R.S. Pero

Allergic pulmonary inflammation in mice is dependent on eosinophil-induced recruitment of effector T cells

The current paradigm surrounding allergen-mediated T helper type 2 (Th2) immune responses in the lung suggests an almost hegemonic role for T cells. Our studies propose an alternative hypothesis implicating eosinophils in the regulation of pulmonary T cell responses. In particular, ovalbumin (OVA)-sensitized/challenged mice devoid of eosinophils (the transgenic line PHIL) have reduced airway levels of Th2 cytokines relative to the OVA-treated wild type that correlated with a reduced ability to recruit effector T cells to the lung. Adoptive transfer of Th2-polarized OVA-specific transgenic T cells (OT-II) alone into OVA-challenged PHIL recipient mice failed to restore Th2 cytokines, airway histopathologies, and, most importantly, the recruitment of pulmonary effector T cells. In contrast, the combined transfer of OT-II cells and eosinophils into PHIL mice resulted in the accumulation of effector T cells and a concomitant increase in both airway Th2 immune responses and histopathologies. Moreover, we show that eosinophils elicit the expression of the Th2 chemokines thymus- and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in the lung after allergen challenge, and blockade of these chemokines inhibited the recruitment of effector T cells. In summary, the data suggest that pulmonary eosinophils are required for the localized recruitment of effector T cells.

Coexpression of IL-5 and Eotaxin-2 in Mice Creates an Eosinophil-Dependent Model of Respiratory Inflammation with Characteristics of Severe Asthma

Mouse models of allergen provocation and/or transgenic gene expression have provided significant insights regarding the cellular, molecular, and immune responses linked to the pathologies occurring as a result of allergic respiratory inflammation. Nonetheless, the inability to replicate the eosinophil activities occurring in patients with asthma has limited their usefulness to understand the larger role(s) of eosinophils in disease pathologies. These limitations have led us to develop an allergen-naive double transgenic mouse model that expresses IL-5 systemically from mature T cells and eotaxin-2 locally from lung epithelial cells. We show that these mice develop several pulmonary pathologies representative of severe asthma, including structural remodeling events such as epithelial desquamation and mucus hypersecretion leading to airway obstruction, subepithelial fibrosis, airway smooth muscle hyperplasia, and pathophysiological changes exemplified by exacerbated methacholine-induced airway hyperresponsiveness. More importantly, and similar to human patients, the pulmonary pathologies observed are accompanied by extensive eosinophil degranulation. Genetic ablation of all eosinophils from this double transgenic model abolished the induced pulmonary pathologies, demonstrating that these pathologies are a consequence of one or more eosinophil effector functions.

Pivotal Advance : Eosinophil infiltration of solid tumors is an early and persistent inflammatory host response

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.

Gαi2-mediated signaling events in the endothelium are involved in controlling leukocyte extravasation

The trafficking of leukocytes from the blood to sites of inflammation is the cumulative result of receptor-ligand-mediated signaling events associated with the leukocytes themselves as well as with the underlying vascular endothelium. Our data show that Gαi signaling pathways in the vascular endothelium regulate a critical step required for leukocyte diapedesis. In vivo studies using knockout mice demonstrated that a signaling event in a non-lymphohematopoietic compartment of the lung prevented the recruitment of proinflammatory leukocytes. Intravital microscopy showed that blockade was at the capillary endothelial surface andex vivo studies of leukocyte trafficking demonstrated that a Gαi-signaling event in endothelial cells was required for transmigration. Collectively, these data suggest that specific Gαi2-mediated signaling between endothelial cells and leukocytes is required for the extravasation of leukocytes and for tissue-specific accumulation.

Mouse eosinophils possess potent antibacterial properties in vivo.

