The present study demonstrates that eosinophils are absent from the skin of CCR3–/– mice and fail to infiltrate their skin following repeated epicutaneous sensitization with OVA. Recruitment of eosinophils to lung parenchyma and into airways following OVA antigen inhalation challenge is also severely impaired in epicutaneously sensitized CCR3–/– mice. Furthermore, epicutaneously sensitized mice fail to develop AHR.
Eosinophils and their product MBP were virtually absent from both sham and OVA-sensitized skin of CCR3–/–
mice (Figure , a and b). Eosinophils were also severely decreased in the lung and BAL fluid of epicutaneously sensitized CCR3–/–
mice challenged with inhalation of OVA (Figure , a and b). However, lack of CCR3 does not affect eosinophil migration in general. Baseline eosinophil numbers in thymus and lung were normal in CCR3–/–
). Furthermore, eosinophils from CCR3–/–
mice instilled into the trachea of WT mice migrate normally to draining lymph nodes (43
). These results suggest that CCR3 plays an essential role in eosinophil recruitment to the skin and the lung.
Eotaxin is an important ligand for CCR3. There is conflicting data regarding the role of eotaxin in eosinophil recruitment to the lung following antigen challenge. In one study, eosinophil numbers in BAL fluid of eotaxin-null mice were reduced by 70% 18 hours, but not 48 hours, after challenge (20
). In another study, eosinophil numbers in BAL fluid of eotaxin-null mice were normal 18 hours after allergen challenge (44
). These data suggest that eotaxin could be redundant in eosinophil recruitment to the lung following antigen challenge. Other CCR3 ligands, such as MCP-3 or RANTES, may also be important for eosinophil recruitment to the lung.
In addition to CCR3, eosinophils may also express CCR1 (45
), IL-8 receptor (46
), and possibly other unidentified chemokine receptors (47
). CCR1 is a receptor for RANTES and MIP-1α. Tissue expression of both of these chemokines is increased in AD and asthma (25
). Nevertheless, our data suggest that CCR1 and IL-8 receptor play a minor role, if any, in eosinophil recruitment into inflamed skin and lung.
A recent study has shown that infusion of eotaxin results in rapid blood eosinophilia and synergizes with IL-5 in eosinophil mobilization (53
), suggesting a role for eotaxin in mobilization of eosinophils from bone marrow. Examination of the physiologic role of eotaxin in eosinophil mobilization using eotaxin-deficient mice has yielded conflicting results. One line of eotaxin-deficient mice showed decreased blood eosinophil counts (20
), and the other line showed normal blood eosinophil counts (44
). Blood eosinophil counts were comparable in CCR3–/–
and WT controls. Given the fact that CCR3 is the only known receptor for eotaxin, these results suggest that eotaxin may not play an important role in the mobilization of eosinophils from the bone marrow in our model. Furthermore, CCR3 deficiency did not interfere with the production of the IL-5 (Figure b), which is well established as a major eosinophil maturation- and mobilization-inducing cytokine.
Mast cells have been reported to express CCR3 as well as other chemokine receptors, which include CCR1, CCR2, CCR5, and CXCR4 (54
). Sham-sensitized skin of CCR3–/–
mice had comparable numbers of mast cells as skin from WT controls (data not shown). This suggests that CCR3 is either redundant or not important for mast cell trafficking to skin. There was no significant increase in mast cell numbers in OVA-sensitized skin sites of WT mice of the 129/BALB/c background or in their CCR3–/–
littermates (data not shown). Therefore the role of CCR3 in the recruitment of mast cells into inflamed skin in our model cannot be determined from the present study.
Th2 but not Th1 cells express CCR3 (15
). Production of IL-4 (Figure a) and IL-5 (Figure b) by splenocytes stimulated with OVA were normal, and serum levels of OVA-specific IgE (Figure3c) were normal in CCR3–/–
mice. These results suggest that CCR3 may not be important for the differentiation of Th2 cells. IL-4 can be expressed by T cells, mast cells, and eosinophils (56
). IL-4 mRNA expression in OVA-sensitized skin is absent in TCRαβ–/–
), but normal in mast cell–deficient (W/Wv
) mice (58
), suggesting that T cells are the major source of skin IL-4 in our model. Expression of the Th2 cytokine, IL-4, in OVA-sensitized skin was comparable in CCR3–/–
mice and WT controls (Figure b), suggesting that CCR3 does not play an important role in the recruitment of Th2 cells to sites of allergic inflammation. In support of this notion is our finding of comparable mRNA expression of the Th2-selective genes, IL-4 (Figure ), IL-5, and GATA-3 (data not shown) in the antigen-challenged lungs of epicutaneously sensitized CCR3–/–
mice and WT controls.
Our present observation that eosinophil recruitment to lung and AHR are both severely diminished in epicutaneously sensitized mice (Figure b and Figure ) does not necessarily mean that the two are causally related. In mice intraperitoneally sensitized with antigen, AHR and lung eosinophilia are often, although not always (59
), dissociated, suggesting that eosinophils may not be important for AHR in this model (60
). We have recently observed that AHR is enhanced, while eosinophil recruitment to lung and BAL is significantly diminished, in CCR3–/–
mice intraperitoneally sensitized with OVA antigen (27
). The different results we obtained in the epicutaneous sensitization and intraperitoneal sensitization models suggest that the mechanisms of development of AHR may differ with different routes of immunization. We have found that mast cells are mobilized to the airway epithelium in intraperitoneally sensitized WT mice (27
), but not in epicutaneously sensitized WT mice, following inhalation challenge (Figure c). Mast cell mobilization into airway epithelium is significantly increased in intraperitoneally sensitized CCR3–/–
), but remains absent in epicutaneously sensitized CCR3–/–
mice (Figure c). These findings suggest that CCR3-independent mobilization of mast cells into the airway epithelium is an important player in AHR in the intraperitoneal sensitization model. In the epicutaneous sensitization model, in the absence of mast cells in airway epithelium CCR3-dependent recruitment of eosinophils may become an important player in AHR. The finding that mast cells are mobilized to the epithelium in intraperitoneally sensitized mice, but not in epicutaneously sensitized mice, suggests that the immune response may differ between the two models. As a matter of fact, the epicutaneous model is more of a predominant Th2 response (24
), while the intraperitoneal model has a stronger Th1 component (63
). Th1 cells in the latter may induce the production of chemokines other than CCR3 ligands (e.g., SDF), which modulate eosinophil and mast cell trafficking. Further work is needed to test this hypothesis.
Taken together, our data suggest CCR3 is essential for eosinophil recruitment to the skin and the lung and for AHR in response to antigen inhalation in epicutaneously sensitized mice. Targeting CCR3 may offer a possible therapy for AD and allergic asthma.