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Defining a Link with Asthma in Mice Congenitally Deficient in Eosinophils

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Science  17 Sep 2004:
Vol. 305, Issue 5691, pp. 1773-1776
DOI: 10.1126/science.1099472

Abstract

Eosinophils are often dominant inflammatory cells present in the lungs of asthma patients. Nonetheless, the role of these leukocytes remains poorly understood. We have created a transgenic line of mice (PHIL) that are specifically devoid of eosinophils, but otherwise have a full complement of hematopoietically derived cells. Allergen challenge of PHIL mice demonstrated that eosinophils were required for pulmonary mucus accumulation and the airway hyperresponsiveness associated with asthma. The development of an eosinophil-less mouse now permits an unambiguous assessment of a number of human diseases that have been linked to this granulocyte, including allergic diseases, parasite infections, and tumorigenesis.

The underlying features of asthma display a marked heterogeneity (1, 2), yet the presence of eosinophils in the airway lumen and lung tissue has been recognized even in the earliest studies (3) and is often regarded as a defining feature of this disease (4, 5). Moreover, the recruitment of eosinophils occurs in animal models of allergen-mediated respiratory inflammation; in particular, mouse models have offered unique opportunities with which to examine detailed pathologic features of this disease. However, the availability of clinical studies and numerous mouse models of asthma have not led to an unambiguous description of eosinophil effector functions in asthma, and questions remain as to the specific role(s), if any, of these leukocytes (6).

A line of mice devoid of eosinophils was created to test hypotheses that link eosinophils and asthma-related pathogenesis. Transgenic mice devoid of eosinophils were created by lineage-specific expression of a cytocidal protein with a promoter fragment identified from studies of secondary granule protein genes expressed in mouse eosinophils (711). A candidate promoter from the gene for eosinophil peroxidase (EPO) was selected on the basis of transfection studies with EPO promoter–luciferase reporter constructs in the eosinophilic cell line AML14.3D10 (12). These studies revealed that upstream sequences from the mouse EPO gene were capable of supporting high-level expression that was unique to eosinophil lineage–committed cells (fig. S1). In addition to mouse EPO-derived sequences, the transgenic construct developed included the diphtheria toxin A (DTA) chain open reading frame (13). The cytocidal character of diphtheria toxin is mediated by the DTA chain (the B chain provides entry into eukaryotic cells) through the catalytic degradation of elongation factor-2 and the subsequent collapse of protein synthesis (14).

Assessment of circulating leukocytes (15) in the resulting EPO-DTA transgenic (PHIL) mice demonstrated that these animals were devoid of eosinophils but otherwise have a full complement of hematopoietically derived cells (Fig. 1A). An examination of splenic lymphoid cells (15) revealed normal numbers of B cells, T cells, and the T lymphocyte CD4+ and CD8+ subtypes (Fig. 1B). Assessments of lung sections and peritoneal cavity exudates from PHIL mice revealed wild-type levels of mast cells (fig. S2, A and B). Moreover, circulating basophils were identified in peripheral blood from PHIL mice (fig. S2C), demonstrating that even a leukocyte lineage sharing a direct common precursor with the eosinophil lineage was unaffected. The specific ablation of eosinophils in PHIL mice also occurred with no effects on either erythropoiesis or the production of platelets (Fig. 1C). A nominal elevation of total white blood cell counts was consistently observed in PHIL mice relative to negative littermates. This increase, however, was not specific to any one cell type and did not elevate circulating cell numbers beyond the normal observable range in wild-type mice.

Fig. 1.

The eosinophil deficiency of PHIL mice is specific and definitive. (A) Peripheral blood of PHIL mice is devoid of eosinophils without effects on the composition of other leukocytes (mean ± SE, n = 17 animals per group). (B) The targeted loss of eosinophils had no effects on lymphocyte subtypes (n = 5 animals per group). (C) The specific ablation of eosinophil lineage–committed cells had no additional effects on other hematopoietic parameters, although a nonspecific marginal increase in the steady-state levels of total circulating white blood cells was observed. (D) Fluorescence-activated cell sorting analyses demonstrated that the marked blood eosinophilia (i.e., the presence of CCR3+ cells) of the IL-5 transgenic line NJ.1726 (16) was completely abolished in NJ.1726/PHIL double transgenic mice. PE, phycoerythrin. (E) Immunohistochemistry (dark purple–stained cells) with eosinophil-specific rabbit polyclonal antisera to MBP demonstrates that tissues or organs with prominent resident populations of eosinophils at baseline in wild-type mice were devoid of these granulocytes in PHIL mice. Scale bar, 100 μm.

