Development and application of novel immunoassays for eosinophil granule major basic proteins to evaluate eosinophilia and myeloproliferative disorders.

BACKGROUND: During eosinophil differentiation, the granule eosinophil major basic protein 1 (eMBP1) is synthesized as a 32-kDa precursor form, referred to as proMBP1, which is processed into the 14-kDa mature form of eMBP1. The prevalence of these two forms of MBP1 in most pathological conditions remains unknown. OBJECTIVE: To develop the immunoassays that differentiate mature eMBP1 and proMBP1 and apply them to analyze their levels in biological fluids from patients with eosinophilia and hematologic disorders. METHODS: We produced a series of monoclonal antibodies and selected pairs capable of discriminating between the two molecular forms of eMBP1. Radioimmunoassay (RIA) was performed to simultaneously quantitate the levels of mature eMBP1 and proMBP1 in secretions from patients with chronic rhinosinusitis (CRS) and sera from patients with hypereosinophilic syndrome (HES) and other myeloproliferative disorders. RESULTS: The novel immunoassays possessed less than 1% crossreactivity between mature eMBP1 and proMBP1. Mature eMBP1, but not proMBP1, was found in nasal secretions of CRS patients. In contrast, elevated serum levels of mature eMBP1 and proMBP1 were observed in approximately 60% and 90% of HES patients, respectively, with proMBP1 present in greater quantities than mature eMBP1. Patients with several myeloproliferative disorders also showed high serum levels of proMBP1 while mature eMBP1 remained at basal levels. CONCLUSION: The novel immunoassays successfully differentiated mature eMBP1 and proMBP1 in human biological fluids. Further studies addressing the clinical correlates of these assays will help to develop biomarkers to diagnose and monitor patients with eosinophilia and myeloproliferative disorders.

Assessment of Lung Eosinophils In Situ Using Immunohistological Staining.

Eosinophils are rare white blood cells that are recruited from circulation to accumulate in the lung in mouse models of allergic respiratory inflammation. In hematoxylin-eosin (HE) stained lungs, eosinophils may be difficult to detect despite their bright eosin staining in the secondary granules. For this reason, antibody-mediated detection of eosinophils is preferable for specific and clearer identification of these cells. Moreover, eosinophils may degranulate, releasing their granule proteins into surrounding tissue, and remnants of cytolysed cells cannot be detected by HE staining. The methods here demonstrate the use of eosinophil-specific anti-mouse antibodies to detect eosinophil granule proteins in formalin-fixed cells both in situ in paraffin-embedded lungs, as well as in cytospin preparations from the lung. These antibody staining techniques enable either colorimetric or fluorescence imaging of eosinophils or their granule proteins with the potential for additional antibodies to be added for detection of multiple molecules.

Mechanism of atopic cataract caused by eosinophil granule major basic protein.

Atopic cataracts develop under the ages of 40 years, after which visual acuity rapidly declines. However, the mechanism underlying the development of atopic cataracts is not yet clear. We focused on the eosinophil granule major basic protein (MBP), which was detected in the aqueous humor of atopic cataracts previously, and which was cytotoxic. Specifically, we investigated its origin in this fluid and its effects on lens epithelial cells (LECs). MBP immunostaining was positive in atopic cataract-derived LECs, but negative in age-related cataract-derived LECs. MBP mRNA was not detected in either type of cataract, but protein was detected in the aqueous humor. Furthermore, the flare values associated with atopic cataracts were higher than those with age-related cataracts. When MBP was purified from eosinophils or recombinant MBP was added to LEC culture medium, cell viability decreased in a concentration-dependent manner, but an MBP antibody neutralized the cytotoxic effect of this protein towards these cells. These results were consistent with the flow of MBP into the aqueous humor from the blood due to a compromised blood-aqueous barrier. Thus, MBP could further penetrate the lens capsule and adhere to LECs, resulting in decreased cell viability and the development of atopic cataracts.

Identification of Piecemeal Degranulation and Vesicular Transport of MBP-1 in Liver-Infiltrating Mouse Eosinophils During Acute Experimental Schistosoma mansoni Infection.

