Cationic proteins from eosinophils bind bone morphogenetic protein receptors promoting vascular calcification and atherogenesis.

AIMS: Blood eosinophil count and eosinophil cationic protein (ECP) concentration are risk factors of cardiovascular diseases. This study tested whether and how eosinophils and ECP contribute to vascular calcification and atherogenesis. METHODS AND RESULTS: Immunostaining revealed eosinophil accumulation in human and mouse atherosclerotic lesions. Eosinophil deficiency in ΔdblGATA mice slowed atherogenesis with increased lesion smooth muscle cell (SMC) content and reduced calcification. This protection in ΔdblGATA mice was muted when mice received donor eosinophils from wild-type (WT), Il4-/-, and Il13-/- mice or mouse eosinophil-associated-ribonuclease-1 (mEar1), a murine homologue of ECP. Eosinophils or mEar1 but not interleukin (IL) 4 or IL13 increased the calcification of SMC from WT mice but not those from Runt-related transcription factor-2 (Runx2) knockout mice. Immunoblot analyses showed that eosinophils and mEar1 activated Smad-1/5/8 but did not affect Smad-2/3 activation or expression of bone morphogenetic protein receptors (BMPR-1A/1B/2) or transforming growth factor (TGF)-ß receptors (TGFBR1/2) in SMC from WT and Runx2 knockout mice. Immunoprecipitation showed that mEar1 formed immune complexes with BMPR-1A/1B but not TGFBR1/2. Immunofluorescence double-staining, ligand binding, and Scatchard plot analysis demonstrated that mEar1 bound to BMPR-1A and BMPR-1B with similar affinity. Likewise, human ECP and eosinophil-derived neurotoxin (EDN) also bound to BMPR-1A/1B on human vascular SMC and promoted SMC osteogenic differentiation. In a cohort of 5864 men from the Danish Cardiovascular Screening trial and its subpopulation of 394 participants, blood eosinophil counts and ECP levels correlated with the calcification scores of different arterial segments from coronary arteries to iliac arteries. CONCLUSION: Eosinophils release cationic proteins that can promote SMC calcification and atherogenesis using the BMPR-1A/1B-Smad-1/5/8-Runx2 signalling pathway.

Detection of Eosinophils in Tissue Sections by Immunohistochemistry.

Eosinophils are bone marrow-derived hematopoietic cells which represent a small subset in the peripheral blood, and under homeostatic conditions predominantly reside in certain organs, such as the gastrointestinal tract. However, eosinophil numbers increase both in the peripheral blood and tissues during allergic inflammation, parasitic infestation, drug reactions, vasculitides, as well as certain hematopoietic neoplasms. Their presence in tissues can be detected by hematoxylin and eosin staining; however, this may be challenging particularly at times of activation and/or degranulation, e.g., during allergic lung inflammation. Thus, detection of eosinophils and/or their released granule proteins is significantly enhanced by immunohistochemistry. This chapter describes methods for the detection of mouse or human eosinophils by using granule protein-specific antibodies in formalin-fixed paraffin-embedded tissue.

Eosinophils in Health and Disease: A State-of-the-Art Review.

Eosinophils play a homeostatic role in the body's immune responses. These cells are involved in combating some parasitic, bacterial, and viral infections and certain cancers and have pathologic roles in diseases including asthma, chronic rhinosinusitis with nasal polyps, eosinophilic gastrointestinal disorders, and hypereosinophilic syndromes. Treatment of eosinophilic diseases has traditionally been through nonspecific eosinophil attenuation by use of glucocorticoids. However, several novel biologic therapies targeting eosinophil maturation factors, such as interleukin (IL)-5 and the IL-5 receptor or IL-4/IL-13, have recently been approved for clinical use. Despite the success of biologic therapies, some patients with eosinophilic inflammatory disease may not achieve adequate symptom control, underlining the need to further investigate the contribution of patient characteristics, such as comorbidities and other processes, in driving ongoing disease activity. New research has shown that eosinophils are also involved in several homeostatic processes, including metabolism, tissue remodeling and development, neuronal regulation, epithelial and microbiome regulation, and immunoregulation, indicating that these cells may play a crucial role in metabolic regulation and organ function in healthy humans. Consequently, further investigation is needed into the homeostatic roles of eosinophils and eosinophil-mediated processes across different tissues and their varied microenvironments. Such work may provide important insights into the role of eosinophils not only under disease conditions but also in health. This narrative review synthesizes relevant publications retrieved from PubMed informed by author expertise to provide new insights into the diverse roles of eosinophils in health and disease, with particular emphasis on the implications for current and future development of eosinophil-targeted therapies.

