Abstract Eosinophil effector functions have been hypothesized to be mediated in part by the release of proteins stored in the secondary granules of mature cells of this leukocyte lineage. Major basic protein-1 (MBP-1) and eosinophil peroxidase (EPO) comprise the majority of protein in these granules. No role has been speculated for these proteins in eosinophilopoiesis and/or the accumulation of mature cells in circulation. Methods: Eosinophilopoiesis and eosinophil accumulation in the periphery were assessed in single and double knockout (MBP-1-/- and/or EPO-/-) mice. Bone marrow and peripheral blood leukocytes were assessed by IHC, FACS and in vitro cell culture assays. Mice were subjected to an acute OVA protocol assessing induced pulmonary pathologies in each of the strains of granule protein knockout mice. Results: We demonstrate that, unlike the single deficiency of MBP-1 or EPO, the absence of both granule proteins resulted in the loss of peripheral blood eosinophils. IHC assessments of bone marrow and spleen demonstrate that eosinophil lineage commitment occurs in these mice. However, these assessments and FACS studies demonstrated a blockade in the maturation of eosinophil-lineage committed cells. This blockade is not rescued by ex vivo culture or by bone marrow engraftment into wild type recipient mice. Similar to other eosinophil-less mouse models, MBP-1-/-/EPO-/- mice also fail to develop pulmonary inflammation in an OVA protocol.

Eosinophil infiltration into tumors has been associated with positive and negative growth human cancers and in mouse models of tumorigenesis. Our previous studies have shown that eosinophils infiltrate tumors and differentially accumulate in the necrotic and capsule regions of tumors. Using various eosinophil-deficient mouse models, we demonstrate that in the absence of eosinophils tumor growth is attenuated. Methods: Tumor growth kinetics were assessed using a B16F10 melanoma subcutaneous cell injection model. Eosinophil chemotaxis was assessed using an ex vivo transwell insert assay system. We compared tumor growth in wild type animals vs. various eosinophil-deficient mouse models that are transgenic line of mice (PHIL) and MBP-/-/EPO-/- double knockout mice that are deficient in eosinophil-specific proteins (major basic protein (MBP) and eosinophil peroxide (EPO)) resulting in a blockade of eosinophilpoiesis. Conclusions: The migration of eosinophils was inhibited through blocking the sphingosine-1-phosphate pathway, suggesting recruitment and accumulation is primarily an inflammatory response. Tumor growth was decreased by ~40-60% in PHIL and in MBP-/-/EPO-/- double knockout mice relative to tumors in wild type animals. In addition, EPO-/- mice, and not MBP-/-mice, replicate this ~40% reduction in tumor size implicating a significant role for EPO in tumor growth kinetics. These data suggest eosinophils respond to inflammatory mediators and mediate augment tumor growth.

SummaryDiscussions of eosinophils are often descriptions of end‐stage effector cells with destructive capabilities mediated predominantly by released cytotoxic cationic granule proteins. Moreover, eosinophils in the medical literature are invariably associated with the pathologies linked with helminth infections or allergic diseases such as asthma. This has led to an almost fatalist view of eosinophil effector functions and associated therapeutic strategies targeting these cells that would make even William of Ockham proud – eosinophil effector functions have physiological consequences that increase patient morbidity/mortality and ‘the only good eosinophils are dead eosinophils’. Unfortunately, the strengths of dogmas are also their greatest weaknesses. Namely, while the repetitive proclamation of dogmatic concepts by authoritative sources (i.e. reviews, meeting proceedings, textbooks, etc.) builds consensus within the medical community and lower the entropies surrounding difficult issues, they often ignore not easily explained details and place diminished importance on alternative hypotheses. The goal of this perspective is twofold: (i) we will review recent observations regarding eosinophils and their activities as well as reinterpret earlier data as part of the synthesis of a new paradigm. In this paradigm, we hypothesize that eosinophils accumulate at unique sites in response to cell turnover or in response to local stem cell activity(ies). We further suggest that this accumulation is part of one or more mechanisms regulating tissue homeostasis. Specifically, instead of immune cells exclusively mediating innate host defence, we suggest that accumulating tissue eosinophils are actually regulators of Local Immunity And/or Remodeling/Repair in both health and disease – the LIAR hypothesis; (ii) we want to be inflammatory (pun intended!) and challenge the currently common perspective of eosinophils as destructive end‐stage effector cells. Our hope is to create more questions than we answer and provoke everyone to spend countless hours simply to prove us wrong! Cite this as: J. J. Lee, E. A. Jacobsen, M. P. McGarry, R. P. Schleimer and N. A. Lee, Clinical & Experimental Allergy, 2010 (40) 563–575