Eosinophils have been long associated with helminthic infections, although their functions in these diseases remain unclear. During schistosomiasis caused by the trematode Schistosoma mansoni, eosinophils are specifically recruited and migrate to sites of granulomatous responses where they degranulate. However, little is known about the mechanisms of eosinophil secretion during this disease. Here, we investigated the degranulation patterns, including the cellular mechanisms of major basic protein-1 (MBP-1) release, from inflammatory eosinophils in a mouse model of S. mansoni infection (acute phase). Fragments of the liver, a major target organ of this disease, were processed for histologic analyses (whole slide imaging), conventional transmission electron microscopy (TEM), and immunonanogold EM using a pre-embedding approach for precise localization of major basic protein 1 (MBP-1), a typical cationic protein stored pre-synthesized in eosinophil secretory (specific) granules. A well-characterized granulomatous inflammatory response with a high number of infiltrating eosinophils surrounding S. mansoni eggs was observed in the livers of infected mice. Moreover, significant elevations in the levels of plasma Th2 cytokines (IL-4, IL-13, and IL-10) and serum enzymes (alanine aminotransferase and aspartate aminotransferase) reflecting altered liver function were detected in response to the infection. TEM quantitative analyses revealed that while 19.1% of eosinophils were intact, most of them showed distinct degranulation processes: cytolysis (13.0%), classical and/or compound exocytosis identified by granule fusions (1.5%), and mainly piecemeal degranulation (PMD) (66.4%), which is mediated by vesicular trafficking. Immunonanogold EM showed a consistent labeling for MBP-1 associated with secretory granules. Most MBP-1-positive granules had PMD features (79.0 ± 4.8%). MBP-1 was also present extracellularly and on vesicles distributed in the cytoplasm and attached to/surrounding the surface of emptying granules. Our data demonstrated that liver-infiltrating mouse eosinophils are able to degranulate through different secretory processes during acute experimental S. mansoni infections with PMD being the predominant mechanism of eosinophil secretion. This means that a selective secretion of MBP-1 is occurring. Moreover, our study demonstrates, for the first time, a vesicular trafficking of MBP-1 within mouse eosinophils elicited by a helminth infection. Vesicle-mediated secretion of MBP-1 may be relevant for the rapid release of small concentrations of MBP-1 under cell activation.

Eosinophil Cationic Protein (ECP), a predictive marker of bullous pemphigoid severity and outcome.

Bullous Pemphigoid (BP) is an inflammatory rare autoimmune bullous dermatosis, which outcome cannot be predicted through clinical investigations. Eosinophils are the main immune infiltrated cells in BP. However, the release of Major Basic Protein (MBP), Eosinophil Derived Neurotoxin (EDN), and Eosinophil Cationic Protein (ECP) upon eosinophil activation has still not been evaluated with respect to BP development. MBP, EDN and ECP were measured by ELISA in serum (n = 61) and blister fluid (n = 20) of patients with BP at baseline, and in serum after 2 months of treatment (n = 41). Eosinophil activation in BP patients was illustrated at baseline by significantly higher MBP, EDN and ECP serum concentrations as compared with control subjects (n = 20), but without distinction according to disease severity or outcome. EDN and ECP values were even higher in the blister fluids (P < 0.01 and P < 0.05, respectively), whereas MBP values were lower (P < 0.001). ECP serum concentration decreased after 60 days of treatment in BP patients with ongoing remission but not in patients who later relapsed (P < 0.05). A reduction of at least 12.8 ng/mL in ECP concentrations provided a positive predictive value for remission of 81%, showing that ECP serum variation could be a useful biomarker stratifying BP patients at risk of relapse.

Frontline Science: Eosinophil-deficient MBP-1 and EPX double-knockout mice link pulmonary remodeling and airway dysfunction with type 2 inflammation.