The loss of eosinophils in PHIL mice was nearly absolute, with only an occasional eosinophil identified in surveys of blood films from 1 of 20 animals examined. This eosinophil deficiency is lifelong and a Mendelian inheritable trait of the line. The specificity of the eosinophil deficiency in PHIL mice was achieved through a cross with interleukin (IL)–5 transgenic animals (16). These IL-5 transgenic mice have circulating eosinophil levels that, in some cases, exceed 100,000 per mm3 of blood, representing ∼50% of all white blood cells. Analyses of blood from double transgenic animals (i.e., mice carrying both the DTA and IL-5 transgenes) again revealed a complete absence of eosinophils (Fig. 1D).

The eosinophil-deficient character of PHIL mice was extended further by immunohistochemistry with antibodies specific for Major Basic Protein (MBP) (15, 17, 18). Tissues with abundant resident populations of eosinophils (i.e., bone marrow, uterus, small intestines, and thymus) in wild-type animals were shown to be devoid of these granulocytes in PHIL mice (Fig. 1E).

PHIL mice were subjected to an acute allergen sensitization/aerosol challenge model of asthma (15) to determine if the presence of eosinophils was causatively linked to the development of disease symptoms. Whereas wild-type mice sensitized/aerosol challenged with chicken ovalbumin (OVA) developed a significant airway eosinophilia [∼50% of bronchoalveolar lavage fluid (BAL) cells], PHIL mice were essentially devoid of eosinophils, with only trace numbers (<0.5%) of eosinophils identifiable in the BAL of one of four OVA-treated animals (Fig. 2A). The loss of eosinophils from the lungs of OVA-treated PHIL mice also extended to tissue-infiltrating cells. Specifically, the lungs of OVA-treated PHIL mice were devoid of eosinophils, unlike the significant tissue eosinophilia that occurred in the areas surrounding the central airways (peribronchial) and the vasculature (perivascular) of OVA-treated wild-type animals (Fig. 2, B to D, and fig. S3). Examination of blood films and bone marrow smears from OVA-treated PHIL animals again revealed only an occasional eosinophil in a fraction of the animals examined.

Fig. 2.

The pulmonary eosinophilia associated with OVA sensitization/aerosol challenge was abolished in PHIL mice. (A) OVA-induced eosinophilia of the airway lumen was lost (decreased by >99%) in PHIL mice (mean ± SE, n = 5 animals per group). *, P < 0.001. (B and C) Assessments of infiltrating eosinophils in (B) wild-type and (C) PHIL mice by immunohistochemistry (dark purple–stained cells) with rabbit polyclonal antisera to mouse MBP revealed that OVA-induced accumulation of eosinophils was also extinguished in PHIL mice. Scale bar, 100 μm. (D) Quantitative assessments of the number of eosinophils infiltrating peribronchial areas (i.e., eosinophils per mm2) demonstrated that OVA-treated PHIL mice were devoid of tissue eosinophils (mean ± SE, n = 5 animals per group). ∅ indicates the absence of eosinophils in any of the sections of any of the mice in the cohort examined.

The targeted ablation of eosinophils had significant effects on allergen-induced pulmonary pathology, suggesting a causative role for these granulocytes. Overall, OVA-induced histopathology in PHIL mice was attenuated relative to OVA-treated wild-type littermates. This lack of pathology was manifested by the reduced airway epithelial hypertrophy in OVA-treated PHIL mice (Fig. 2, B and C). In addition, assessment of airway mucins by periodic acid–Schiff (PAS) staining (15) demonstrated that OVA-induced goblet cell metaplasia/mucus accumulation (GM/MA) in PHIL mice was significantly reduced (Fig. 3, A to C). A quantitative assessment of the staining in these tissue sections revealed a 68% reduction of PAS staining in PHIL mice relative to OVA-treated wild-type animals (Fig. 3D). However, the GM/MA observed in OVA-treated PHIL mice was still significant when compared to allergen-naive animals. This observation suggests that eosinophils contribute to, and are necessary for, the levels of pathology observed in wild-type mice, but that they are not alone sufficient to account for these wild-type levels. That is, both eosinophil-dependent and -independent mechanisms exist in the lung that elicit GM/MA after allergen challenge.

