Eotaxin is required for the baseline level of tissue eosinophils

Abstract

Eotaxin is an eosinophil-selective chemokine that is constitutively expressed in a variety of organs such as the intestine. Previous studies have demonstrated that the recruitment of eosinophils during inflammation is partially dependent on eotaxin, but the function of constitutive eotaxin during homeostasis has not been examined. To elucidate the biological role of this molecule, we now examine tissue levels of eosinophils in healthy states in wild-type and eotaxin-deficient mice. The lamina propria of the jejunum of wild-type mice is demonstrated to express eotaxin mRNA, but not mRNA for the related monocyte chemoattractant proteins. Wild-type mice contained readily detectable eosinophils in the lamina propria of the jejunum. In contrast, mice genetically deficient in eotaxin had a large selective reduction in the number of eosinophils residing in the jejunum. The reduction of tissue eosinophils was not limited to the jejunum, because a loss of thymic eosinophils was also observed in eotaxin-deficient mice. These studies demonstrate that eotaxin is a fundamental regulator of the physiological trafficking of eosinophils during healthy states. Because a variety of chemokines are constitutively expressed, their involvement in the baseline trafficking of leukocytes into nonhematopoietic tissue should now be considered.

Figure 1

Northern analysis of chemokine expression in the jejunum. Total RNA (20 μg) from the jejunum of wild-type mice was electrophoresed and transferred to a membrane that was hybridized under conditions of high stringency with cDNA probes for murine eotaxin, MCP-1, MCP-2, MCP-3, MCP-5, RANTES, and 28S ribosomal protein (37) by using methods previously described (17). Control RNA is derived from the lung of interleukin-4-overexpressing clara cell lung transgenic mice. Each numbered lane represents a different animal. Autoradiographs were exposed for 1–4 days. The chemokine cDNA probes were previously described (8), except for MCP-2, which has recently been cloned (M.N.S. and A.D.L., unpublished data).

Figure 2

In situ hybridization of eotaxin mRNA localization in the jejunum. Microscopy of frozen tissue sections hybridized with radiolabeled antisense (a, b, d, and e) or sense (c and f) eotaxin cRNA probes. The sections were washed under high-stringency conditions and autoradiographed for 40 days. Bright-field photomicrographs (a and d) of the same dark-field sections (b, c, e, and f) are shown. (Original magnification is ×36 in a–c and ×90 in d–f.)

Figure 3

High magnification of eotaxin mRNA localization in the lamina propria of the jejunum. Bright-field (A and C) and dark-field (B) microscopy of jejunum hybridized with a radiolabeled eotaxin antisense cRNA probe. In A and B, taken in the same field, hybridization signals are located predominantly at the neck of the villus (arrowheads). In a higher-power magnification (C), hybridization signals are predominantly localized to an aggregation of mononuclear cells (arrows). An eosinophil is indicated (arrowhead) adjacent to the mononuclear cell aggregation. (Original magnification is ×460 in A and B and ×1360 in C.)

Figure 4

Eosinophil distribution in the jejunum of wild-type and eotaxin-deficient mice. The jejunum is stained with Wright’s–Giemsa stain (a–d) and the eosinophils are indicated in the submucosa and lamina propria by arrows. Wild-type (+/+) and eotaxin-deficient (−/−) mice are indicated. The jejunum in wild-type mice (e–g) and eotaxin-deficient mice (h) is stained with anti-MBP, and immunoreactivity is detected by nickel/cobalt enhancement of peroxidase activity. Eosinophils are indicated by arrows. (Original magnification is ×500 in a–d, ×30 in e, ×60 in f and h, and ×125 in g.)

Figure 5

Quantitation of eosinophils in the jejunum. Eosinophils were enumerated from tissue sections that were stained with anti-MBP antiserum. The data are presented for wild-type (+/+) and eotaxin-deficient (−/−) mice. Two eotaxin-deficient mouse lines are indicated by the • and ▪ data points. The horizontal line represents the mean.

Figure 6

Photomicrographs of thymic eosinophils. Tissue sections were analyzed with HAE staining (a and b), Wright’s–Giemsa staining (c and d), cyanide-resistant endogenous peroxidase activity (e and f), or anti-MBP immunoreactivity (g and h). The data are presented for wild-type (+/+; Left) and eotaxin-deficient (−/−; Right) mice. The arrows point to eosinophils.