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Weihrauch T, Melo RCN, Gray N, Voehringer D, Weller PF, Raap U. Eosinophil extracellular vesicles and DNA traps in allergic inflammation. FRONTIERS IN ALLERGY 2024; 5:1448007. [PMID: 39148911 PMCID: PMC11324581 DOI: 10.3389/falgy.2024.1448007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2024] [Accepted: 07/23/2024] [Indexed: 08/17/2024] Open
Abstract
Eosinophil granulocytes, a specialized subset of white blood cells, have traditionally been associated with allergic responses and parasitic infections. However, recent research has unveiled their versatile roles in immune regulation beyond these classical functions. This review highlights the emerging field of eosinophil biology, with a particular focus on their release of extracellular vesicles (EVs) and extracellular DNA traps (EETs). It further explores potential implications of eosinophil-derived EVs and EETs for immune responses during inflammatory diseases. The release of EVs/EETs from eosinophils, which also affects the eosinophils themselves, may influence both local and systemic immune reactions, affecting the pathophysiology of conditions such as airway inflammation, chronic rhinosinusitis and atopic dermatitis.
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Affiliation(s)
- Tobias Weihrauch
- Division of Experimental Allergy and Immunodermatology, Faculty of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
| | - Rossana C N Melo
- Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences (ICB), Federal University of Juiz de Fora, UFJF, Juiz de Fora, Brazil
| | - Natalie Gray
- Division of Experimental Allergy and Immunodermatology, Faculty of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
- Division of Anatomy, Faculty of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
| | - David Voehringer
- Department of Infection Biology, University Hospital Erlangen, Erlangen, Germany
- FAU Profile Center Immunomedicine (FAU I-MED), Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Peter F Weller
- Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, United States
| | - Ulrike Raap
- Division of Experimental Allergy and Immunodermatology, Faculty of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
- Research Center for Neurosensory Science, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
- University Clinic of Dermatology and Allergy, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
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Melo RCN, Silva TP. Eosinophil activation during immune responses: an ultrastructural view with an emphasis on viral diseases. J Leukoc Biol 2024; 116:321-334. [PMID: 38466831 DOI: 10.1093/jleuko/qiae058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2023] [Revised: 02/17/2024] [Accepted: 02/21/2024] [Indexed: 03/13/2024] Open
Abstract
Eosinophils are cells of the innate immune system that orchestrate complex inflammatory responses. The study of the cell biology of eosinophils, particularly associated with cell activation, is of great interest to understand their immune responses. From a morphological perspective, activated eosinophils show ultrastructural signatures that have provided critical insights into the comprehension of their functional capabilities. Application of conventional transmission electron microscopy in combination with quantitative assessments (quantitative transmission electron microscopy), molecular imaging (immunoEM), and 3-dimensional electron tomography have generated important insights into mechanisms of eosinophil activation. This review explores a multitude of ultrastructural events taking place in eosinophils activated in vitro and in vivo as key players in allergic and inflammatory diseases, with an emphasis on viral infections. Recent progress in our understanding of biological processes underlying eosinophil activation, including in vivo mitochondrial remodeling, is discussed, and it can bring new thinking to the field.
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Affiliation(s)
- Rossana C N Melo
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Rua José Lourenço Kelmer, campus, Juiz de Fora, MG, 36036-900, Brazil
| | - Thiago P Silva
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Rua José Lourenço Kelmer, campus, Juiz de Fora, MG, 36036-900, Brazil
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Rodrigo-Muñoz JM, Gil-Martínez M, Naharro-González S, Del Pozo V. Eosinophil-derived extracellular vesicles: isolation and classification techniques and implications for disease pathophysiology. J Leukoc Biol 2024; 116:260-270. [PMID: 38836652 DOI: 10.1093/jleuko/qiae133] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2023] [Revised: 05/17/2024] [Accepted: 06/04/2024] [Indexed: 06/06/2024] Open
Abstract
Eosinophils are leukocytes characterized by their ability to release granule content that is highly rich in enzymes and proteins. Besides the antihelminthic, bactericidal, and antiviral properties of eosinophils and their secretory granules, these also play a prominent role in the pathophysiology of diseases such as asthma, eosinophilic esophagitis, and other hypereosinophilic conditions by causing tissue damage and airway hyperresponsiveness. Although this cell was first recognized mainly for its capacity to release granule content, nowadays other capabilities such as cytokine secretion have been linked to its physiology, and research has found that eosinophils are not only involved in innate immunity, but also as orchestrators of immune responses. Nearly 10 yr ago, eosinophil-derived extracellular vesicles (EVs) were first described; since then, the EV field has grown exponentially, revealing their vital roles in intracellular communication. In this review, we synthesize current knowledge on eosinophil-derived EVs, beginning with a description of what they are and what makes them important regulators of disease, followed by an account of the methodologies used to isolate and characterize EVs. We also summarize current understanding of eosinophil-derived vesicles functionality, especially in asthma, the disease in which eosinophil-derived EVs have been most widely studied, describing how they modulate the role of eosinophils themselves (through autocrine signaling) and the way they affect airway structural cells and airway remodeling. Deeper understanding of this cell type could lead to novel research in eosinophil biology, its role in other diseases, and possible use of eosinophil-derived EVs as therapeutic targets.
