Chronic aeroallergen during infancy enhances eotaxin-3 expression in airway epithelium and nerves

Abstract

We have documented that exposure of rhesus monkeys to house dust mite aeroallergen during postnatal development resulted in significant recruitment of eosinophils into the airway mucosa (Clin Exp Allergy 33:1686-1694, 2003). Because eosinophils were not uniformly distributed throughout the five conducting airway generations examined, we speculated that trafficking within anatomic microenvironments of the lung is mediated by differential chemokine expression. To address this question, we used quantitative real-time RT-PCR to evaluate the related eosinophilic chemokines, eotaxin (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26) within isolated airways of infant monkey lung. Overall, chemokine mRNA expression levels in house dust mite-exposed airways were as follows: eotaxin-3 > eotaxin > eotaxin-2. Immunofluorescence staining for eotaxin-3 and CC chemokine receptor 3 (CCR3) showed positive cells within epithelium and peripherally located nerve fiber bundles of the airway wall. Epithelial volume of eotaxin-3 within the trachea correlated with epithelial volume of major basic protein. CCR3+ and MHC Class II+ dendritic cells, but not eosinophils or mast cells, co-localized within eotaxin-3+ nerve fiber bundles. We conclude that localized expression of eotaxin-3 plays an important role in the recruitment of diverse CCR3+ cell populations to different anatomic microenvironments within the infant airway in response to chronic allergen exposure.

Figure 1.

Effect of house dust mite exposure on gene expression levels for eotaxin, eotaxin-2, and eotaxin-3 within four different airway generations: comparison with distribution of eosinophils. (A and B) Volume of major basic protein (eosinophils) within four different airway generations of infant monkeys (filtered air, n = 5; HDM, n = 6). Columns represent the volume of major basic protein–positive cells within either epithelial (A) and interstitial (B) compartments sampled from trachea, proximal airway, midlevel airway, and respiratory bronchiole, as previously reported (17). T, trachea; P, proximal airway; M, midlevel airway; R, respiratory bronchiole. All data are represented as mean ± SEM and analyzed by two-way ANOVA. *P = 0.0012 as compared with filtered air control animals, **P = 0.0017 as compared with filtered air control animals. (C–E) Columns represent the average relative fold change of eotaxin (C), eotaxin-2 (D), and eotaxin-3 (E) mRNA for indicated genes by RT-PCR analysis using isolated airway generations from HDM-exposed infant monkeys (n = 5–6) after normalization with 18S mRNA levels, relative to filtered air control infant monkeys (n = 4–5). RT-PCR analysis for each monkey and airway generation was repeated over 2–3 experiments; control monkeys represent a pool of 12–15 separate replications by RT-PCR. The dotted line represents the point at which expression of experimental versus control is equivalent (no change in expression). *** P = 0.0285 by one-way ANOVA.

Figure 2.

Effect of HDM exposure on eotaxin-3 protein expression within different airway generations and compartments. (A–D) Immunofluorescence staining of cryosections obtained from midlevel airways, a representative filtered air control infant monkey (A and B) is compared with a representative HDM-exposed infant monkey (C and D). Cryosections were double labeled for eotaxin-3 (green, A and C) and PGP 9.5 (red staining, B and D). Arrows point to large nerve fiber bundles peripherally located within the airway wall. Scale bar = 50 μm. (E and F) Abundance and distribution of eotaxin-3–immunoreactive cells within five different airway generations of infant monkeys (filtered air, n = 5; HDM, n = 6). Columns represent the volume of eotaxin-3–positive cells within epithelial (E) and interstitial (F) compartments sampled from cryopreserved blocks of the left caudal lobe. Blocks Tr 1, 3, 5, and 7 correspond to the trachea (Tr) and regions from the most proximal (1) to distal (7) airways of the lobe. All data are represented as mean ± SEM. *P = 0.0003 by two-way ANOVA as compared with filtered air control animals.

Figure 2.