Eosinophils and the release of cationic granule proteins have long been implicated in the development of the type 2-induced pathologies linked with respiratory inflammation. Paradoxically, the ablation of the two genes encoding the most abundant of these granule proteins, major basic protein-1 (MBP-1) and eosinophil peroxidase (EPX), results in a near collapse of eosinophilopoiesis. The specificity of this lineage ablation and the magnitude of the induced eosinopenia provide a unique opportunity to clarify the importance of eosinophils in acute and chronic inflammatory settings, as well as to identify potential mechanism(s) of action linked with pulmonary eosinophils in those settings. Specifically, we examined these issues by assessing the induced immune responses and pathologies occurring in MBP-1-/-/EPX-/- mice after 1) ovalbumin sensitization/provocation in an acute allergen-challenge protocol, and 2) crossing MBP-1-/-/EPX-/- mice with a double-transgenic model of chronic type 2 inflammation (i.e., I5/hE2). Acute allergen challenge and constitutive cytokine/chemokine expression each induced the accumulation of pulmonary eosinophils in wild-type controls that was abolished in the absence of MBP-1 and EPX (i.e., MBP-1-/-/EPX-/- mice). The expression of MBP-1 and EPX was also required for induced lung expression of IL-4/IL-13 in each setting and, in turn, the induced pulmonary remodeling events and lung dysfunction. In summary, MBP-1-/-/EPX-/- mice provide yet another definitive example of the immunoregulatory role of pulmonary eosinophils. These results highlight the utility of this unique strain of eosinophil-deficient mice as part of in vivo model studies investigating the roles of eosinophils in health and disease settings.

Dose-response relationship of specific allergen exposure-induced immunological tolerance: a mouse model.

BACKGROUND: It is believed that adequate allergen preimmunization exposure could induce immunologic tolerance. The purpose of this study was to investigate the dose-dependent mechanisms related to antigen-specific tolerance induction in a mouse model. METHODS: Mice were assigned to 5 groups: the control (Cont) group received phosphate-buffered saline (PBS) preimmunization exposure and PBS sham immunization; the other 4 groups were exposed preimmunization to PBS (PBS group) or ovalbumin (OVA) (first mucosal doses: 1.25%, 2.5%, or 5% wt/vol aerosol from days -3 to -1) prior to OVA immunization. The OVA-immunized mice received intraperitoneal doses of 20 µg OVA (on days 1, 7, and 14), and then a second set of mucosal doses with 0.5% wt/vol OVA aerosol (on days 18 to 20). After assessment of airway hyperresponsiveness (AHR), the mice were euthanized and their blood, bronchoalveolar lavage fluids (BALFs), and lung tissues were collected for further analyses. RESULTS: OVA-immunized mice exposed to OVA preimmunization had reduced AHR and immunoglobulin E production when compared to the PBS group. OVA preimmunization exposure inhibited eosinophilic inflammation in lung tissues. The proportions of BALF eosinophil counts from the groups exposed to OVA preimmunization were significantly decreased when compared with those exposed to PBS preimmunization. The balance of T helper 2 (Th2) and T regulatory (Treg) cytokines in BALFs were additionally observed in this mouse model. CONCLUSION: Our results suggest that preimmunization exposure to an appropriate dose of a specific antigen could suppress allergic airway inflammation by induction of immunological tolerance.

A Novel In-Frame Deletion in the Leucine Zipper Domain of C/EBPε Leads to Neutrophil-Specific Granule Deficiency.

Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by neutrophil dysfunction, bilobed neutrophil nuclei and lack of neutrophil-specific granules. Defects in a myeloid-specific transcription factor, CCAAT/enhancer binding protein-ε (C/EBPε), have been identified in two cases in which homozygous frameshift mutations led to loss of the leucine zipper domain. In this study, we report a 55-y-old woman affected with SGD caused by a novel homozygous 2-aa deletion (ΔRS) in the leucine zipper domain of the C/EBPε gene. The patient showed characteristic neutrophil abnormalities and recurrent skin infections; however, there was no history of deep organ infections. Biochemical analysis revealed that, in contrast to the two frameshift mutations, the ΔRS mutant maintained normal cellular localization, DNA-binding activity, and dimerization, and all three mutants exhibited marked reduction in transcriptional activity. The ΔRS mutant was defective in its association with Gata1 and PU.1, as well as aberrant cooperative transcriptional activation of eosinophil major basic protein. Thus, the ΔRS likely impairs protein-protein interaction with other transcription factors, resulting in a loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function, and indicate that multiple molecular mechanisms lead to SGD.

IL-33 activates eosinophils of visceral adipose tissue both directly and via innate lymphoid cells.