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Affiliation(s)
- José Manuel Rodrigo-Muñoz
- Immunoallergy Laboratory, Immunology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Avda. Reyes Católicos, 228040 Madrid, Spain
- CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), Av. Monforte de Lemos, 3-5. Pabellón 11. Planta 0, 28029 Madrid, Spain
| | - Marta Gil-Martínez
- Immunoallergy Laboratory, Immunology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Avda. Reyes Católicos, 228040 Madrid, Spain
- CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), Av. Monforte de Lemos, 3-5. Pabellón 11. Planta 0, 28029 Madrid, Spain
| | - Sara Naharro-González
- Immunoallergy Laboratory, Immunology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Avda. Reyes Católicos, 228040 Madrid, Spain
| | - Victoria Del Pozo
- Immunoallergy Laboratory, Immunology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Avda. Reyes Católicos, 228040 Madrid, Spain
- CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), Av. Monforte de Lemos, 3-5. Pabellón 11. Planta 0, 28029 Madrid, Spain
- Universidad Autónoma de Madrid, Ciudad Universitaria de Cantoblanco, 28049 Madrid, Spain
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Neves VH, Palazzi C, Bonjour K, Ueki S, Weller PF, Melo RCN. In Vivo ETosis of Human Eosinophils: The Ultrastructural Signature Captured by TEM in Eosinophilic Diseases. Front Immunol 2022; 13:938691. [PMID: 35874692 PMCID: PMC9301467 DOI: 10.3389/fimmu.2022.938691] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2022] [Accepted: 06/15/2022] [Indexed: 12/23/2022] Open
Abstract
Eosinophilic diseases, also termed eosinophil-associated diseases (EADs), are characterized by eosinophil-rich inflammatory infiltrates and extensive eosinophil degranulation with clinically relevant organ pathology. Recent evidence shows that eosinophil cytolytic degranulation, that is, the release of intact, membrane-delimited granules that arises from the eosinophil cytolysis, occurs mainly through ETosis, meaning death with a cytolytic profile and extrusion of nucleus-originated DNA extracellular traps (ETs). The ultrastructural features of eosinophil ETosis (EETosis) have been studied mostly in vitro after stimulation, but are still poorly understood in vivo. Here, we investigated in detail, by transmission electron microscopy (TEM), the ultrastructure of EETosis in selected human EADs affecting several tissues and organ systems. Biopsies of patients diagnosed with eosinophilic chronic rhinosinusitis/ECRS (frontal sinus), ulcerative colitis/UC (intestine), and hypereosinophilic syndrome/HES (skin) were processed for conventional TEM. First, we found that a large proportion of tissue-infiltrated eosinophils in all diseases (~45-65% of all eosinophils) were undergoing cytolysis with release of free extracellular granules (FEGs). Second, we compared the morphology of tissue inflammatory eosinophils with that shown by in vitro ETosis-stimulated eosinophils. By applying single-cell imaging analysis, we sought typical early and late EETosis events: chromatin decondensation; nuclear delobulation and rounding; expanded nuclear area; nuclear envelope alterations and disruption; and extracellular decondensed chromatin spread as ETs. We detected that 53% (ECRS), 37% (UC), and 82% (HES) of all tissue cytolytic eosinophils had ultrastructural features of ETosis in different degrees. Eosinophils in early ETosis significantly increased their nuclear area compared to non-cytolytic eosinophils due to excessive chromatin decondensation and expansion observed before nuclear envelope disruption. ETosis led not only to the deposition of intact granules, but also to the release of eosinophil sombrero vesicles (EoSVs) and Charcot-Leyden crystals (CLCs). Free intact EoSVs and CLCs were associated with FEGs and extracellular DNA nets. Interestingly, not all cytolytic eosinophils in the same microenvironment exhibited ultrastructure of ETosis, thus indicating that different populations of eosinophils might be selectively activated into this pathway. Altogether, our findings captured an ultrastructural signature of EETosis in vivo in prototypic EADs highlighting the importance of this event as a form of eosinophil degranulation and release of inflammatory markers (EoSVs and CLCs).