Mechanisms of toxicity mediated by neutrophil and eosinophil granule proteins.

Neutrophils and eosinophils are granulocytes which are characterized by the presence of granules in the cytoplasm. Granules provide a safe storage site for granule proteins that play important roles in the immune function of granulocytes. Upon granulocytes activation, diverse proteins are released from the granules into the extracellular space and contribute to the fight against infections. In this article, we describe granule proteins of both neutrophils and eosinophils able to kill pathogens and review their anticipated mechanism of antimicrobial toxicity. It should be noted that an excess of granules protein release can lead to tissue damage of the host resulting in chronic inflammation and organ dysfunction.

Neutrophil and Eosinophil Granule Proteins as Potential Biomarkers of Assessing Disease Activity and Severity in Patients With Ulcerative Colitis.

BACKGROUND: Colonoscopy can assess disease activity and severity of ulcerative colitis (UC) accurately, but it is invasive and costly. Role of noninvasive biomarkers of intestinal inflammation in evaluation of patients with UC is not well understood. In this study, we assessed fecal eosinophil cationic protein (FECP), fecal myeloperoxidase (FMPO), and fecal calprotectin (FC) as surrogate markers of disease activity and severity in patients with UC, and then evaluated effect of the combination of these markers. METHODS: Sixty-three UC patients and 59 cases of age-matched controls were investigated. All patients underwent clinical, endoscopic, and histological assessment for disease activity and severity. Fecal samples were analyzed for FECP, FC, and FMPO. RESULTS: All three fecal biomarkers were elevated in patients compared with controls (P = 0.000). Significant differences were found between inactive UC and controls (P = 0.000). Cases with severe UC had significantly higher FECP levels than those with mild UC (p < 0.05), but there were no significant differences in FC and FMPO levels among disease severity groups. All three biomarkers showed positive correlation with Ulcerative Colitis Activity Index (UCAI). The areas under the ROC curve of FECP, FC, and FMPO were 0.939, 0.783, and 0.785, respectively. Sensitivity and specificity of fecal biomarkers in assessing disease activity were FECP-88.46%, 89.47%; FC-80.77%, 68.42%; and FMPO-84.62%, 63.16%. CONCLUSIONS: All three fecal biomarkers could be used as surrogate markers for assessing disease activity of UC, and FECP provided superior discrimination than FMPO and FC. Moreover, FECP could distinguish between mild disease and severe disease group.

The Eosinophil in Health and Disease: from Bench to Bedside and Back.

Historically, eosinophils have been considered as end-stage cells involved in host protection against parasitic infection and in the mechanisms of hypersensitivity. However, later studies have shown that this multifunctional cell is also capable of producing immunoregulatory cytokines and soluble mediators and is involved in tissue homeostasis and modulation of innate and adaptive immune responses. In this review, we summarize the biology of eosinophils, including the function and molecular mechanisms of their granule proteins, cell surface markers, mediators, and pathways, and present comprehensive reviews of research updates on the genetics and epigenetics of eosinophils. We describe recent advances in the development of epigenetics of eosinophil-related diseases, especially in asthma. Likewise, recent studies have provided us with a more complete appreciation of how eosinophils contribute to the pathogenesis of various diseases, including hypereosinophilic syndrome (HES). Over the past decades, the definition and criteria of HES have been evolving with the progress of our understanding of the disease and some aspects of this disease still remain controversial. We also review recent updates on the genetic and molecular mechanisms of HES, which have spurred dramatic developments in the clinical strategies of diagnosis and treatment for this heterogeneous group of diseases. The conclusion from this review is that the biology of eosinophils provides significant insights as to their roles in health and disease and, furthermore, demonstrates that a better understanding of eosinophil will accelerate the development of new therapeutic strategies for patients.

Expression of Mas-related gene X2 on mast cells is upregulated in the skin of patients with severe chronic urticaria.