Eosinophils are multifunctional leukocytes involved in allergic reactions as well as adipose tissue regulation. IL-5 is required for eosinophil survival; however, the in vivo mechanisms of eosinophil regulation are not fully understood. A tg mouse model with il5 promoter-driven EGFP expression was established for detecting the IL-5-producing cells in vivo. Il5-egfp tg mice expressed high levels of EGFP in gonadal adipose tissue (GAT) cells. EGFP(+) cells in GAT were mainly group 2 innate lymphoid cells (ILCs). IL-33 preferentially expanded EGFP(+) cells and eosinophils in GAT in vivo. EGFP(+) ILCs were found to upregulate prg2 mRNA expression in GAT eosinophils. These results demonstrate that ILCs activate eosinophils in GAT. The blockage of IL-33Rα, on the other hand, did not impair EGFP(+) ILC numbers but did impair eosinophil numbers in vivo. GAT eosinophils expressed IL-33Rα and IL-33 expanded eosinophil numbers in CD90(+) cell-depleted mice. IL-33 was further observed to induce the expression of retnla and epx mRNA in eosinophils. These findings demonstrate that IL-33 directly activates eosinophils in GAT, and together with our other findings described above, our findings show that IL-33 has dual pathways via which it activates eosinophils in vivo: a direct activation pathway and a group 2 ILC-mediated pathway.

Eosinophil granule proteins: form and function.

Experimental and clinical data strongly support a role for the eosinophil in the pathogenesis of asthma, allergic and parasitic diseases, and hypereosinophilic syndromes, in addition to more recently identified immunomodulatory roles in shaping innate host defense, adaptive immunity, tissue repair/remodeling, and maintenance of normal tissue homeostasis. A seminal finding was the dependence of allergic airway inflammation on eosinophil-induced recruitment of Th2-polarized effector T-cells to the lung, providing a missing link between these innate immune effectors (eosinophils) and adaptive T-cell responses. Eosinophils come equipped with preformed enzymatic and nonenzymatic cationic proteins, stored in and selectively secreted from their large secondary (specific) granules. These proteins contribute to the functions of the eosinophil in airway inflammation, tissue damage, and remodeling in the asthmatic diathesis. Studies using eosinophil-deficient mouse models, including eosinophil-derived granule protein double knock-out mice (major basic protein-1/eosinophil peroxidase dual gene deletion) show that eosinophils are required for all major hallmarks of asthma pathophysiology: airway epithelial damage and hyperreactivity, and airway remodeling including smooth muscle hyperplasia and subepithelial fibrosis. Here we review key molecular aspects of these eosinophil-derived granule proteins in terms of structure-function relationships to advance understanding of their roles in eosinophil cell biology, molecular biology, and immunobiology in health and disease.

Eosinophil major basic protein activates human cord blood mast cells primed with fibroblast membranes by integrin-ß1.

BACKGROUND: Mast cell (MC) - eosinophil (Eos) activating cross-talk might be critical for the severity and chronicity of allergy. Among soluble mediators, eosinophil major basic protein (MBP), a hallmark of allergy, is particularly important because it was shown to activate specific MC subtypes. We previously demonstrated that MBP activates IgE-desensitized rat MC and human lung and cord blood-derived MC (CBMC) after priming with fibroblast membranal stem cell factor. However, a distinct mechanism for this activation was missing. Therefore, we aimed to investigate it. METHODS: Major basic protein-1 activation of CBMC primed with fibroblast-derived membranes (FBM) was measured by ß-hexosaminidase and tryptase release. Chemical cross-linking followed by micrometric flow cytometry probed direct interactions. Antibodies neutralized integrin-ß1 and recognized its active form. Pertussis toxin (Ptx) was used to decrease integrin-ß1 active form expression. Hematopoietic cell kinase (Hck) was identified by immunoprecipitation (IP) and silenced by siRNA. RESULTS: Major basic protein-1-induced CBMC activation is mediated partly by MBP1-integrin-ß1 interaction on the MC surface. FBM prime CBMC via a G protein, as confirmed by Ptx, to shift integrin-ß1 to its active form. Following MBP1 binding, integrin-ß1 binds Hck that further transduces the activation signal. MC priming with FBM leads to up-regulation in Hck protein level. MC integrin-ß1 neutralization inhibits MBP1-induced activation and uptake. Hck silencing results with reduced MBP1-induced activation. CONCLUSIONS: Fibroblast-derived membranes, integrin-ß1, and Hck are involved in MBP1-induced activation of CBMC and therefore represent a distinct mechanism for this activation. This finding might implicate integrin-ß1 and Hck as targets for decreasing MC - Eos activating cross-talk in allergy.