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Affiliation(s)
- Vitor H. Neves
- Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil
| | - Cinthia Palazzi
- Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil
| | - Kennedy Bonjour
- Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil
- Laboratory of Molecular and Morphological Pathology, Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
| | - Shigeharu Ueki
- Department of General Internal Medicine and Clinical Laboratory Medicine, Graduate School of Medicine, Akita University, Akita, Japan
| | - Peter F. Weller
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States
| | - Rossana C. N. Melo
- Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States
- *Correspondence: Rossana C. N. Melo,
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Bonjour K, Palazzi C, Silva TP, Malta KK, Neves VH, Oliveira-Barros EG, Neves I, Kersten VA, Fortuna BT, Samarasinghe AE, Weller PF, Bandeira-Melo C, Melo RCN. Mitochondrial Population in Mouse Eosinophils: Ultrastructural Dynamics in Cell Differentiation and Inflammatory Diseases. Front Cell Dev Biol 2022; 10:836755. [PMID: 35386204 PMCID: PMC8979069 DOI: 10.3389/fcell.2022.836755] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Accepted: 02/25/2022] [Indexed: 12/20/2022] Open
Abstract
Mitochondria are multifunctional organelles of which ultrastructure is tightly linked to cell physiology. Accumulating evidence shows that mitochondrial remodeling has an impact on immune responses, but our current understanding of the mitochondrial architecture, interactions, and morphological changes in immune cells, mainly in eosinophils, is still poorly known. Here, we applied transmission electron microscopy (TEM), single-cell imaging analysis, and electron tomography, a technique that provides three-dimensional (3D) views at high resolution, to investigate mitochondrial dynamics in mouse eosinophils developing in cultures as well as in the context of inflammatory diseases characterized by recruitment and activation of these cells (mouse models of asthma, H1N1 influenza A virus (IAV) infection, and schistosomiasis mansoni). First, quantitative analyses showed that the mitochondrial area decrease 70% during eosinophil development (from undifferentiated precursor cells to mature eosinophils). Mitophagy, a consistent process revealed by TEM in immature but not in mature eosinophils, is likely operating in mitochondrial clearance during eosinophilopoiesis. Events of mitochondria interaction (inter-organelle membrane contacts) were also detected and quantitated within developing eosinophils and included mitochondria-endoplasmic reticulum, mitochondria-mitochondria, and mitochondria-secretory granules, all of them significantly higher in numbers in immature compared to mature cells. Moreover, single-mitochondrion analyses revealed that as the eosinophil matures, mitochondria cristae significantly increase in number and reshape to lamellar morphology. Eosinophils did not change (asthma) or reduced (IAV and Schistosoma infections) their mitochondrial mass in response to inflammatory diseases. However, asthma and schistosomiasis, but not IAV infection, induced amplification of both cristae numbers and volume in individual mitochondria. Mitochondrial cristae remodeling occurred in all inflammatory conditions with the proportions of mitochondria containing only lamellar or tubular, or mixed cristae (an ultrastructural aspect seen just in tissue eosinophils) depending on the tissue/disease microenvironment. The ability of mitochondria to interact with granules, mainly mobilized ones, was remarkably captured by TEM in eosinophils participating in all inflammatory diseases. Altogether, we demonstrate that the processes of eosinophilopoiesis and inflammation-induced activation interfere with the mitochondrial dynamics within mouse eosinophils leading to cristae remodeling and inter-organelle contacts. The understanding of how mitochondrial dynamics contribute to eosinophil immune functions is an open interesting field to be explored.