BACKGROUND: Wheal reactions to intradermally injected neuropeptides, such as substance P (SP) and vasoactive intestinal peptide, are significantly larger and longer lasting in patients with chronic urticaria (CU) than in nonatopic control (NC) subjects. Mas-related gene X2 (MrgX2) has been identified as a receptor for basic neuropeptides, such as SP and vasoactive intestinal peptide. Mast cell (MC) responsiveness to eosinophil mediators contributes to the late-phase reaction of allergy. OBJECTIVE: We sought to compare the frequency of MrgX2 expression in skin MCs from patients with CU and NC subjects and to identify the receptor for basic eosinophil granule proteins on human skin MCs. METHODS: MrgX2 expression was investigated by using immunofluorescence in skin tissues from NC subjects and patients with severe CU and on skin-derived cultured MCs. MrgX2 expression in human MCs was reduced by using a lentiviral small hairpin RNA silencing technique. Ca(2+) influx was measured in CHO cells transfected with MrgX2 in response to eosinophil granule proteins. Histamine and prostaglandin D2 levels were measured by using enzyme immunoassays. RESULTS: The number of MrgX2(+) skin MCs and the percentage of MrgX2(+) MCs in all MCs in patients with CU were significantly greater than those in NC subjects. Eosinophil infiltration in urticarial lesions was observed in 7 of 9 patients with CU. SP, major basic protein, and eosinophil peroxidase, but not eosinophil-derived neurotoxin, induced histamine release from human skin MCs through MrgX2. CONCLUSION: MrgX2 might be a new target molecule for the treatment of wheal reactions in patients with severe CU.

Effect of RNA interference therapy on the mice eosinophils CCR3 gene and granule protein in the murine model of allergic rhinitis.

OBJECTIVE: To observe the clinical manifestations of allergic rhinitis mice and the expression changes of the eosinophils CCR3 and the granule protein mRNA in the bone marrow, peripheral blood and nasal lavage fluid. METHODS: Twenty-four BALB/c mice were randomly divided into the control group, PBS therapy group, siRNA therapy group and the CCR3 siRNA therapy group (n=6). Allergic rhinitis model were sensitized and stimulated by ovalbunfin, and CCR3 siRNA therapy group were administered with CCR3 transnasally before stimulated. The levels of the eosinophils CCR3, MBP, ECP and EPO in bone marrow, peripheral blood and nasal lavage fluid were detected by RT-PCR. RESULTS: Compared to the control group and CCR3 siRNA therapy group, the nasal mucosa of the PBS therapy group and siRNA therapy group developed epithalaxy, goblet cells hyperplasia, squamous epithelium metaplasia, epithelium necrosis, lamina propria and submucosa gland hyperplasia, vasodilatation, tissue edema, and the characterized eosinophil infiltration. RT-PCR indicated that the CCR3 mRNA, MBP, ECP and EPO expression in bone marrow, peripheral blood and nasal lavage fluid of the CCR3 siRNA therapy group was lower than the PBS therapy group and siRNA therapy group (P<0.05). CONCLUSIONS: The RNA interference therapy to CCR3 by local administration pernasal can suppress the process of the development, migration and invasion of the allergic rhinitis eosinophil, thus can reduce the effect of eosinophils and then reduce the inflammation effect of the allergic rhinitis. It may be a new treatment for respiratory tract allergic inflammation.

The expression of protease-activated receptors in chronic rhinosinusitis.

BACKGROUND: A recent study suggested that protease-activated receptors (PARs) are involved in allergic respiratory diseases, such as asthma. Chronic rhinosinusitis (CRS) is one of the most common chronic airway diseases, but little is understood about its pathogenesis. The purpose of this study was to compare the expression and distribution of PARs in biopsy specimens obtained from CRS and control patients. METHODS: Biopsy specimens were obtained from 7 pituitary tumor patients as controls, 8 CRS patients with aspirin-tolerant asthma (ATA), 7 CRS patients with aspirin-induced asthma (AIA), and 7 CRS patients without asthma (CRS). Sections were stained for PAR-1, PAR-2, PAR-3 and PAR-4 using specific polyclonal antibodies. Staining was scored semiquantitatively for both intensity and distribution. To confirm the presence of PARs on inflammatory cells, double staining with eosinophil cationic protein (EG2) and elastase was also performed. RESULTS: Both the epithelium and the infiltrating inflammatory cells in the CRS with asthma groups showed significant upregulation of the expression of PAR-2 and PAR-3 compared with the CRS without asthma group and the control group. In the patients with CRS complicated by asthma, eosinophils were increased among PAR-2- and PAR-3-positive cells. In the patients with CRS not complicated by asthma, neutrophils were increased among PAR-2-positive cells. CONCLUSIONS: Differences in the expression of PAR-2 and PAR-3 on epithelial cells, eosinophils and neutrophils may be involved in the pathogenesis of CRS. CRS may be able to be treated by targeting PAR-2 and PAR-3.

