Cormier, Stephania A.

Abstract Paradigms of eosinophil effector function in the lungs of asthma patients invariably depend on activities mediated by cationic proteins released from secondary granules during a process collectively referred to as degranulation. In this study, we generated knockout mice deficient for eosinophil peroxidase (EPO) to assess the role(s) of this abundant secondary granule protein in an OVA-challenge model. The loss of EPO had no effect on the development of OVA-induced pathologies in the mouse. The absence of phenotypic consequences in these knockout animals extended beyond pulmonary histopathologies and airway changes, as EPO-deficient animals also displayed OVA-induced airway hyperresponsiveness after provocation with methacholine. In addition, EPO-mediated oxidative damage of proteins (e.g., bromination of tyrosine residues) recovered in bronchoalveolar lavage from OVA-treated wild-type mice was <10% of the levels observed in bronchoalveolar lavage recovered from asthma patients. These data demonstrate that EPO activities are inconsequential to the development of allergic pulmonary pathologies in the mouse and suggest that degranulation of eosinophils recruited to the lung in this model does not occur at levels comparable to those observed in humans with asthma.

Abstract The relationship between eosinophils and the development of Ag-induced pulmonary pathologies, including airway hyper-responsiveness, was investigated using mice deficient for the secondary granule component, major basic protein-1 (mMBP-1). The loss of mMBP-1 had no effect on OVA-induced airway histopathologies or inflammatory cell recruitment. Lung function measurements of knockout mice demonstrated a generalized hyporeactivity to methacholine-induced airflow changes (relative to wild type); however, this baseline phenotype was observable only with methacholine; no relative airflow changes were observed in response to another nonspecific stimulus (serotonin). Moreover, OVA sensitization/aerosol challenge of wild-type and mMBP-1−/− mice resulted in identical dose-response changes to either methacholine or serotonin. Thus, the airway hyper-responsiveness in murine models of asthma occurs in the absence of mMBP-1.

Abstract Asthma and mouse models of allergic respiratory inflammation are invariably associated with a pulmonary eosinophilia; however, this association has remained correlative. In this report, a causative relationship between eosinophils and allergen-provoked pathologies was established using eosinophil adoptive transfer. Eosinophils were transferred directly into the lungs of either naive or OVA-treated IL-5−/− mice. This strategy resulted in a pulmonary eosinophilia equivalent to that observed in OVA-treated wild-type animals. A concomitant consequence of this eosinophil transfer was an increase in Th2 bronchoalveolar lavage cytokine levels and the restoration of intracellular epithelial mucus in OVA-treated IL-5−/− mice equivalent to OVA-treated wild-type levels. Moreover, the transfer also resulted in the development of airway hyperresponsiveness. These pulmonary changes did not occur when eosinophils were transferred into naive IL-5−/− mice, eliminating nonspecific consequences of the eosinophil transfer as a possible explanation. Significantly, administration of OVA-treated IL-5−/− mice with GK1.5 (anti-CD4) Abs abolished the increases in mucus accumulation and airway hyperresponsiveness following adoptive transfer of eosinophils. Thus, CD4+ T cell-mediated inflammatory signals as well as signals derived from eosinophils are each necessary, yet alone insufficient, for the development of allergic pulmonary pathology. These data support an expanded view of T cell and eosinophil activities and suggest that eosinophil effector functions impinge directly on lung function.

Abstract The eosinophil-associated ribonuclease (Ear) family in the mouse consists of thirteen genes, eleven of which encode RNases that have physical/functional properties similar to the human Ears, eosinophil-derived neurotoxin and eosinophil cationic protein. The expression of Ear genes in the mouse is confined to sites of known eosinophilopoiesis, with the exception of the lung. Two Ear genes, Ear1 and Ear2, are predominantly expressed in the lungs of naive mice. Total Ear gene expression and RNase activity in bronchoalveolar lavage fluid increases significantly upon the induction of pulmonary inflammation using an ovalbumin (OVA) model of allergic sensitization and challenge. Interestingly, pulmonary Ear11 transcripts, which are absent in naive mice, accumulate as a consequence of OVA-mediated TH2 inflammation in the lung. The induction of Ear11 expression is dependent on the presence of T cells, in particular, CD4+ T lymphocytes. This effect is likely the result of the elaboration of TH2 cytokine levels, because pulmonary instillation of interleukin-4 or interleukin-13 induces the accumulation of Ear11 transcripts in naive animals. This study demonstrates that despite an allergen-mediated pulmonary eosinophilia and earlier studies showing that Ears are constituents of eosinophil secondary granules, alveolar macrophages are a significant source of these RNases in lungs of OVA-treated mice.

Abstract Tumor-associated eosinophilia has been observed in numerous human cancers and several tumor models in animals; however, the details surrounding this eosinophilia remain largely undefined and anecdotal. We used a B16-F10 melanoma cell injection model to demonstrate that eosinophil infiltration of tumors occurred from the earliest palpable stages with significant accumulations only in the necrotic and capsule regions. Furthermore, the presence of diffuse extracellular matrix staining for eosinophil major basic protein was restricted to the necrotic areas of tumors, indicating that eosinophil degranulation was limited to this region. Antibody-mediated depletion of CD4+ T cells and adoptive transfer of eosinophils suggested, respectively, that the accumulation of eosinophils is not associated with T helper cell type 2-dependent immune responses and that recruitment is a dynamic, ongoing process, occurring throughout tumor growth. Ex vivo migration studies have identified what appears to be a novel chemotactic factor(s) released by stressed/dying melanoma cells, suggesting that the accumulation of eosinophils in tumors occurs, in part, through a unique mechanism dependent on a signal(s) released from areas of necrosis. Collectively, these studies demonstrate that the infiltration of tumors by eosinophils is an early and persistent response that is spatial-restricted. It is more important that these data also show that the mechanism(s) that elicit this host response occur, independent of immune surveillance, suggesting that eosinophils are part of an early inflammatory reaction at the site of tumorigenesis.

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.