Molecular analysis of non-specific protection against murine malaria induced by BCG vaccination.

Although the effectiveness of BCG vaccination in preventing adult pulmonary tuberculosis (TB) has been highly variable, epidemiologic studies have suggested that BCG provides other general health benefits to vaccinees including reducing the impact of asthma, leprosy, and possibly malaria. To further evaluate whether BCG immunization protects against malarial parasitemia and to define molecular correlates of this non-specific immunity, mice were vaccinated with BCG and then challenged 2 months later with asexual blood stage Plasmodium yoelii 17XNL (PyNL) parasites. Following challenge with PyNL, significant decreases in parasitemia were observed in BCG vaccinated mice relative to naïve controls. To identify immune molecules that may be associated with the BCG-induced protection, gene expression was evaluated by RT-PCR in i) naïve controls, ii) BCG-vaccinated mice, iii) PyNL infected mice and iv) BCG vaccinated/PyNL infected mice at 0, 1, 5, and 9 days after the P. yoelii infection. The expression results showed that i) BCG immunization induces the expression of at least 18 genes including the anti-microbial molecules lactoferrin, eosinophil peroxidase, eosinophil major basic protein and the cathelicidin-related antimicrobial peptide (CRAMP); ii) an active PyNL infection suppresses the expression of important immune response molecules; and iii) the extent of PyNL-induced suppression of specific genes is reduced in BCG-vaccinated/PyNL infected mice. To validate the gene expression data, we demonstrated that pre-treatment of malaria parasites with lactoferrin or the cathelicidin LL-37 peptide decreases the level of PyNL parasitemias in mice. Overall, our study suggests that BCG vaccination induces the expression of non-specific immune molecules including antimicrobial peptides which may provide an overall benefit to vaccinees by limiting infections of unrelated pathogens such as Plasmodium parasites.

Protection of montelukast on OVA-induced eosinophilic gastroenteritis via modulating IL-5, eotaxin-1 and MBP expression.

The aim of this study was to further explore the possible mechanisms of montelukast on oral mice ovalbumin-induced eosinophilic gastroenteritis in a mouse model. The results indicated that montelukast could prevent levels of eotaxin-1 and interleukin-5 in intestinal mucosa homogenate when compared with model group. Interestingly, the increase of major basic protein expression in jejunal tissue also was attenuated by treated with montelukast.

Major basic protein from eosinophils and myeloperoxidase from neutrophils are required for protective immunity to Strongyloides stercoralis in mice.

Eosinophils and neutrophils contribute to larval killing during the primary immune response, and neutrophils are effector cells in the secondary response to Strongyloides stercoralis in mice. The objective of this study was to determine the molecular mechanisms used by eosinophils and neutrophils to control infections with S. stercoralis. Using mice deficient in the eosinophil granule products major basic protein (MBP) and eosinophil peroxidase (EPO), it was determined that eosinophils kill the larvae through an MBP-dependent mechanism in the primary immune response if other effector cells are absent. Infecting PHIL mice, which are eosinophil deficient, with S. stercoralis resulted in development of primary and secondary immune responses that were similar to those of wild-type mice, suggesting that eosinophils are not an absolute requirement for larval killing or development of secondary immunity. Treating PHIL mice with a neutrophil-depleting antibody resulted in a significant impairment in larval killing. Naïve and immunized mice with neutrophils deficient in myeloperoxidase (MPO) infected with S. stercoralis had significantly decreased larval killing. It was concluded that there is redundancy in the primary immune response, with eosinophils killing the larvae through an MBP-dependent mechanism and neutrophils killing the worms through an MPO-dependent mechanism. Eosinophils are not required for the development or function of secondary immunity, but MPO from neutrophils is required for protective secondary immunity.