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Affiliation(s)
- Kennedy Bonjour
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, Juiz de Fora, Brazil
| | - Cinthia Palazzi
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, Juiz de Fora, Brazil
| | - Thiago P Silva
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, Juiz de Fora, Brazil
| | - Kássia K Malta
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, Juiz de Fora, Brazil
| | - Vitor H Neves
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, Juiz de Fora, Brazil
| | - Eliane G Oliveira-Barros
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, Juiz de Fora, Brazil
| | - Igor Neves
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, Juiz de Fora, Brazil
| | - Victor A Kersten
- Laboratory of Inflammation, Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Bruno T Fortuna
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, Juiz de Fora, Brazil
| | - Amali E Samarasinghe
- Division of Pulmonology, Allergy-Immunology and Sleep, Department of Pediatrics, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, United States
| | - Peter F Weller
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States
| | - Christianne Bandeira-Melo
- Laboratory of Inflammation, Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
| | - Rossana C N Melo
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, Juiz de Fora, Brazil.,Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States
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6
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Label-free imaging and evaluation of characteristic properties of asthma-derived eosinophils using optical diffraction tomography. Biochem Biophys Res Commun 2022; 587:42-48. [PMID: 34864394 DOI: 10.1016/j.bbrc.2021.11.084] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2021] [Revised: 11/14/2021] [Accepted: 11/23/2021] [Indexed: 12/15/2022]
Abstract
Optical diffraction tomography (ODT), an emerging imaging technique that does not require fluorescent staining, can measure the three-dimensional distribution of the refractive index (RI) of organelles. In this study, we used ODT to characterize the pathological characteristics of human eosinophils derived from asthma patients presenting with eosinophilia. In addition to morphological information about organelles appearing in eosinophils, including the cytoplasm, nucleus, and vacuole, we succeeded in imaging specific granules and quantifying the RI values of the granules. Interestingly, ODT analysis showed that the RI (i.e., molecular density) of granules was significantly different between eosinophils from asthma patients and healthy individuals without eosinophilia, and that vacuoles were frequently found in the cells of asthma patients. Our results suggest that the physicochemical properties of eosinophils derived from patients with asthma can be quantitatively distinguished from those of healthy individuals. The method will provide insight into efficient evaluation of the characteristics of eosinophils at the organelle level for various diseases with eosinophilia.
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Cañas JA, Rodrigo-Muñoz JM, Gil-Martínez M, Sastre B, del Pozo V. Exosomes: A Key Piece in Asthmatic Inflammation. Int J Mol Sci 2021; 22:963. [PMID: 33478047 PMCID: PMC7835850 DOI: 10.3390/ijms22020963] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2020] [Revised: 01/13/2021] [Accepted: 01/18/2021] [Indexed: 02/07/2023] Open
Abstract
Asthma is a chronic disease of the airways that has an important inflammatory component. Multiple cells are implicated in asthma pathogenesis (lymphocytes, eosinophils, mast cells, basophils, neutrophils), releasing a wide variety of cytokines. These cells can exert their inflammatory functions throughout extracellular vesicles (EVs), which are small vesicles released by donor cells into the extracellular microenvironment that can be taken up by recipient cells. Depending on their size, EVs can be classified as microvesicles, exosomes, or apoptotic bodies. EVs are heterogeneous spherical structures secreted by almost all cell types. One of their main functions is to act as transporters of a wide range of molecules, such as proteins, lipids, and microRNAs (miRNAs), which are single-stranded RNAs of approximately 22 nucleotides in length. Therefore, exosomes could influence several physiological and pathological processes, including those involved in asthma. They can be detected in multiple cell types and biofluids, providing a wealth of information about the processes that take account in a pathological scenario. This review thus summarizes the most recent insights concerning the role of exosomes from different sources (several cell populations and biofluids) in one of the most prevalent respiratory diseases, asthma.
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Affiliation(s)
- José A. Cañas
- Immunoallergy Laboratory, Immunology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD), Avenida Reyes Católicos, 2, 28040 Madrid, Spain; (J.A.C.); (J.M.R.-M.); (M.G.-M.)
- CIBER de Enfermedades Respiratorias (CIBERES), Av. de Monforte de Lemos, 3-5, 28029 Madrid, Spain
| | - José M. Rodrigo-Muñoz
- Immunoallergy Laboratory, Immunology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD), Avenida Reyes Católicos, 2, 28040 Madrid, Spain; (J.A.C.); (J.M.R.-M.); (M.G.-M.)
- CIBER de Enfermedades Respiratorias (CIBERES), Av. de Monforte de Lemos, 3-5, 28029 Madrid, Spain
| | - Marta Gil-Martínez
- Immunoallergy Laboratory, Immunology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD), Avenida Reyes Católicos, 2, 28040 Madrid, Spain; (J.A.C.); (J.M.R.-M.); (M.G.-M.)
| | - Beatriz Sastre
- Immunoallergy Laboratory, Immunology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD), Avenida Reyes Católicos, 2, 28040 Madrid, Spain; (J.A.C.); (J.M.R.-M.); (M.G.-M.)