RATIONALE: Eosinophils are a major leukocyte recruited to the lungs in both mouse allergen provocation models and patients with asthma. However, a link between these cells and the pathological changes occurring in the lung remains obscure although eosinophil effector functions have been hypothesized to be mediated, in part, by the release of secreted cationic proteins sequestered in cytoplasmic granules (i.e., the degranulation of activated eosinophils recruited to the lung).METHODS: Gene knockout lines of mice deficient for the two eosinophil major basic proteins (MBP-1, -2) were generated (strain: 129) by homologous recombination in ES cells and used in an acute allergen sensitization/aerosol challenge protocol to define causative links with pulmonary allergic infammation.RESULTS: This study demonstrates that despite observations showing that allergen challenge mouse models display only nominal levels of eosinophil degranulation the loss of the eosinophil granule protein MBP-2 abolished allergen-induced airway hyperresponsiveness (AHR). The loss of AHR in MBP-2-/- mice occurred with out concominant loss of other allergen-induced pulmonary pathologies (e.g., goblet cell metaplasia), suggesting a specific link between this pathophysiologic response and MBP-2 mediated activities. Interestingly, the MBP-2 associated effects on AHR were specific to this major basic protein isoform as the loss of the more abundant granule protein MBP-1 had no effects on pulmonary pathologies, including AHR.CONCLUSIONS: The identification of a causative link between AHR and the release of an eosinophil secondary granule protein (i.e., degranluation) provides a an important link between allergen-induced pathologies and this long suspected eosinophil-mediated activity in the lung. RATIONALE: Eosinophils are a major leukocyte recruited to the lungs in both mouse allergen provocation models and patients with asthma. However, a link between these cells and the pathological changes occurring in the lung remains obscure although eosinophil effector functions have been hypothesized to be mediated, in part, by the release of secreted cationic proteins sequestered in cytoplasmic granules (i.e., the degranulation of activated eosinophils recruited to the lung). METHODS: Gene knockout lines of mice deficient for the two eosinophil major basic proteins (MBP-1, -2) were generated (strain: 129) by homologous recombination in ES cells and used in an acute allergen sensitization/aerosol challenge protocol to define causative links with pulmonary allergic infammation. RESULTS: This study demonstrates that despite observations showing that allergen challenge mouse models display only nominal levels of eosinophil degranulation the loss of the eosinophil granule protein MBP-2 abolished allergen-induced airway hyperresponsiveness (AHR). The loss of AHR in MBP-2-/- mice occurred with out concominant loss of other allergen-induced pulmonary pathologies (e.g., goblet cell metaplasia), suggesting a specific link between this pathophysiologic response and MBP-2 mediated activities. Interestingly, the MBP-2 associated effects on AHR were specific to this major basic protein isoform as the loss of the more abundant granule protein MBP-1 had no effects on pulmonary pathologies, including AHR. CONCLUSIONS: The identification of a causative link between AHR and the release of an eosinophil secondary granule protein (i.e., degranluation) provides a an important link between allergen-induced pathologies and this long suspected eosinophil-mediated activity in the lung.

