We hypothesized that MCP-1 and its receptor, CCR2, are independently required for the development of Aspergillus-antigen-induced allergic asthma. We found that wild-type C57BL/6 mice challenged with Aspergillus antigen developed robust Th2 responses associated with pulmonary inflammation, AHR, mucus production and fibrosis. Surprisingly, neither MCP-1 nor CCR2 was critical for the development of these lung pathologies, since robust responses were also seen in mice with deletions of genes encoding either protein. These results demonstrate that neither MCP-1 nor CCR2 are required for the development of experimental allergic asthma induced by exposure to Aspergillus antigen.
Our results stand in contrast to some previous reports showing important roles for MCP-1 or CCR2 in other models of allergic asthma [25
]. Although the precise explanation of these differences is not clear, there are several experimental factors that may contribute. For example, the choice of antigen and the route of sensitization differ between models. We used antigens prepared from Aspergillus
, an important allergen in some people with asthma, and administered it exclusively to the respiratory tract, presumably a relevant route for sensitization in asthma. Previous studies have used ovalbumin [25
] or cockroach antigen [27
] and have used intraperitoneal antigen injections to sensitize prior to antigen challenge. CCR2-deficient mice have been shown to have defects in recruitment of antigen-presenting cells to the peritoneum [21
], suggesting that CCR2 could be important for sensitization when antigen is administered to the peritoneum. Another factor that differs between studies is timing. We studied mice at 4 days after the final allergen challenge, when all aspects of the Aspergillus
antigen-induced experimental asthma phenotype are present. Campbell et al.
found that the administration of MCP-1 antibody could inhibit AHR in cockroach antigen sensitized and challenged mice at very early time points (1 and 8 h post challenge) but not later (24 h after challenge) [27
]. The effect on AHR at 1 and 8 h was ascribed to MCP-1's ability to activate mast cells, which are important in some asthma models but not in others [36
]. Genetic background may also be an important factor, since mouse strains vary widely in their response to airway antigen challenge [37
]. Previous experimental asthma studies involving CCR2-deficient mice have used mice of mixed genetic backgrounds [25
], whereas we used mice that had been backcrossed nine times to C57BL/6 and therefore have a more homogenous genetic background. Some of the specifics of our experimental system may therefore account for the lack of a requirement for MCP-1 and CCR2. However, MacLean et al.
] used an allergic asthma model involving ovalbumin, intraperitoneal sensitization, and mice of mixed genetic backgrounds and found that CCR2-deficient mice had intact responses to allergen challenge. This indicates that the lack of a requirement for CCR2 is not unique to a single asthma model. It also highlights the difficulty in pinpointing the experimental factors that account for the diverse results reported by various investigators.
Of note, neither MCP-1 nor CCR2 was critical for inflammatory cell migration to the lungs after Aspergillus
antigen challenge. We found that Aspergillus
antigen-induced monocyte recruitment (as measured by counting BAL fluid macrophages) was intact in both MCP-1- and CCR2-deficient mice. While intact alveolar macrophage recruitment in response to airway instillation of Saccharopolyspora rectivirgula
has been reported in CCR2-deficient mice [38
], other in vivo
models have demonstrated requirements for MCP-1 and CCR2 in monocyte/macrophage recruitment [19
]. Our finding indicates that other chemoattractants are sufficient for maximal monocyte/macrophage recruitment in this Aspergillus
antigen model. In support of this observation, a recent microarray-based analysis of gene expression changes in a similar asthma model found that 14 different chemokines (including MCP-1/JE) were induced by Aspergillus
antigen challenge [43
]. However, we did find that MCP-1 and CCR2 may have indirect effects on eosinophil recruitment in response to Aspergillus
antigen. While there was marked eosinophil recruitment to the lungs in MCP-1- and CCR2-deficient mice, there was a trend toward fewer eosinophils than in wild-type mice. Since MCP-1 is not a chemoattractant for eosinophils (which lack CCR2), this trend suggests that MCP-1 may have indirect effects on eosinophil recruitment in this model. A more dramatic decrease of eosinophil recruitment has been seen following neutralization of MCP-1 in another model, but that effect was associated with other signs of impaired Th2 immunity [33
]. Although there may be some role for MCP-1 and CCR2 in eosinophil recruitment, robust inflammatory responses to Aspergillus
antigen occurred even in the complete absence of either of these molecules.
In contrast to our results indicating a robust Th2 response in MCP-1- and CCR2-deficient mice after Aspergillus
antigen challenge, diminished Th2 cytokine production has been reported in studies of MCP-1 neutralization or deletion in different models [19
]. In studies involving CCR2-deficient mice, the results have been more heterogenous, suggesting that CCR2 deletion may increase [25
], decrease [24
], or have no effect on Th2 responses [26
]. As mentioned previously, the explanation for these different Th2 responses in CCR2-deficient mice is not clear, and may suggest that complex pathways involving other CCR2 ligands or MCP-1 receptors [46
] are operational in different models of inflammation. However, if these pathways exist and were important in the model we used, we would have expected to find that deletion of MCP-1 and CCR2 had different effects. Instead, we observed that MCP-1- and CCR2-deficient mice were similar in all respects, including cytokine production, IgE production, and AHR. Our results support the idea that the role of MCP-1 and CCR2 in the development of allergic responses may be dependent upon the experimental model used.
The role of MCP-1 and CCR2 in the development of allergen-induced airway fibrosis has not been extensively explored. Previous findings of increased pulmonary fibrosis in CCR2-deficient mice compared to wild-type mice after treatment with Aspergillus
conidia were accompanied by neutrophilic inflammation and the inability of CCR2-deficient mice to clear the organism normally [28
]. Consequently, the persistence of Aspergillus
organisms in the airway may have altered the fibrotic response. Other studies involving different experimental systems have suggested that MCP-1 and CCR2 may directly or indirectly contribute to the development of fibrosis. Gharaee-Kermani et al. [47
] found that MCP-1 directly induced increased production of collagen by cultured fibroblasts, although the role of CCR2 was not explored in that report. MCP-1 and CCR2 may also indirectly influence fibrosis via their effects on inflammatory cells. Previous studies showed that CCR2-deficient mice developed less pulmonary fibrosis in response to three different stimuli, including intratracheal bleomycin instillation, than did wild-type mice [48
]; however, those studies did not test the requirement for MCP-1 in the development of fibrosis. In C57BL/6 mice, bleomycin induces a robust inflammatory response that consists of neutrophils and lymphocytes, with a smaller component of eosinophils [50
], in contrast to our allergen model. Thus, it is possible that the relative abundance or types of recruited cells in response to a particular airway challenge greatly influence the character or extent of lung fibrosis mediated by MCP-1 or CCR2.. Therefore, based on these previously published results we might have expected MCP-1 and CCR2 to be critical to the development of allergen-mediated fibrosis. However, we found that MCP-1-deficient and CCR2-deficient mice each developed marked fibrosis following Aspergillus
antigen challenge, similar to wild-type mice. Our result, in contrast to the reported requirement for CCR2 in the development of bleomycin-induced pulmonary fibrosis, suggests that different cell types and mediators may be operational in allergen-induced airway fibrosis than those observed in bleomycin-induced lung fibrosis.