ABSTRACT Eosinophils are best known as the predominant cellular infiltrate associated with asthma and parasitic infections. Recently, numerous studies have documented the presence of Toll-like receptors (TLRs) on the surfaces of eosinophils, suggesting that these leukocytes may participate in the recognition and killing of viruses and bacteria. However, the significance of this role in the innate immune response to bacterial infection is largely unknown. Here we report a novel role for eosinophils as antibacterial defenders in the host response. Isolated mouse eosinophils possessed antipseudomonal properties in vitro. In vivo, interleukin-5 transgenic mice, which have profound eosinophilia, demonstrated improved clearance of Pseudomonas aeruginosa introduced into the peritoneal cavity. The findings of improved bacterial clearance following adoptive transfer of eosinophils, and impaired bacterial clearance in mice with a congenital eosinophil deficiency, established that this effect was eosinophil specific. The data presented also demonstrate that eosinophils mediate this antibacterial effect in part through the release of cationic secondary granule proteins. Specifically, isolated eosinophil granules had antibacterial properties in vitro, and administration of eosinophil granule extracts significantly improved bacterial clearance in vivo. These data suggest a potent yet underappreciated antibacterial role for eosinophils in vivo, specifically for eosinophil granules. Moreover, the data suggest that the administration of eosinophil-derived products may represent a viable adjuvant therapy for septic or bacteremic patients in the intensive care unit.

Post-translational Tyrosine Nitration of Eosinophil Granule Toxins Mediated by Eosinophil Peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO-/-, gp91phox-/-, and NOS-/- mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.

A3 Adenosine Receptor Signaling Influences Pulmonary Inflammation and Fibrosis

Adenosine is a signaling molecule produced during conditions that cause cellular stress or damage. This signaling pathway is implicated in the regulation of pulmonary disorders through the selective engagement of adenosine receptors. The goal of this study was to examine the involvement of the A(3) adenosine receptor (A(3)R) in a bleomycin model of pulmonary inflammation and fibrosis. Results demonstrated that A(3)R-deficient mice exhibit enhanced pulmonary inflammation that included an increase in eosinophils. Accordingly, there was a selective up-regulation of eosinophil-related chemokines and cytokines in the lungs of A(3)R-deficient mice exposed to bleomycin. This increase in eosinophil numbers was accompanied by a decrease in the amount of extracellular eosinophil peroxidase activity in lavage fluid from A(3)R-deficient mice exposed to bleomycin, an observation suggesting that the A(3)R is necessary for eosinophil degranulation in this model. Despite an increase in inflammatory metrics associated with A(3)R-deficient mice treated with bleomycin, there was little difference in the degree of pulmonary fibrosis. Examination of fibrotic mediators demonstrated enhanced transforming growth factor (TGF)-beta1 expression, but not a concomitant increase in TGF-beta1 activity. This was associated with the loss of expression of matrix metalloprotease 9, an activator of TGF-beta1, in alveolar macrophages and airway mast cells in the lungs of A(3)R-deficient mice. Together, these results suggest that the A(3)R serves antiinflammatory functions in the bleomycin model, and is also involved in regulating the production of mediators that can impact fibrosis.

Homologous recombination into the eosinophil peroxidase locus generates a strain of mice expressing Cre recombinase exclusively in eosinophils

Eosinophils are generally linked to innate host defense against helminths, as well as the pathologies associated with allergic diseases, such as asthma. Nonetheless, the activities of eosinophils remain poorly understood, which in turn, has prevented detailed definitions of their role(s) in health and disease. Homologous recombination in embryonic stem cells was used to insert a mammalianized Cre recombinase in the ORF encoding Epx. This knock‐in strategy overcame previous inefficiencies associated with eosinophil‐specific transgenic approaches and led to the development of a knock‐in strain of mice (eoCRE), capable of mediating recombination of “floxed” reporter cassettes in >95% of peripheral blood eosinophils. We also showed that this Cre expression was limited exclusively to eosinophil‐lineage committed cells with no evidence of Cre‐mediated toxicity. The efficiency and specificity of Cre expression in eoCRE mice were demonstrated further in a cross with a knock‐in mouse containing a “(flox‐stop‐flox)” DTA cassette at the ROSA26 locus, generating yet another novel, eosinophil‐less strain of mice. The development of eoCRE mice represents a milestone in studies of eosinophil biology, permitting eosinophil‐specific gene targeting and overexpression in the mouse as part of next‐generation studies attempting to define eosinophil effector functions.