The oesophageal string test: a novel, minimally invasive method measures mucosal inflammation in eosinophilic oesophagitis.

OBJECTIVE: Eosinophil predominant inflammation characterises histological features of eosinophilic oesophagitis (EoE). Endoscopy with biopsy is currently the only method to assess oesophageal mucosal inflammation in EoE. We hypothesised that measurements of luminal eosinophil-derived proteins would correlate with oesophageal mucosal inflammation in children with EoE. DESIGN: The Enterotest diagnostic device was used to develop an oesophageal string test (EST) as a minimally invasive clinical device. EST samples and oesophageal mucosal biopsies were obtained from children undergoing upper endoscopy for clinically defined indications. Eosinophil-derived proteins including eosinophil secondary granule proteins (major basic protein-1, eosinophil-derived neurotoxin, eosinophil cationic protein, eosinophil peroxidase) and Charcot-Leyden crystal protein/galectin-10 were measured by ELISA in luminal effluents eluted from ESTs and extracts of mucosal biopsies. RESULTS: ESTs were performed in 41 children with active EoE (n=14), EoE in remission (n=8), gastro-oesophageal reflux disease (n=4) and controls with normal oesophagus (n=15). EST measurement of eosinophil-derived protein biomarkers significantly distinguished between children with active EoE, treated EoE in remission, gastro-oesophageal reflux disease and normal oesophagus. Levels of luminal eosinophil-derived proteins in EST samples significantly correlated with peak and mean oesophageal eosinophils/high power field (HPF), eosinophil peroxidase indices and levels of the same eosinophil-derived proteins in extracts of oesophageal biopsies. CONCLUSIONS: The presence of eosinophil-derived proteins in luminal secretions is reflective of mucosal inflammation in children with EoE. The EST is a novel, minimally invasive device for measuring oesophageal eosinophilic inflammation in children with EoE.

Eosinophils increase neuron branching in human and murine skin and in vitro.

Cutaneous nerves are increased in atopic dermatitis, and itch is a prominent symptom. We studied the functional interactions between eosinophils and nerves in human and mouse skin and in culture. We demonstrated that human atopic dermatitis skin has eosinophil granule proteins present in the same region as increased nerves. Transgenic mice in which interleukin-5 (IL-5) expression is driven by a keratin-14 (K14) promoter had many eosinophils in the epidermis, and the number of nerves was also significantly increased in the epidermis. In co-cultures, eosinophils dramatically increased branching of sensory neurons isolated from the dorsal root ganglia (DRG) of mice. This effect did not occur in DRG neurons co-cultured with mast cells or with dead eosinophils. Physical contact of the eosinophils with the neurons was not required, and the effect was not blocked by an antibody to nerve growth factor. DRG neurons express eotaxin-1, ICAM-1 and VCAM-1, which may be important in the recruitment, binding, and activation of eosinophils in the region of cutaneous nerves. These data indicate a pathophysiological role for eosinophils in cutaneous nerve growth in atopic dermatitis, and suggest they may present a therapeutic target in atopic dermatitis and other eosinophilic skin conditions with neuronal symptoms such as itch.

Eosinophil extracellular DNA traps in skin diseases.

BACKGROUND: In the skin, eosinophils are found in a broad spectrum of diseases, including infectious diseases. OBJECTIVE: In this study, we investigated whether eosinophil extracellular traps, structures containing DNA in association with eosinophil granule proteins able to bind and kill bacteria, are present in the skin under various pathologic conditions. METHODS: Immunofluorescence staining was performed on sections of paraformaldehyde-fixed and paraffin-embedded skin biopsy tissues of 25 different eosinophilic skin diseases by using propidium iodide and an antibody to eosinophil cationic protein. Slides were evaluated by laser scanning microscopy. RESULTS: Eosinophils releasing DNA together with eosinophil cationic protein were detected in infectious skin diseases such as ectoparasitosis and larva migrans. Further, we observed the extracellular DNA structures in allergic/reactive diseases (Wells syndrome, hypereosinophilic syndrome, positive reaction of atopy patch test, allergic contact dermatitis, drug hypersensitivity) and in autoimmune diseases (bullous pemphigoid, pemphigus foliaceus, dermatitis herpetiformis). The average number of eosinophils releasing DNA in the skin was usually below 10%, although in Wells syndrome the proportion was up to 30%. In areas with clusters of eosinophils, up to 50% of the eosinophils were seen to generate eosinophil extracellular traps. CONCLUSION: Eosinophil extracellular traps are seen in both infectious and noninfectious inflammatory skin diseases and are particularly common in Wells syndrome.