The identification and characterization of mouse basophils have historically been hampered by the extreme rarity of this cell type. Virtually no photomicrographs of hematologically stained (eg, Wright-Giemsa) examples of mouse basophils exist in the literature. However, 4 recent studies in the past 2 years have used flow cytometry and a defined set of cell-surface markers to identify and subsequently isolate mouse “basophils,” including the publication of stained cytospin preparations of these cells. Surprisingly, a reevaluation of the data from all 4 of the studies revealed several issues of concern that suggest that the cells under study are not necessarily basophils. Nonetheless, we propose that these studies do provide the foundation for a reevaluation of the defining characteristics of a basophil and/or provide support for the provocative conclusion that a new previously overlooked leukocyte subtype has been identified. The purpose of this commentary is to revisit these previously published studies, highlight the relevant issues, and provide a different perspective in the hope of developing a consensus within the research community as to the true identity of the “basophils” described in these studies.

RATIONALE: The lack of a mouse models of asthma that faithfully replicates key symptoms associated with human allergic pulmonary disease, including the degranulation of eosinophils, has limited the usefulness of these models and, in turn, progress in the development of successful therapies for this syndrome. METHODS: Transgenic mice expressing IL-5 partially reproduce many of the pathologies associated with asthma and microarray data comparing gene expression in the lungs of allergen treated wild type vs. eosinophil-less mice (transgenic line: PHIL) demonstrated that in the absence of eosinophils pulmonary eotaxin-2 expression was elevated by more than an order of magnitude. Collectively, these observations suggested that IL-5 and eotaxin-2 may elicit unique responses and to test this hypothesis, we generated double transgenic (I5/E2) mice systemically expressing IL-5 in mature T cells and locally expressing eotaxin-2 in lung epithelial cells. RESULTS: We show I5/E2 mice develop a near-complete array of pulmonary pathologies representative of asthma, highlighted by extensive eosinophil degranulation, airway obstruction, and exacerbated methacholine-induced airway hyperresponsiveness. The genetic ablation of all eosinophils from I5/E2 mice (though breeding with PHIL) demonstrated that the induced pulmonary pathologies are directly the result of eosinophil activities. CONCLUSIONS: Unlike established allergen models, this novel transgenic line reproduces many of the changes occurring in asthma patients. In particular, the ability of I5/E2 mice to replicate the eosinophil activities occurring in asthma patients offers a unique opportunity to study asthma using a rodent model and fuels expectations of valid extrapolation to humans.

RATIONALE: Allergen-mediated inflammatory responses are associated with a massive expansion in the number of accumulating tissue eosinophils that often also display evidence of activation/degranulation. Unfortunately, because functionally sensitive eosinophil-specific antibodies useful in immunohistochemistry (IHC) platforms are rare, high throughput assays to assess archived clinical biopsies have been out of reach.METHODS: We have developed a novel anti-eosinophil peroxidase (EPO) mouse monoclonal antibody by sensitizing EPO knockout mice. This antibody is capable of reliably detecting both tissue eosinophils and evidence of released EPO using the most common archived clinical samples - formalin-fixed paraffin-embedded tissues.RESULTS: A standardized anti-EPO based IHC assay was used to examine gastrointestinal biopsies from patients who displayed physical symptoms suggestive of allergic inflammation (i.e., peripheral eosinophilia, atopy, diarrhea/flushing with certain trigger foods) but whose pathology reports were otherwise inconclusive. This new IHC assay confirmed that in many of these cases eosinophils were present and their numbers were not particularly elevated. However, and in contrast to the "near-normal" numbers of intact eosinophils in these cases, the EPO-based IHC assay revealed evidence for extensive and often regionally-specific eosinophil degranulation. Specifically, this assay was capable of detecting and visualizing the presence of both enucleated cytoplasmic fragments and large numbers of individual free eosinophil secondary granules.CONCLUSIONS: The availability of this new anti-EPO monoclonal antibody-based assay provides the pathology community with an invaluable resource for high throughput IHC screens of biopsies to aide in the assessments of allergic patients. RATIONALE: Allergen-mediated inflammatory responses are associated with a massive expansion in the number of accumulating tissue eosinophils that often also display evidence of activation/degranulation. Unfortunately, because functionally sensitive eosinophil-specific antibodies useful in immunohistochemistry (IHC) platforms are rare, high throughput assays to assess archived clinical biopsies have been out of reach. METHODS: We have developed a novel anti-eosinophil peroxidase (EPO) mouse monoclonal antibody by sensitizing EPO knockout mice. This antibody is capable of reliably detecting both tissue eosinophils and evidence of released EPO using the most common archived clinical samples - formalin-fixed paraffin-embedded tissues. RESULTS: A standardized anti-EPO based IHC assay was used to examine gastrointestinal biopsies from patients who displayed physical symptoms suggestive of allergic inflammation (i.e., peripheral eosinophilia, atopy, diarrhea/flushing with certain trigger foods) but whose pathology reports were otherwise inconclusive. This new IHC assay confirmed that in many of these cases eosinophils were present and their numbers were not particularly elevated. However, and in contrast to the "near-normal" numbers of intact eosinophils in these cases, the EPO-based IHC assay revealed evidence for extensive and often regionally-specific eosinophil degranulation. Specifically, this assay was capable of detecting and visualizing the presence of both enucleated cytoplasmic fragments and large numbers of individual free eosinophil secondary granules. CONCLUSIONS: The availability of this new anti-EPO monoclonal antibody-based assay provides the pathology community with an invaluable resource for high throughput IHC screens of biopsies to aide in the assessments of allergic patients.