Effect of HDM exposure on eotaxin-3 protein expression within different airway generations and compartments. (A–D) Immunofluorescence staining of cryosections obtained from midlevel airways, a representative filtered air control infant monkey (A and B) is compared with a representative HDM-exposed infant monkey (C and D). Cryosections were double labeled for eotaxin-3 (green, A and C) and PGP 9.5 (red staining, B and D). Arrows point to large nerve fiber bundles peripherally located within the airway wall. Scale bar = 50 μm. (E and F) Abundance and distribution of eotaxin-3–immunoreactive cells within five different airway generations of infant monkeys (filtered air, n = 5; HDM, n = 6). Columns represent the volume of eotaxin-3–positive cells within epithelial (E) and interstitial (F) compartments sampled from cryopreserved blocks of the left caudal lobe. Blocks Tr 1, 3, 5, and 7 correspond to the trachea (Tr) and regions from the most proximal (1) to distal (7) airways of the lobe. All data are represented as mean ± SEM. *P = 0.0003 by two-way ANOVA as compared with filtered air control animals.

Figure 3.

Effect of HDM exposure on CCR3 protein expression within different airway generations and compartments. (A and B) Immunofluorescence staining for CCR3; a midlevel airway from a representative HDM-exposed infant monkey is shown. The dotted line in A defines the epithelial compartment. The dotted line in B surrounds two small nerve fiber bundles peripherally located within the airway wall. Scale bar = 20 μm. (C and D) Abundance and distribution of CCR3-immunoreactive cells within five different airway generations of infant monkeys (filtered air, n = 5; HDM, n = 6). Columns represent the volume of CCR3+ cells within epithelial (C) and interstitial (D) compartments sampled from cryopreserved blocks of the left caudal lobe. Blocks Tr 1, 3, 5, and 7 correspond to the trachea (Tr) and regions from the most proximal (1) to distal (7) airways of the lobe. All data are represented as mean ± SEM. *P = 0.0025 by two-way ANOVA as compared with filtered control animals.

Figure 3.

Effect of HDM exposure on CCR3 protein expression within different airway generations and compartments. (A and B) Immunofluorescence staining for CCR3; a midlevel airway from a representative HDM-exposed infant monkey is shown. The dotted line in A defines the epithelial compartment. The dotted line in B surrounds two small nerve fiber bundles peripherally located within the airway wall. Scale bar = 20 μm. (C and D) Abundance and distribution of CCR3-immunoreactive cells within five different airway generations of infant monkeys (filtered air, n = 5; HDM, n = 6). Columns represent the volume of CCR3+ cells within epithelial (C) and interstitial (D) compartments sampled from cryopreserved blocks of the left caudal lobe. Blocks Tr 1, 3, 5, and 7 correspond to the trachea (Tr) and regions from the most proximal (1) to distal (7) airways of the lobe. All data are represented as mean ± SEM. *P = 0.0025 by two-way ANOVA as compared with filtered control animals.

Figure 4.

Immunofluorescence staining for CCR3+ cells and eosinophils within airway nerves of HDM-exposed infant monkeys. Adjacent cryosections from a midlevel airway of a representative HDM-exposed infant monkey were double immunostained for eotaxin-3 (A) and PGP 9.5 (B) or individually stained for CCR3 (C) or major basic protein (D). Large arrows point to the nerve fiber bundle in corresponding sections, small arrowheads point to CCR3 positive cells. Scale bar = 20 μm.

Figure 5.

Immunofluorescence staining of CCR3+ cells and CD117/c-kit expression within airway nerves of HDM-exposed infant monkeys. Cryosections from a midlevel airway of a representative HDM-exposed infant monkey were double immunostained for CD117/c-kit and CCR3. Both CD117/c-kit (A) and CCR3 (B) expression were detected in a representative airway nerve bundle; arrowheads in B point to three CCR3+ cells. Isotype control immunostaining for CD117/c-kit (C) and CCR3 (D) antibodies are shown. Overlay of CD117/c-kit and CCR3 staining (E) shows no overlap in expression. As a positive control, CCR3+ eosinophils (white arrows) and a CD117/c-kit–positive mast cell (arrow with asterisk) are present within the interstitial region of the same cryosection (F). Scale bar = 20 μm.

Figure 6.

Immunofluorescence staining of MHC Class II–positive cells within airway nerves of HDM-exposed infant monkeys. Cryosections from a midlevel airway of a representative filtered air control (A and B) or HDM-exposed (C and D) monkey were double immunostained for MHC Class II (A and C) and PGP 9.5 (B and D). Arrows point to MHC Class II–positive cells in the periphery of nerve bundles (A) or within nerve bundles (C). Scale bar = 20 μm.