Dissociation by steroids of eosinophilic inflammation from airway hyperresponsiveness in murine airways

Airway inflammation and hyperresponsiveness (AHR) are recognised as major characteristics of bronchial asthma; however, their relationship is still poorly understood. Exposure to allergen causes an increase in airway responsiveness that is associated with an influx of inflammatory cells, particularly eosinophils, into the airways in allergic humans [1] and sensitised mice [2], which suggests a causal relationship between airway inflammation and AHR [3, 4]. However, there is also much published literature suggesting that there is no relationship between allergic airway inflammation and AHR.

In this study we wished to determine whether there was a dissociation between the effective dose of a steroid, dexamethasone, needed to affect antigen-induced AHR in comparison with that needed to affect airway inflammation in the mouse. We have previously described a murine model of asthma that includes non-specific AHR and associated eosinophilia in the airways [5]. In the present study we followed the same sensitising and challenging protocol but decided to determine AHR in conscious, spontaneously breathing, unrestrained mice by whole-body plethysmography [6‐9]. Airway responsiveness was expressed as enhanced pause (P _enh), a calculated value, which is an indirect measurement that is correlated with measurement of airway resistance, impedance and intrapleural pressure in the same animal [6]. This method was chosen instead of our previously used invasive method because it might offer several potential advantages: it is technically less demanding, it allows repeated measurements over a long period and it avoids the use of anaesthetic and mechanical ventilation. However, one possible disadvantage is that one cannot rule out a contribution by the nose and upper respiratory tract to the parameters measured. This method of antigen-induced airway inflammation and AHR is very similar to that of Dohi et al. [9] in which they report a strong correlation between P _enh and eosinophil number in bronchoalveolar lavage (BAL) fluid.

In this study we have shown for the first time that there is dissociation between the dose of steroid needed to affect antigen-induced BAL and lung tissue eosinophilia and that needed to affect AHR. The ED₅₀ dose of dexamethasone required to inhibit AHR is higher than that needed to inhibit eosinophilia. It is possible that eosinophilia has to be completely inhibited to have an effect on AHR; indeed, at 1 mg/kg dexamethasone, eosinophil infiltration into the BAL fluid following challenge is almost completely blocked and at the same dose AHR is also completely reversed. Lung tissue eosinophilia, however, is only inhibited by about 50% at 1 mg/kg dexamethasone, which further indicates the dissociation between eosinophilia and AHR. De Bie et al. [11] showed that dexamethasone (0.5 mg/kg) inhibited both antigen-induced AHR and airway eosinophilia in the mouse; however, using similar doses we found only an effect on eosinophilia. In the study by De Bie et al. [11] they administered the steroid intraperitoneally and employed a different way of measuring AHR, which might account for the difference.

Throughout the literature there are reports of various interventions that affect both allergic AHR and eosinophilia. Antibodies against interleukin-5 (IL-5) have been shown to inhibit both AHR and eosinophilia in the mouse [12‐14]. Both allergic AHR and eosinophilia have been shown to be reduced in the following cases: in mice deficient in ICAM-1 (intercellular cell-adhesion molecule-1) [15] by treatment with an anti-B7-2 (CD86) monoclonal antibody [7, 16] and with an anti-CTLA4-IgG [17]; in Vβ8⁺-deficient mice and BALB/c mice treated with antibodies against Vβ8 [18]; in mice lacking a functioning 5-lipoxygenase enzyme [19]; in interferon-β-treated mice [20]; in IL-12 treated mice [21, 22]; and in mice treated with an immunosuppressive agent, FK-506 [8].

There are reports of interventions inhibiting allergic eosinophilia but not AHR: in humans, an IL-5-blocking monoclonal antibody [23]; in mice, antibodies against IL-5 [24‐26] and IL-5 knockout animals [27]. Other interventions have been shown to have the reverse effect, inhibiting allergic AHR without affecting eosinophilia: antibodies against interferon-γ in mice [26], antibodies against IL-16 in mice [28], IL-10-deficient mice [29] and mast-cell-deficient mice [24, 25]; Tournoy et al. [30] showed that by lowering the allergic challenge eosinophilia was lost but AHR remained.

Treatment with dexamethasone inhibited other leukocytes measured in the lung with ED₅₀ values comparable to those determined for eosinophilia (Table 1). This would suggest that these inflammatory cells are also not associated with AHR; indeed, neutrophil numbers in the BAL fluid and tissue were not reduced to unchallenged levels by any dose of steroid used here (Table 2), whereas AHR was completely reversed. The involvement in AHR of other leukocytes measured here cannot be completely ruled out because it might be necessary to completely inhibit their infiltration into the lung before any impact on AHR is observed. Increased levels of macrophages, monocytes and lymphocytes in the lung were only completely inhibited at 1 mg/kg of dexamethasone, which is the corresponding dose needed to block AHR.

There is therefore a wealth of literature on the association between allergic eosinophilia and AHR that is sometimes confusing and contradictory. This is the first study that has addressed this question with a range of doses of corticosteroid, compounds known to block AHR and eosinophilia in all animal models of asthma and to affect inflammation and AHR in asthmatics in a clinical setting. We feel that this novel pharmacological approach has revealed a clear dissociation between eosinophilia and AHR in the same animal and this concurs with a study in humans showing no correlation between AHR and the number of inflammatory cells in sputum or bronchoalveolar lavage [31]. These data question the rationale that many pharmaceutical and biotechnology companies have adopted in embarking on drug discovery programmes that target the eosinophil activation/infiltration signalling pathways (e.g. IL-5, VLA-4 and CCR-3).

These data suggest that other factors, such as airway wall remodelling, activation state of the eosinophils, T-cell activation or autonomic dysfunction, might be more important in the development of AHR.

Dissociation was observed between the dose of steroid needed to affect AHR compared with that required to affect inflammation, suggesting that AHR is not a direct consequence of eosinophilia. This novel pharmacological approach has revealed a clear dissociation between eosinophilia and AHR by using steroids that are the mainstay of asthma therapy. If the eosinophil is not associated with AHR, as this result suggests, the information described here is vitally important given that many pharmaceutical companies are engaged in developing low-molecular-mass compounds that target eosinophil activation/recruitment for the treatment of asthma.