WDM EPONs not only allow for cautious pay-as-you-grow upgrades of single-channel TDM EPONs but also avoid linearly increasing polling cycle times for an increasing number of ONUS. In this article, we first provide a comprehensive overview of the state of the art of TDM EPONs and recently reported dynamic bandwidth allocation algorithms, including decentralized scheduling schemes. After reviewing previous work on WDM EPONs, we address the requirements of WDM upgraded EPONs and make recommendations on an evolutionary WDM upgrade at the architecture, protocol, and dynamic bandwidth allocation algorithm levels, taking backward compatibility with MPCP and future-proofness against arbitrary WDM ONU structures into account. We describe and compare online and offline scheduling paradigms for WDM EPONs. Our simulation results indicate that online scheduling can achieve lower delays, especially at high loads. We outline areas of future research on WDM EPONs.

RATIONALE: The allergen provocation of an eosinophil-less transgenic line of mice demonstrated that eosinophils are required for OVA-induced pulmonary pathologies, including the development of airway hyperresponsiveness (AHR). This causative relationship suggests a link between eosinophil effector functions and the underlying immune responses in the lung following allergen challenge. METHODS: B cell activities (e.g., OVA-specific antibodies) BAL cytokine levels, and T cell functions in an acute OVA sensitized/aerosol challenged model of pulmonary inflammation were assessed in eosinophil-less transgenic mice (line: PHIL). RESULTS: OVA sensitization/aerosol challenge of PHIL mice showed that the loss of eosinophils in these animals was associated with dramatic reductions in induced pulmonary pathologies, including goblet metaplasia/mucus accumulation. More importantly, assessments of immune inflammatory parameters showed that the loss of eosinophils are accompanied with significant reductions in allergen-mediated pulmonary Th2 responses. CONCLUSIONS: The dependency of allergen-induced pulmonary pathologies on eosinophils suggests that these granulocytes participate at a fundamental level in underlying inflammatory responses following allergen sensitization/aerosol challenge. The data suggest that either cell autonomous expression of Th2 inflammatory signals by eosinophils themselves or eosinophil-mediated elaboration of lymphocyte activities are critical components of allergen-induced responses in the lung.