Fig. 3.

The specific loss of eosinophils in PHIL mice resulted in a significant reduction in OVA-induced GM/MA. Representative lung sections after PAS staining are shown for (A) saline control and (B) OVA sensitized/OVA aerosol challenged wild-type mice in comparison to (C) OVA sensitized/OVA aerosol challenged PHIL mice. The sections of each panel show early-branching central conducting airways, whereas the insets show smaller, more distal bronchioles. Scale bars, 100 μm. (D) Quantitative assessments of airway epithelial mucus content showed a marked decrease (relative to wild type) in PHIL mice (mean ± SE, 5 to 10 animals per group). All evaluations of histopathology were performed in duplicate as independent observer-blinded assessments. *, P < 0.05.

The association between allergen-induced pulmonary eosinophilia and the development of lung dysfunction in both asthma patients and mouse models has been, at best, a collection of confusing and often contradictory observations. The lack of symptom improvement in asthma patients after administration of antibodies to IL-5 exemplifies the ambiguous character of clinical studies that attempt to ablate eosinophils (6). Mouse models purporting to ablate eosinophils are also ambiguous, as they either do not completely eliminate pulmonary eosinophils or they elicit the loss of eosinophils by mechanisms that do not differentiate between effects on eosinophils and other potentially important cellular targets (1922). However, measurements of lung function after OVA sensitization/aerosol challenge of PHIL mice (15) showed that methacholine-induced airway hyperresponsiveness was dependent on the presence of eosinophils (Fig. 4). Moreover, the specific loss of eosinophils also led to improvement of other pulmonary function parameters associated with the distal regions of the lung (fig. S4).

Fig. 4.

In the absence of eosinophils, OVA-induced airway hyperresponsiveness does not develop. Lung function was assessed as airway resistance (Rn) in response to aerosolized methacholine, in saline-treated (WT/Saline) and OVA sensitized/OVA aerosol challenged (WT/OVA) wild-type mice in comparison to saline-treated (PHIL/Saline) and OVA sensitized/OVA aerosol challenged (PHIL/OVA) PHIL mice (n = 5 to 10 animals per group). Asterisks indicate a significant difference (P < 0.01) between WT/OVA and either WT/Saline, PHIL/Saline, or PHIL/OVA mice.

The lack of observable phenotypes in knock-out mice deficient for the abundant secondary granule proteins MBP-1 (18) and EPO (17) suggests that activities other than degranulation, including antigen presentation (23), the release of small molecule mediators of inflammation [e.g., the synthesis and release of eicosanoid mediators of inflammation (24)], and immune regulation of the pulmonary microenvironment through either modulations of T cell activities (21) or eosinophil-derived cytokine and/or chemokine expression (25) are likely to be the relevant effector functions. Eosinophil-derived cytokine and/or chemokine expression, in particular, is noteworthy as it may account for the chronic and seemingly self-sustaining character of allergic pulmonary inflammation, which often leads to lung remodeling events (26, 27). Significant decreases of Th2 cytokine levels in BAL of OVA-treated PHIL mice (28) lend support to this hypothesis and suggest that a prominent eosinophil effector function in the lung is localized immune regulation.

This study shows that eosinophil activities are important contributory factors leading to symptoms that are classically defined as hallmark features of asthma. More importantly, these data provide validation of earlier studies that independently concluded that a causative link exists between eosinophils and allergic pulmonary pathologies (22, 29). The dependency of allergen-induced pulmonary pathologies on eosinophils suggests that these granulocytes participate at a significant level in underlying inflammatory responses. Regardless of the ultimate definition of the causative activities mediated by eosinophils, the challenge of future studies will be to develop confirmatory clinical studies to unambiguously define the role(s) and extent of eosinophil effector functions in asthma patients. The results of such studies will not only widen our understanding of the principle causes of asthma, but are also likely to lead to targeted therapeutic approaches previously dismissed and/or overlooked.

Supporting Online Material

www.sciencemag.org/cgi/content/full/305/5691/1773/DC1

Materials and Methods

Figs. S1 to S4

References and Notes

References and Notes

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