL/6 mice. Although there are strain-related differences in the extent, for example, of changes in lung resistance versus compliance (24
), or MCh reactivity (24
), AHR remains a consistent outcome of sensitization and short-term airway challenge. Extending airway challenge with the addition of four or eight more exposures over an additional 2 to 4 wk resulted in a marked attenuation of AHR in both strains of mice. In BALB/c mice, AHR was virtually abolished after seven challenges, whereas C57BL/6 mice showed a progressive decrease in AHR with increasing challenges. These decreases in AHR were observed throughout the MCh dose–response curve and in measures of both Rl
These significant decreases in AHR occurred despite the maintenance of a significant increase in numbers of BAL fluid and lung tissue eosinophils through seven inhalation challenges with allergen. At face value, the data suggest the absence of a correlation between lung and BAL fluid eosinophilia and persistence of AHR. By 11 challenges, this dissociation was no longer apparent, as airway function normalized. The role of eosinophils in AHR is controversial in many species, including humans (29
) and mice (31
). Even after sensitization and short-term challenge, discrepancies have been reported (33
). We and others (31
) have suggested that eosinophils and IL-5 are linked to AHR, in contrast to another report (35
), at least when sensitization is followed by limited airway challenge (one to three intranasal or nebulized challenges). Moreover, with respect to the temporal sequence of these events, lung tissue eosinophilia has been better correlated with AHR than BAL fluid eosinophil numbers (24
). Exceptions to the association of AHR and BAL fluid eosinophilia may be attributed to differences in the challenge protocol and readouts of lung function (36
), although the exact reason(s) remain unclear. In a previous study, we also demonstrated that after secondary challenge, the role of eosinophils in the development of AHR became more complicated, and, to some extent, eosinophil independent (37
The persistence of lung and BAL fluid eosinophilia in the absence of AHR after seven challenges could be the result of several possibilities. One possibility is that the data simply reflect differences in the kinetics of individual responses. A second possibility is that eosinophilia is not linked to development of AHR; this is possible but less likely on the basis of the data discussed above. These discrepancies are reinforced by two contradictory reports using “eosinophil-less” mice (38
). A third alternative is that the eosinophils persist for longer periods in the lung tissue (34
), but are no longer activated so as to result in AHR. As there are no good markers of eosinophil activation, this question remains open, and could explain the shift in kinetics or dissociation of eosinophilia and AHR. Since other factors (eotaxin-1, RANTES, and GM-CSF) affecting eosinophil recruitment, survival, and activation were elevated, at least after seven challenges, this may account for the persistent eosinophilia but an eosinophil activation factor present after short-term challenge, such as IL-5, was no longer available. However, our results showed that the levels of RANTES or GM-CSF in BAL fluid were not correlated to the persistence of AHR in mice after repeated OVA challenges.
It is also striking that other responses implicated in the development of AHR also persisted after repetitive challenges despite the normalization of lung function in response to inhaled MCh. CD4+
T cells are thought to play a central role in the development of AHR and lung inflammation (40
). Depletion of CD4+
T cells during the sensitization phase prevented the subsequent development of these responses (41
T cells are also responsible for the production, at least in part, of the Th2 cytokines IL-4, IL-5, and IL-13 (11
). Virtually all the pathophysiological manifestations of allergen-induced airway inflammation and AHR can be accounted for by these cytokines (42
). After increasing numbers of challenges, the levels of these Th2 cytokines in BAL fluid were significantly reduced despite persistent elevation of CD4+
T cells in lung tissue. If CD4+
T cells are primarily responsible for Th2 cytokine production, one implication is that after long-term airway challenge, the functional activity of these lung T cells or their phenotype is altered. Although Th2 CD4+
T cells may be essential in the induction phase of allergic responses in the lung, their role over time may become modified (45
). It is also possible that with repeated challenges they become “tolerized”—that is, no longer responsive to allergen— although we have no direct data supporting this conclusion.
Despite the decreases in levels of IL-13 after seven challenges, goblet cell metaplasia/mucus hyperproduction persisted. However, after 11 challenges, this increase in PAS-positive cell numbers was also resolving. IL-13 appears essential for goblet cell metaplasia and mucus production (44
). Rather than involving an undefined pathway maintaining this response over seven challenges, the results likely reflect a simple shift in kinetics, with levels of IL-13 falling more rapidly than the disappearance of PAS-positive cells. As observed with eosinophils, continued monitoring for an additional 2 wk showed a loss of these PAS-positive cells.
In contrast to the decreases in Th2 cytokine production, increases were seen in other cytokine levels. IL-12 levels were increased in the BAL fluid of BALB/c and C57BL/6 mice, but with different kinetics. IL-12 is an important regulator of the balance between Th1 and Th2 cells (46
). IL-12 induces production of IFN-γ (47
), but we saw no concomitant increases in the levels of IFN-γ in BAL fluid. In similar murine models, IL-12 administration did inhibit lung eosinophilia and AHR, as well as decrease IL-4 and IL-5, and increase IFN-γ (48
). IL-12 effects may also be IFN-γ independent (49
). However, it seems unlikely that the dissociation between AHR and lung and BAL fluid eosinophilia after seven challenges was related to IL-12 levels. This dissociation was not seen after 11 challenges in both strains, and elevated IL-12 levels at this time point were associated with the inhibition of AHR as well as the attenuation of lung and BAL fluid eosinophilia.