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disease characterized by defects in trafficking of intracellular vesicles to lysosome-related organelles such as melanosomes, lung lamellar bodies and platelet dense granules. The ashen ( rab27a ) mouse coat color mutant maintained at Roswell Park Cancer Institute (ash-Roswell) has been proposed as an animal model of HPS based upon prolonged bleeding times, decreased platelet aggregation and greatly decreased platelet dense granule components, including serotonin and ADP. However, human patients with Rab27a deficiency (Griscelli Syndrome) and other isolates of mouse ashen mutants do not exhibit prolonged bleeding suggesting an additional mutation in a gene affecting platelets but not melanocytes in the ash-Roswell mouse. In order to identify this mutation, a backcross between ash-Roswell and the PWK control strain was analyzed. In 390 backcross progeny, deficient platelet serotonin and diluted coat color ( Rab27a ) phenotypes segregated independently and in the proportions expected for single gene traits. The gene for serotonin deficiency was narrowed to a 2.1Mb region of mouse chromosome 10 containing 15 genes. None of these genes have been implicated previously in platelet storage pool deficiency. These studies indicate this genomic region contains a gene which uniquely controls platelet dense granules and is a candidate gene for a new form of human HPS.

RATIONALE: Animal models of asthma seldom replicate all aspects of human disease. Although mouse models do display, eosinophil infiltration, mucus secretion and airway hypersensitivity, they rarely reproduce epithelium desquamation, eosinophil degranulation, mucus plug formation, permanent hyper-responsiveness to methacholine and significant airway remodelling. Our goal was to develop a mouse model that would faithfully replicate characteristic pathologies present in patients with severe asthma.METHODS: Two transgenic mice were created: one that constitutively expresses IL-5 in T cells and the one that expresses eotaxin-2 in the lung. These two strains were crossed to produce the double transgenic mouse model of asthma under study. In this model we assessed lung histopathology, cytokine levels, and lung function, which were compared with data obtained from asthma patients.RESULTS: Systemic expression of IL-5 and local expression of eotaxin-2 produced phenotypes seen in only asthma patients. In particular, histology displayed characteristic eosinophilic inflammation in the lungs with significant eosinophil degranulation. Moreover, airway morphology featured epithelial desquamation, sub-epithelium thickening, smooth muscle hyperplasia/hypertrophy, and airway remodelling. Significant goblet cell metaplasia led to mucus accumulation in airway lumen and airway obstruction. Levels of Th2 cytokines were also elevated. Lung function in these animals was considerably impaired as indicated by bronchial hypersensitivity and increased airway resistance. Severity of lung pathologies increased with age.CONCLUSIONS: Synergistic effect of elevated levels of IL-5 and eotaxin-2 in the mouse results in a phenotype that closely resemble asthma in humans and should prove to be invaluable in the characterization of this disease. RATIONALE: Animal models of asthma seldom replicate all aspects of human disease. Although mouse models do display, eosinophil infiltration, mucus secretion and airway hypersensitivity, they rarely reproduce epithelium desquamation, eosinophil degranulation, mucus plug formation, permanent hyper-responsiveness to methacholine and significant airway remodelling. Our goal was to develop a mouse model that would faithfully replicate characteristic pathologies present in patients with severe asthma. METHODS: Two transgenic mice were created: one that constitutively expresses IL-5 in T cells and the one that expresses eotaxin-2 in the lung. These two strains were crossed to produce the double transgenic mouse model of asthma under study. In this model we assessed lung histopathology, cytokine levels, and lung function, which were compared with data obtained from asthma patients. RESULTS: Systemic expression of IL-5 and local expression of eotaxin-2 produced phenotypes seen in only asthma patients. In particular, histology displayed characteristic eosinophilic inflammation in the lungs with significant eosinophil degranulation. Moreover, airway morphology featured epithelial desquamation, sub-epithelium thickening, smooth muscle hyperplasia/hypertrophy, and airway remodelling. Significant goblet cell metaplasia led to mucus accumulation in airway lumen and airway obstruction. Levels of Th2 cytokines were also elevated. Lung function in these animals was considerably impaired as indicated by bronchial hypersensitivity and increased airway resistance. Severity of lung pathologies increased with age. CONCLUSIONS: Synergistic effect of elevated levels of IL-5 and eotaxin-2 in the mouse results in a phenotype that closely resemble asthma in humans and should prove to be invaluable in the characterization of this disease.

