A large body of data from animal models and asthmatic patients demonstrates that the complement system is activated during the development of AHR (3
). In support of a specific role for activation of the alternative pathway in the full development of altered airway function, we previously showed that therapies which specifically block this pathway prevent the development of experimental asthma (3
). The mechanisms by which the alternative pathway is activated in this model have not been identified. In particular, it was unclear why fH, an effective endogenous inhibitor of the alternative pathway, did not prevent alternative pathway activation during the development of asthma. We hypothesized that the effectiveness of endogenous fH was limited, at least in part, by the nature of its interaction with activating surfaces during the development of AHR and inflammation. In the current study, we addressed this question directly by employing agents that could either enhance or block the interaction of fH with tissue surfaces.
The results demonstrated that endogenous fH did limit alternative complement pathway-mediated injury during the development of AHR, but that this protection was incomplete. We showed that fH gained access to the airways during the development of AHR, and was present in significant quantities in the BAL fluid following provocative challenge of previously sensitized and challenged mice. When the interaction of fH with tissue surfaces was blocked with rH 19-20 in mice that had established allergic airway disease, the levels of C3a in BAL fluid were significantly higher than in vehicle-treated mice, demonstrating that fH does limit complement activation on activating surfaces in the airways during the development of AHR. rH 19-20 does not block fluid phase fH function (15
); therefore, enhanced C3a generation in its presence suggests that the relevant effects are due to surface activation. Mice treated with rH 19-20 also developed a greater degree of AHR, increased infiltration of the airways with eosinophils, goblet cell metaplasia along the airways, and higher levels of IL-5 and IL-13 in the BAL fluid. Treatment with rH 19-20 without allergen challenge did not have an effect on airway responses, however, indicating that fH is not functionally necessary to control airway function or inflammation in the absence of the allergic insult.
In order to test whether improved targeting of fH to activating surfaces could overcome the limitations of endogenous fH, we employed a recombinant complement inhibitor that incorporates the complement regulatory region of fH but also contains the binding region of CR2 for tissue-fixed iC3b/C3d. In this way, the inhibitory potential of fH was modulated through direct targeting to C3 activation fragments. CR2-fH has previously been shown to provide alternative-pathway specific local control of complement activation both in vitro
and in vivo
). Functionally, treatment with this agent during primary allergen challenge significantly reduced the development of AHR, the number of eosinophils in BAL fluid, and goblet cell metaplasia in the airways. Treatment with CR2-fH also decreased the levels of the Th2 cytokines IL-5 and IL-13 in BAL fluid. Conversely, in mice treated with CR2-fH, levels of the Th1 cytokine IFN-γ were increased. Similar inhibitory effects of CR2-fH treatment on the development of AHR and eosinophilic airway inflammation were observed in Rw sensitized and challenged mice. The overall degree of protection in mice treated with CR2-fH was equivalent to that obtained with another targeted complement inhibitor, CR2-Crry, which can control both the classical and alternative pathways. This suggests that fH, when targeted to sites of C3 cleavage, can prevent complement-mediated injury induced by allergen sensitization and challenge as effectively as a broader spectrum inhibitor that blocks both the classical and the alternative pathways.
Given the effectiveness of CR2-fH in preventing the development of AHR and airway inflammation in the primary challenge model, we also sought to determine whether this agent can be used to suppress established allergic airway inflammation induced by a single, provocative allergen challenge in mice that were previously sensitized and challenged. Similar to the findings in the primary allergen challenge model, when treated with CR2-fH at the time of provocative allergen challenge in mice with established allergic airway disease, the degree of airway responsiveness to inhaled methacholine (MCh), the number of eosinophils and the levels of Th2-type cytokines in BAL fluid were significantly decreased, while IFN-γ levels in BAL fluid were increased. As with the primary model, CR2-fH was equally effective as CR2-Crry at controlling these outcomes. These results demonstrated that the CR2-fH was effective at controlling allergen-induced exacerbation of lung allergic responses.
These findings highlight three important points: 1) the limitations of endogenous fH to completely prevent alternative complement pathway-mediated injury in the primary and secondary challenge models was primarily due to deficiencies in the delivery of the molecule to the site where it can function most effectively, 2) targeted complement inhibitors can effectively prevent the full spectrum of allergen-induced changes in AHR, airway inflammation, and Th2 cytokine production, and 3) therapeutic complement inhibitors can ameliorate injury in both the primary (preventing development) and secondary (established disease) models of AHR. The iC3b/C3d binding region of CR2 has been successfully used to develop several targeted complement inhibitors (14
). These targeted inhibitors appear to work at lower concentrations than untargeted complement inhibitors and may pose a lower risk of infection (15
). The efficacy of these agents in both the primary and secondary models of asthma was particularly informative establishing the role of the alternative pathway of complement activation in asthma and the potential for therapeutic intervention.
Mutations in fH have been associated with the development of several diseases, including age-related macular degeneration, atypical hemolytic uremic syndrome, and membranoproliferative glomerulonephritis type 2 (10
). Even in the presence of fully functional fH, however, uncontrolled alternative pathway activation contributes to several different diseases including asthma (24
). The inability of endogenous fH to limit complement activation on the disparate tissues involved in these diseases may be due to the nature of the interaction of fH with the activating surfaces in these diseases. Endogenous fH contains C3b binding regions, but protection of a given surface by fH is also believed to depend upon fH interacting with other surface molecules, such as sialic acid (7
). Thus, the relative degree of activation of the alternative pathway on a particular surface appears to be a function of the interaction between fH and the repertoire of molecules expressed by that surface. It is also possible that the failure of endogenous fH to fully prevent alternative pathway activation in these models was due to an insufficient concentration of fH within the airways. Nevertheless, the systemic administration of CR2-fH provided even greater protection from alternative pathway-mediated injury than that provided by the endogenous fH.
We describe the effectiveness of two CR2-targeted complement inhibitors in preventing the development of AHR and airway inflammation in both primary and secondary models of experimental asthma. The targeted delivery of fH was as effective in these models as the targeted delivery of Crry, indicating that the complement regulatory region of endogenous fH was capable of preventing complement-mediated injury in this disease when directed to sites of complement activation. CR2-fH may have fewer side effects than CR2-Crry since it does not block the classical pathway of complement. Nevertheless, the efficacy of both CR2-fH and CR2-Crry in these models identified the general effectiveness of using CR2 to target complement inhibitors to sites of complement activation. Future approaches could include the targeted delivery of complement regulatory proteins to specific molecular targets expressed within the lung (35
). Given some of the limitations and concerns with current therapies, targeted complement inhibitory therapies hold promise in the treatment of severe asthma.