Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue eosinophilia

Abstract

The chemokines are a large group of chemotactic cytokines that regulate leukocyte trafficking and have recently been shown to inhibit human immunodeficiency virus entry into cells. Eotaxin is a C-C chemokine implicated in the recruitment of eosinophils in a variety of inflammatory disorders and, unlike all other eosinophil chemoattractants, is eosinophil specific. However, given the large number of chemoattractants that have activities on eosinophils, it is unclear whether eotaxin has an important role in vivo. Furthermore, it remains unclear why there is constitutive expression of eotaxin in healthy states in the absence of eosinophilic inflammation. To begin to determine the significance of eotaxin at baseline and during eosinophil-mediated disease processes, we have used targeted gene disruption to generate mice that are deficient in eotaxin. Such mice demonstrate that eotaxin enhances the magnitude of the early (but not late) eosinophil recruitment after antigen challenge in models of asthma and stromal keratitis. Surprisingly, a role for eotaxin in regulating the constitutive number of eosinophils in the peripheral circulation is also demonstrated. These results indicate a contributory role for eotaxin in the generation of peripheral blood and antigen-induced tissue eosinophilia.

Figure 1

Generation of eotaxin-disrupted mice. Shown in (A) is the eotaxin genomic locus, the targeting vector, and the targeted null locus. Vertical rectangles represent exons; black regions in the exons are deleted by the targeting strategy. The targeting vector contains a neomycin-resistant gene (neo) and a herpes simplex virus thymidine kinase (HSV–TK) gene in tandem. In (B), Southern analysis of BamHI-digested genomic DNA from the offspring of a heterozygous cross. Wt and null indicate wild-type and targeted locus, respectively. (C) shows a Northern analysis of skin total RNA (20 mcg) from homozygote null (−/−), heterozygote (+/−), and wild-type mice (+/+). The Northern blot was hybridized with a full-length murine eotaxin cDNA or 28S rRNA probe. Restriction enzyme abbreviations: N, NotI; B, BamHI; Xh, XhoI; R5, EcoRV; X, XbaI.

Figure 2

Eosinophil count in the peripheral blood of naive mice. The absolute eosinophil count in the peripheral blood is shown for wild-type (+/+) and eotaxin null (−/−) mice. The results are expressed as mean ± SEM for +/+ (n = 12) and −/− (n = 14); P = 0.007.

Figure 3

Allergen induced airway eosinophilia. Mice were sensitized systemically to OVA and underwent inhaled OVA challenge. At 18 h after allergen challenge, the lungs were assessed for eotaxin mRNA expression (A) and the eosinophil count in the bronchoalveolar fluid (B). Northern blot analysis of total RNA evaluating eotaxin expression in eotaxin null (−/−) and wild-type mice (+/+). The hybridization of a 28S rRNA cDNA probe is also shown. Each lane represents a different mouse. In (B), the number of eosinophils in the lung fluid is shown as mean ± SEM for wild-type (n = 10), eotaxin null (n = 13), or control unsensitized mice (n = 3); P = 0.005 between +/+ and −/−.

Figure 4

Antigen-induced corneal eosinophilia. Wild-type and eotaxin null mice received four subcutaneous immunizations with antigens from the parasitic helminth O. volvulus. Animals were subsequently injected intracorneally with parasite antigen and sacrificed 1 d later. 5-μm sections of the cornea were immunostained with rabbit sera to eosinophil major basic protein and eosinophils in the corneal stroma were counted. Data are shown as mean ± SEM for wild-type (n = 13) and eotaxin null (n = 14) mice with differences between groups being significant (P = 0.02).