Effects of respiratory syncytial virus infection and major basic protein derived from eosinophils in pulmonary alveolar epithelial cells (A549).

RSV (respiratory syncytial virus)-induced pneumonia and bronchiolitis may be associated with hyperresponsive conditions, including asthma. Eosinophilic proteins such as MBP (major basic protein) may also be associated with the pathophysiology of asthma. To elucidate the roles of RSV infection and MBP in the pathogenesis of pneumonia with hyperresponsiveness, we investigated the effects of RSV infection and MBP on A549 (alveolar epithelial) cells. CPE (cytopathic effects) in A549 cells were observed by microscopy. Apoptosis and cell death was evaluated by flow cytometric analysis and modified MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. We also measured 15 types of cytokines and chemokines in A549 cell supernatants. Although RSV alone did not affect the CPE of A549, high concentrations of MBP resulted in cell death within 24 h. Combinations of RSV and MBP synergistically induced cell death. In A549 cells infected with RSV alone, the release of GM-CSF (granulocyte-macrophage colony-stimulating factor) was significantly enhanced compared with control cells (no infection). In the cells treated with MBP alone, the production of IL (interleukin)-2, 4, 5, 7, 10, 12, 13, 17, IFN (interferon)-γ, GM-CSF, G-CSF (granulocyte colony-stimulating factor) and MIP (macrophage inflammatory protein)-1ß was significantly increased compared with control cells. Notably, the levels of GM-CSF and IL-17 in RSV/MBP-treated cells were significantly higher than those treated with MBP alone. These results suggest that MBP synergistically enhanced the release of various cytokines/chemokines and the cell death of RSV-infected A549 cells, indicating that MBP may be closely associated with the pathophysiology of allergic reactions in bronchiolitis/pneumonia due to RSV.

Suppression of EAE by oral tolerance is independent of endogenous IFN-beta whereas treatment with recombinant IFN-beta ameliorates EAE.

IFN-beta is anticipated to have an important function in mucosal tolerance, as it is one of the major cytokines produced by plasmacytoid dendritic cells, and has recently been suggested as central to the maintenance of mucosal homeostasis. Here, we have investigated whether oral tolerance is dependent on endogenous IFN-beta by feeding low-dose self-antigen myelin basic protein to IFN-beta(-/-) mice with subsequent induction of experimental autoimmune encephalomyelitis (EAE). Our study shows that oral tolerance was readily induced in IFN-beta(-/-) mice compared with their wild-type littermates (IFN-beta(+/+)). The non-self-antigen ovalbumin induced oral tolerance in both groups. These data indicate that endogenous IFN-beta is not required for induction of oral tolerance, whereas delivery of recombinant IFN-beta results in significant reduction in clinical score of EAE. Oral tolerance induction was associated with lower production of antigen-specific IFN-gamma, no shift toward antigen-specific Th2, Th17 or TGF-beta response was observed. Oral tolerance in IFN-beta(-/-) mice was also associated with the induction of regulatory and memory T cells in the mucosal-associated immune organs, however this was not a prerequisite for establishment of oral tolerance.

Eosinophils oxidize damage-associated molecular pattern molecules derived from stressed cells.

Eosinophils (Eos) are found at increased numbers within necrotic areas of tumors. We show that necrotic material from cell lysates containing damage-associated molecular pattern molecules induce eosinophil degranulation (release of major basic protein and eosinophil peroxidase) and enhance their oxidative burst while the stimulatory capacity of cell lysates is significantly diminished following oxidation. High mobility group box 1 (HMGB1), a prototypic damage-associated molecular pattern molecule, released following necrosis but not apoptosis, induced a similar effect on Eos. Additionally, we demonstrate that HMGB1 enhances eosinophil survival and acts as a chemoattractant. Consistently, we show that Eos express an HMGB1 receptor, the receptor for advanced glycation end product, and that anti-receptor for advanced glycation end product could diminish the HMGB1-mediated effects. Of all tested biologic activities, Eos respond most sensitively to the presence of necrotic material including HMGB1 with generation of peroxide. We postulate that Eos "sense" necrotic cell death, migrating to and responding to areas of tissue injury/necrosis. Oxidation of cell lysates reduces their biologic activity when compared with native lysates. We postulate that eosinophil-associated modulation of immunity within tumor and other damaged tissues may be primarily by promoting oxidative degradation of necrotic material. Novel therapeutic strategies may be considered by advancing oxidative denaturation of released necrotic material using Eos or other aerobic strategies.

