Fundamental signals that regulate eosinophil homing to the gastrointestinal tract

Abstract

The histological identification of increased eosinophils in the gastrointestinal tract occurs in numerous clinical disorders; however, there is a limited understanding of the mechanisms regulating eosinophil trafficking into this mucosal surface. The results presented in this study characterize the processes regulating eosinophil homing into the gastrointestinal tract at baseline. Eosinophils were found to be present in the lamina propria of 19-day-old embryos and germ-free adult mice at concentrations comparable to those present in non-germ-free adult mice. Furthermore, eosinophil gastrointestinal levels were not altered by increasing circulating eosinophils after pulmonary allergen challenge. Gastrointestinal eosinophil levels were partially reduced in mice deficient in recombinase activating gene-1 (RAG-1), IL-5, or the beta common chain (betac), but these reductions paralleled reductions in circulating eosinophils. In contrast, mice deficient in eotaxin had a marked reduction in gastrointestinal eosinophils but normal levels of eosinophils in the hematopoietic compartments. Furthermore, eotaxin was important for regulating eosinophil levels, even in the presence of high levels of IL-5. These investigations demonstrate eosinophil homing into the gastrointestinal tract during embryonic development occurring independently of viable intestinal flora. Furthermore, eotaxin is identified as the primary regulator of eosinophil gastrointestinal homing under homeostatic states, and may therefore have a fundamental role in innate immune responses.

Figure 1

Immunohistochemical identification of eosinophils in different segments of the gastrointestinal tract of mice. Eosinophils are found in the lamina propria of the stomach (a), duodenum (b), jejunum (c), ileum (d), colon (e), and cecum (f). In the small intestine (b–d), eosinophils are predominantly located in the crypt region. In the duodenum (b), eosinophils are also seen throughout the length of the villus. Eosinophils were not routinely seen in the mucosal or serosal layers. These representative photomicrographs were taken from 129 SvEv mice. Each section is immunostained with anti-MBP antiserum and processed by nickel/cobalt enhancement of peroxidase activity. Eosinophils are indicated by arrows. ×125.

Figure 2

Quantitation of gastrointestinal eosinophil numbers in embryonic and perinatal mice. The number of eosinophils (a and c) and CD45⁺ cells (b and d) residing in the small intestine of wild-type mice was determined in day- 19 embryos and in postnatal mice during the first 2 weeks of life. In a and b, cell levels were normalized to the villus/crypt unit. In c and d, cell numbers were normalized to the unit area (mm²) of the lamina propria. The mean ± SEM for littermate 129 SvEv mice (n = 5–12 mice in each group) is shown. In b, there was no significant difference between any of the groups (P > 0.05).

Figure 3

Eosinophil levels after pulmonary allergen challenge. Mice (129 SvEv) were challenged with Aspergillus fumigatus allergen intranasally 3 days per week for 3 weeks. Eighteen hours after the last allergen exposure, the mice were sacrificed and the number of eosinophils in the blood (a), BALF (b), and jejunum (c) was analyzed. The data are expressed as mean ± SEM (n = 15–18 mice.

Figure 4

Northern analysis of chemokine expression in the gastrointestinal tract. Total RNA (20 μg) from various gastrointestinal segments of wild-type 129 SvEv 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 MIP-1α, using methods described previously (17). Control RNA is derived from the lungs of IL-4 transgenic Clara cell lung in transgenic mice (39). Each lane represents RNA from a different animal, except the lanes for the tongue and esophagus, which were pooled from multiple animals. Autoradiographs were exposed for 1–2 days. The chemokine cDNA probes were described previously (16), except for MCP-2, which has recently been cloned (M.N. Sarafi and A.D. Luster, unpublished data). The ethidium bromide–stained gel (EtBr) is also shown.

Figure 5

Quantitation of eosinophils in different gastrointestinal segments of wild-type and eotaxin-deficient mice. Eosinophils were counted from tissue sections of wild-type (+/+) and eotaxin-deficient (–/–) mice immunostained with anti-MBP antiserum. Eosinophils are normalized per squared millimeter. Each data point represents an individual mouse. The horizontal line is the mean value. Levels of eosinophils indicated near the ordinate were undetectable.

Figure 6

Gastrointestinal eosinophils in mice carrying a combination of the CD2 IL-5 transgene and the wild-type or eotaxin gene-targeted allele. The level of eosinophils in the jejunum of littermate mice that are transgenic (Tg) or wild-type (WT) for IL-5, and carrying the wild-type (+/+) or homozygous deletion (–/–) of eotaxin, is shown. Each data point represents an individual mouse. The horizontal line is the mean value.

Figure 7

Quantitation of gastrointestinal eosinophils in mice deficient in IL-5 or βc. The number of eosinophils residing in the small intestine (jejunum) of IL-5 (a) and βc (b) gene-targeted mice was compared with that in control wild-type mice. Each data point represents the number of eosinophils in 1 mouse. The horizontal line is the mean value.

Figure 8

Quantitation of gastrointestinal eosinophil numbers in RAG-1–deficient mice. The number of eosinophils residing in the jejunum of RAG-1 gene-targeted mice was compared with that in control wild-type mice. Each data point represents the number of eosinophils in 1 mouse, and the horizontal line is the mean value.