Common to both strains after long-term challenge was the increase in BAL fluid IL-10 levels. These IL-10 levels were sustained for a further 2 wk (11 challenges). In general, IL-10 is considered to be an antiinflammatory factor and the release of IL-10 in asthma serves to downregulate the inflammatory reaction associated with this disorder (50
). IL-10 is produced by CD4+
regulatory T cells, which have been shown to play a critical role in several models of tolerance (53
). Although in our study it is not clear that tolerance was induced by long-term challenge, the increases in IL-10 with repeated challenge could have contributed to the loss of AHR, the reduction in lung BAL fluid eosinophilia, and perhaps the prevention of eosinophil activation. We also considered “tolerance” from the point of view of oral tolerance developing as a result of licking of the pelt over time with repeated exposures. Initial experiments using repeated intranasal challenges did not alter the character of the responses, but simply shifted the kinetics by 1 to 2 wk (data not shown).
Considerable efforts have been made by several laboratories to develop a murine model of chronic allergic airway inflammation and AHR (55
Table E1 in the online supplement). The presumption in these studies is that chronic allergic airway inflammation, expressing an eosinophilic signature, is maintained by long-term (repetitive) airway allergen exposure, which leads to structural airway remodeling and, as a result, persistent AHR. Although the experimental designs varied considerably between the studies (i.e., strains of mice, type of allergen, route of sensitization, and duration or number of airway challenges), the endpoint measurements were common and included AHR, airway eosinophilia, and airway tissue remodeling (goblet cell metaplasia, collagen deposition, and thickness of the airway smooth muscle layer). Of significance in evaluating and comparing these studies is the implication of a specific component in the ability to elicit chronic and persistent changes. Thus, the strain of mouse, the specific allergen, the dose and number of allergen challenges, the route of administration, and the intervals between exposures all have been interpreted to explain a successful outcome. There are also considerable differences in what constitutes a persistent change in, for example, lung function or eosinophilic inflammation, where for the most part, all have shown attenuation of both of these responses with increasing allergen exposure, with rare exceptions (60
). The demonstration of increased collagen deposition, smooth muscle hypertrophy, or limited airway eosinophilia with increasing allergen exposure, findings we did not observe, is also subject to interpretation. Careful morphometry, avoidance of tangential sections, and exclusion of areas around blood vessels are absolutely necessary to show such chronic changes, especially in the face of normalization of lung function.
Concern has also been raised that commercial preparations of OVA are contaminated with endotoxin and that endotoxin coadministration with OVA creates a state of tolerance (61
). Somewhat to the contrary, endotoxin contamination has also been stated to be necessary for Th2 responses, at least in some strains of mice (62
). We have seen little difference in results when using preparations of OVA assayed to be “endotoxin free.”
A number of studies have focused on the functional diversity of DCs in the lung parenchyma and airways (63
), and the role these cells play in the priming and activation of T lymphocytes (65
). The important contribution of DCs in asthma pathogenesis has also been described (16
). The role of DCs after chronic or repeated allergen challenge has not been well defined. In the present study, we determined whether alterations in DC function might account for the progressive decreases in AHR and airway inflammation after increasing challenges, while numbers and expression of CD80 and CD86 were significantly decreased after 11 versus 3 challenges. Further, we investigated whether there were changes in DC numbers or phenotype in the peribronchial lymph nodes, and none were detected. It is unclear at present whether the decrease in number and accessory molecule expression of lung mDCs was responsible for the T-cell unresponsiveness to allergen after increased challenges and whether this was linked to the increase in IL-10 seen after 11 challenges. A number of reports suggested that IL-10 attenuates the differentiation and maturation of DCs in vitro
, especially mDCs (67
). Immature mDCs, generated under these high IL-10 conditions, may induce T-cell tolerance (69
), thus playing a role in the resolution of AHR and airway allergic inflammation after repeated allergen challenge. de Heer and coworkers reported that pDCs provided intrinsic protection against inflammatory responses to antigen (19
). However, in our experiments, pDCs (CD11c+
) were not increased in the lung parenchyma or draining lymph nodes. To directly address the issue of the role of lung mDCs in the resolution of AHR and allergic airway inflammation, transfer experiments were performed. Transfer of antigen-pulsed BMDCs to mice receiving 11 allergen challenges restored responsiveness to allergen, with development of AHR and eosinophilic inflammation. These data suggest that DCs can sustain T-cell–mediated allergic responses in the airways. The mechanism(s) contributing to the restoration of AHR and airway eosinophilia by instillation of mDCs after repeated allergen challenges are not clear. One possibility is that transferred DCs reactivated previously stimulated, antigen-specific T cells. Alternatively, and perhaps more likely, transferred DCs could activate newly primed T cells. BMDCs have the potential to generate primed T cells from naive T cells. Another possibility is that DCs directly cause AHR. Several reports have demonstrated that myeloid DCs have the capacity to produce several cytokines, not only IL-12 and IL-10, but also IL-13, which may directly contribute to airway smooth muscle hypercontractility.
In summary, this study demonstrates that repeated allergen challenge in two strains of mice results in the loss of AHR to inhaled MCh despite the persistence of airway eosinophilia, at least for a period beyond normalization of lung function. Together with the loss of AHR, there were significant decreases in Th2 cytokine production but consistent and sustained increases in IL-10 and IL-12 levels. Repeated challenge was associated with a reduction in the number of lung mDCs, and transfer of antigen-pulsed DCs reversed this attenuation of AHR and eosinophilic inflammation. This decrease in allergic responsiveness with repeated challenge has plagued the development of a true chronic model of allergen exposure in mice. Nonetheless, the data reveal potential avenues of intervention in preventing some of the long-term consequences of repeated allergen exposure.