Rationale Previous studies have shown that eosinophils accumulate within many epithelial tumors. Nonetheless, the basis of this accumulation or the specific localization of eosinophils within these tumors remains obscure. Methods C57BL/6J mice were injected (s.c. ) with B16 melanoma cells and tumors harvested at days 10 and 16 for immunohistochemistry using antibodies to eosinophil major basic protein (MBP). Additional groups of tumor bearing mice were examined, including recipients of adoptively transferred eosinophils, and mice administered mAbs to deplete CD4 + T-cells and eosinophils. Results Eosinophils accumulate differentially in tumors, occurring in the necrotic and capsule areas and noticeably absent from viable regions. Interestingly, necrotic areas appeared in tumors of mice devoid of eosinophils. This accumulation was an active process as adoptively transferred ( i.v. ) eosinophils were recruited to necrotic areas within 24 hours of transfer. Transwell chemotaxis assays failed to detect tumor cell-released chemoattractants. Despite an association with Th2 responses, eosinophil accumulation also occurred in mice devoid of CD4 + T cells. Conclusions Eosinophils are actively recruited to solid tumors and differentially accumulate in the necrotic and capsule areas independent of Th2 responses (i.e., in the absence of CD4 + T cells). In the absence of released chemoattractants and a direct link between eosinophils and tumor cell death, the mechanism(s) and consequence(s) of this accumulation is unclear.

Rationale The availability of eosinophil-specific antibodies capable of identifying infiltrating eosinophils in formalin-fixed paraffin-embedded FF-PE) tissues from humans and most animal model systems is limited. However, the omnipresence of eosinophils in mammals suggests that antibody reagents targeting conserved regions of granule proteins from any one mammal may have pan-species specificity. Methods Eosinophil secondary granule proteins (ESGPs) were purified from eosinophils of IL-5 transgenic mice and used, together with Freund's adjuvant, as the antigen to immunize rabbits. The resulting polyclonal serum was assessed by ELISA and immunohistochemistry with FF-PE sections. Results The polyclonal anti-mouse ESGP antisera displayed a unique ability to detect infiltrating tissue eosinophils from all of the mammalian species tested, including mice, rats, non-human primates, and guinea pigs. In addition, it was shown that this antisera was capable of specifically detecting eosinophils lung sections from asthma patients, even in cases where the blocks were collected more than 20 years ago. Conclusions Sequence conservation exists between one or more of the mouse ESGP genes and orthologues in other mammals as to allow pan-species epitope recognition by mouse total ESGP polyclonal antisera. This reagent uniquely recognizes eosinophils regardless of the species examined, including FF-PE human biopsy samples. This reagent is a novel and previously unavailable tool for the study of diseases associated with eosinophils.

Rationale The activation state of eosinophils and its relationship to allergen-induced pulmonary pathologies has remained unclear. This report describes a link between eosinophil CD69 cell surface levels in the lung, activation, and allergen-provoked pathologies in mice. Methods Purified blood (i.e., CD69 − ) eosinophils were transferred ( viz. , intratracheal instillation) into the lungs of either naive or ovalbumin (OVA)-treated IL-5 −/− mice and subsequent changes in CD69 cell surface expression were determined by FACS analysis. In some cohorts, mice OVA-treated mice were concurrently administered mAbs to ablate CD4 + T cells. This strategy resulted in a pulmonary eosinophilia in both groups of mice equivalent to that observed in OVA-treated wild-type animals. Results Reconstitution of the pulmonary eosinophilia of OVA-treated IL-5 −/− mice lead to the development of pulmonary pathologies equivalent to those observed in OVA-treated wild-type mice. This occurred only in OVA-treated mice and did not occur following transfer of eosinophils to naive animals. Significantly, the development of pathologies was associated with CD69 expression on the transferred eosinophils. This activation marker did not appear on eosinophils transferred to naive IL-5 −/− mice. Moreover, this CD69 expression on eosinophils was T cell dependent and did not occur in mice treated with GK1.5 (anti-CD4) antibodies. Conclusions Thus, pulmonary pathologies are dependent, in part, on CD4 + T cell-mediated inflammatory signals that lead not only to eosinophil recruitment but also eosinophil activation as characterized by expression of CD69. These data support an expanded view of T cell and eosinophil activities and suggest that eosinophil effector functions impinge directly on lung function.