Mechanisms of eosinophilia in the pathogenesis of hypereosinophilic disorders.

The increased numbers of activated eosinophils in the blood and tissues that typically accompany hypereosinophilic disorders result from a variety of mechanisms. Exciting advances in translating discoveries achieved from mouse models and molecular strategies to the clinic have led to a flurry of new therapeutics specifically designed to target eosinophil-associated diseases. So far, this form of hypothesis testing in humans in vivo through pharmacology generally has supported the paradigms generated in vitro and in animal models, raising hopes that a spectrum of novel therapies soon may become available to help those who have eosinophil-associated diseases.

Eosinophil-mediated cholinergic nerve remodeling.

Eosinophils are observed to localize to cholinergic nerves in a variety of inflammatory conditions such as asthma, rhinitis, eosinophilic gastroenteritis, and inflammatory bowel disease, where they are also responsible for the induction of cell signaling. We hypothesized that a consequence of eosinophil localization to cholinergic nerves would involve a neural remodeling process. Eosinophil co-culture with cholinergic IMR32 cells led to increased expression of the M2 muscarinic receptor, with this induction being mediated via an adhesion-dependent release of eosinophil proteins, including major basic protein and nerve growth factor. Studies on the promoter sequence of the M2 receptor indicated that this induction was initiated at a transcription start site 145 kb upstream of the gene-coding region. This promoter site contains binding sites for a variety of transcription factors including SP1, AP1, and AP2. Eosinophils also induced the expression of several cholinergic genes involved in the synthesis, storage, and metabolism of acetylcholine, including the enzymes choline acetyltransferase, vesicular acetylcholine transferase, and acetylcholinesterase. The observed eosinophil-induced changes in enzyme content were associated with a reduction in intracellular neural acetylcholine but an increase in choline content, suggesting increased acetylcholine turnover and a reduction in acetylcholinesterase activity, in turn suggesting reduced catabolism of acetylcholine. Together these data suggest that eosinophil localization to cholinergic nerves induces neural remodeling, promoting a cholinergic phenotype.

Intragranular vesiculotubular compartments are involved in piecemeal degranulation by activated human eosinophils.

Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30-60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.

Do serum levels of eosinophil granule-derived protein change in patients undergoing pelvic radiotherapy?

AIMS: Eosinophils have an important role in the pathogenesis of inflammatory bowel disease, with faecal levels of the eosinophil granule proteins, eosinophil cationic protein (ECP) and eosinophil protein X (EPX) reflecting disease activity. Eosinophil crypt abscesses are a characteristic histological finding in acute gastrointestinal radiation-induced mucosal damage. This pilot study aimed to investigate changes in serum levels of ECP/EPX during pelvic radiotherapy. MATERIALS AND METHODS: Patients with no history of inflammatory bowel disease, starting a 5-week course of pelvic radiotherapy, had serum ECP/EPX levels measured before radiotherapy and during the fourth week of treatment. Bowel toxicity was graded at week 4 using the Common Toxicity Criteria Scale. RESULTS: Fifteen patients who were to undergo adjuvant radiotherapy for gynaecological cancer were recruited. The mean serum levels of ECP and EPX before treatment were 17.3 microg/l (range 2.0-49.3 microg/l) and 37.3 microg/l (range 12.0-94.0 microg/l), respectively. The mean serum levels during week 4 of radiotherapy for ECP and EPX were 43.0 microg/l (range 2.4-164.0 microg/l) and 38.7 microg/l (range 9.0-79.0 microg/l), respectively. Serum ECP levels increased at week 4 compared with levels before radiotherapy (P = 0.02). Acute bowel toxicity was seen in 12 patients (80%) at week 4: Grade 1 in 25% patients and Grade 2 in 75%. In this small study, no correlation was seen between acute bowel toxicity at week 4 and serum ECP or EPX levels. CONCLUSIONS: Serum ECP levels increase in response to pelvic irradiation. This may reflect the known involvement of eosinophils in the acute response to radiotherapy. Further study is required to determine when levels start to rise and their relationship to the degree of acute bowel toxicity.