Biology of the eosinophil.

In this review, we aim to put in perspective the biology of a multifunctional leukocyte, the eosinophil, by placing it in the context of innate and adaptive immune responses. Eosinophils have a unique contribution in initiating inflammatory and adaptive responses, due to their bidirectional interactions with dendritic cells and T cells, as well as their large panel of secreted cytokines and soluble mediators. The mechanisms and consequences of eosinophil responses in experimental inflammatory models and human diseases are discussed.

The role of eosinophil major basic protein in angiogenesis.

BACKGROUND: Eosinophil-derived major basic protein (MBP) plays an active role in allergic inflammation and tissue remodelling. However, its role in angiogenesis has not been established as yet. Therefore our objective was to investigate whether MBP exhibits any direct pro-angiogenic effects. METHODS: Rat aortic endothelial cells and human umbilical vascular endothelial cells were cultured with different concentrations of MBP and their viability (Trypan blue exclusion test), proliferation (thymidine incorporation) and capillary-like structure formation (matrigel assay) were investigated in vitro. The angiogenic activity of MBP was then tested in vivo using the chick chorio allantoic membrane (CAM) assay. RESULTS: Subcytotoxic concentrations of MBP induce endothelial cell proliferation and enhance the pro-mitogenic effect of vascular endothelial growth factor (VEGF), but do not affect their VEGF release. MBP promotes capillarogenesis by endothelial cells seeded on matrigel and sprouting formation in the CAM assay. Furthermore, we have shown that the pro-angiogenic effect of MBP is not due to its cationic charge since stimulation of the CAMs with the synthetic polycation, poly-L-arginine does not induce any angiogenic effects. CONCLUSIONS: These data demonstrate that MBP has pro-angiogenic effects in vitro and in vivo, providing a novel mechanism whereby MBP can participate in tissue inflammation and remodelling in atopic diseases.

IL-17E upregulates the expression of proinflammatory cytokines in lung fibroblasts.

BACKGROUND: IL-17E is a new TH2 cytokine that promotes airway eosinophilia in mice. IL-17E proinflammatory activity has been proposed to involve induction of cytokine and chemokine production. Recruitment of inflammatory cells may be mediated by tissue-resident cells. OBJECTIVE: This study aimed to evaluate whether fibroblasts represent a target of IL-17E for the production of eosinophil active mediators in the lung. METHODS: Expression of IL-17B receptor (IL-17BR), a receptor for IL-17E, was evaluated by immunofluorescent staining, Western blot, and real-time PCR in human primary lung fibroblasts. Mediator production was analyzed by using real-time PCR and ELISA after stimulation of fibroblasts with IL-17E alone or in combination with TNF-alpha and TGF-beta1. Expression of IL-17E and of eosinophil major basic protein was evaluated by immunohistochemistry in bronchial biopsies from subjects with asthma. RESULTS: Human primary lung fibroblasts constitutively expressed IL-17BR. IL-17BR mRNA levels were increased in cells stimulated with TNF-alpha and decreased with TGF-beta1. IL-17E slightly upregulated CC chemokine ligand (CCL)-5, CCL-11, GM-CSF, and CXC chemokine ligand (CXCL)-8 mRNA in fibroblasts. Moreover, IL-17E and TNF-alpha synergistically induced GM-CSF and CXCL-8 mRNA. IL-17E also potentiated the upregulation of CXCL-8 transcripts observed with TGF-beta1. In contrast, TGF-beta1 decreased IL-17E-induced CCL-11 mRNA. The capacity of IL-17E to enhance GM-CSF and CXCL-8 responses to TNF-alpha was accompanied by production and secretion of both proteins by lung fibroblasts. Finally, IL-17E was detected in asthma in eosinophil-infiltrated bronchial submucosa. CONCLUSION: IL-17E may contribute to the induction and maintenance of eosinophilic inflammation in the airways by acting on lung fibroblasts. This study supports a role for IL-17E in asthma pathophysiology.