Rationale The refractory character of mouse eosinophils with respect to activation/degranulation has limited the utility of these animals as models of eosinophil effector functions (EEFs) in the lung and, therefore, human diseases. The effects of ectopic expression of both IL-5 and eotaxin-2 were assessed to develop a model system that duplicates the EEFs that occur in the lungs of asthma patients. Methods The effects of eotaxin-2 and IL-5 were assessed either through intratracheal instillation in OVA-treated mice or by constitutive expression in transgenic animals. Results Instillation of either eotaxin-1 or -2 into the lungs of OVA-treated mice results in eosinophil degranulation and the release of secondary granule proteins. The observed effects of eotaxin-2 were particularly significant. Constitutive expression of either eotaxin-2 or IL-5 in transgenic mice had limited, but defined, effect on the lung, including a nominal tissue eosinophilia without an increase in BAL eosinophils or induced histopathology. Nonetheless, mice of each line displayed altered airway responses to methacholine. In contrast, transgenic mice expressing both IL-5 and eotaxin-2 developed a dramatic tissue and airway eosinophilia. This eosinophilia was also accompanied by evidence of degranulation and the release of secondary granule proteins as well as equally dramatic pulmonary histopathologies and lung dysfunction. Conclusions The synergistic effect of expressing both eotaxin-2 and IL-5 leads to the recruitment/activation of eosinophils and, in turn, to pulmonary pathologies. These data suggest that lung EEFs may require both stimuli and that these activities may contribute to pulmonary pathologies.

Rationale Tissue eosinophilia is an accepted hallmark feature of parasite infections and allergen-mediated inflammatory responses. We have developed a number of polyclonal and monoclonal antibodies specific for mouse eosinophils and their associated products to facilitate studies of this leukocyte and the use of mouse models of human diseases. Methods Eosinophil secondary granule proteins, including major basic protein (MBP), eosinophil peroxidase (EPO), and eosinophil associated ribonucleases (EARs), were purified from eosinophils derived from IL-5 transgenic mice. These purified proteins were used in immunization and screening protocols to generate rabbit polyclonal antisera as well as monoclonal antibodies in rats and mice. Results Rabbit polyclonal antisera reactive to MBP and EARs were generated and shown to be useful reagents in western blots of cell/tissue extracts, immuno-electron microscopy, and immunohistochemistry/immunofluorescence using formalin-fixed paraffin-embedded tissue sections. Moreover, a series of rat and mouse monoclonal antibodies reactive to MBP, EPO, and EARs were produced and shown also to function in the venues noted above. Many of these antibodies also cross-react with human and other non-rodent eosinophil targets. Finally, specific monoclonal antibodies were selected for their use in an immunoblot ELISA format which has yielded a quantitative assay to measure the levels of released granule components in compartments such as the airway lumen (i.e., BAL). Conclusions The availability of multiple eosinophil-specific antibody reagents provides the community with invaluable resources for the characterization of eosinophils and their role(s) in disease model systems. Information regarding specific reagents, as well as their availability, is possible through a newly established website http://www.eosinophils.org.

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.

RATIONALE:The eosinophil is part of innate immunity, accumulating in response to parasitic infections, allergic and asthmatic inflammation, and may be part of the immune response triggered during tumorgenesis and viral infection.Although the activities mediated by eosinophils are numerous, degranulation is believed to be a prominent effector function.In particular, the release of cationic proteins has been suggested to mediate tissue damage and elicit cell agonist activities on other proinflammatory cells.Unfortunately, the study of these proteins is hindered by the low abundance of the eosinophil and the lack of technologies to study these macromolecules.