- CIBER de Enfermedades Respiratorias (CIBERES), Av. de Monforte de Lemos, 3-5, 28029 Madrid, Spain
| | - Victoria del Pozo
- Immunoallergy Laboratory, Immunology Department, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD), Avenida Reyes Católicos, 2, 28040 Madrid, Spain; (J.A.C.); (J.M.R.-M.); (M.G.-M.)
- CIBER de Enfermedades Respiratorias (CIBERES), Av. de Monforte de Lemos, 3-5, 28029 Madrid, Spain
- Faculty of Medicine, Universidad Autónoma de Madrid, 28029 Madrid, Spain
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Loktionov A. Eosinophils in the gastrointestinal tract and their role in the pathogenesis of major colorectal disorders. World J Gastroenterol 2019; 25:3503-3526. [PMID: 31367153 PMCID: PMC6658389 DOI: 10.3748/wjg.v25.i27.3503] [Citation(s) in RCA: 62] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/06/2019] [Revised: 05/22/2019] [Accepted: 05/31/2019] [Indexed: 02/06/2023] Open
Abstract
Eosinophils are currently regarded as versatile mobile cells controlling and regulating multiple biological pathways and responses in health and disease. These cells store in their specific granules numerous biologically active substances (cytotoxic cationic proteins, cytokines, growth factors, chemokines, enzymes) ready for rapid release. The human gut is the main destination of eosinophils that are produced and matured in the bone marrow and then transferred to target tissues through the circulation. In health the most important functions of gut-residing eosinophils comprise their participation in the maintenance of the protective mucosal barrier and interactions with other immune cells in providing immunity to microbiota of the gut lumen. Eosinophils are closely involved in the development of inflammatory bowel disease (IBD), when their cytotoxic granule proteins cause damage to host tissues. However, their roles in Crohn's disease and ulcerative colitis appear to follow different immune response patterns. Eosinophils in IBD are especially important in altering the structure and protective functions of the mucosal barrier and modulating massive neutrophil influx to the lamina propria followed by transepithelial migration to colorectal mucus. IBD-associated inflammatory process involving eosinophils then appears to expand to the mucus overlaying the internal gut surface. The author hypothesises that immune responses within colorectal mucus as well as ETosis exerted by both neutrophils and eosinophils on the both sides of the colonic epithelial barrier act as additional pathogenetic factors in IBD. Literature analysis also shows an association between elevated eosinophil levels and better colorectal cancer (CRC) prognosis, but mechanisms behind this effect remain to be elucidated. In conclusion, the author emphasises the importance of investigating colorectal mucus in IBD and CRC patients as a previously unexplored milieu of disease-related inflammatory responses.
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9
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Dias FF, Amaral KB, Malta KK, Silva TP, Rodrigues GSC, Rosa FM, Rodrigues GOL, Costa VV, Chiarini-Garcia H, Weller PF, Melo RCN. Identification of Piecemeal Degranulation and Vesicular Transport of MBP-1 in Liver-Infiltrating Mouse Eosinophils During Acute Experimental Schistosoma mansoni Infection. Front Immunol 2018; 9:3019. [PMID: 30619361 PMCID: PMC6306457 DOI: 10.3389/fimmu.2018.03019] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2018] [Accepted: 12/06/2018] [Indexed: 12/11/2022] Open
Abstract
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.
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Affiliation(s)
- Felipe F Dias
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil
| | - Kátia B Amaral
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil
| | - Kássia K Malta
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil
| | - Thiago P Silva
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil
| | - Gabriel S C Rodrigues
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil
| | - Florence M Rosa
- Laboratory of Parasitology, Department of Parasitology, Microbiology and Immunology, Federal University of Juiz de Fora, Juiz de Fora, Brazil
| | - Gisele O L Rodrigues
- Laboratory of Immunopharmacology, Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil
| | - Vivian V Costa
- Center for Drug Research and Development of Pharmaceuticals, Federal University of Minas Gerais, Belo Horizonte, Brazil.,Research Group in Arboviral Diseases, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil
| | - Hélio Chiarini-Garcia
- Laboratory of Reproduction and Structural Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil
| | - Peter F Weller
- Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School Boston, Boston, MA, United States
| | - Rossana C N Melo
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil.,Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School Boston, Boston, MA, United States
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10
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Carmo LAS, Bonjour K, Spencer LA, Weller PF, Melo RCN. Single-Cell Analyses of Human Eosinophils at High Resolution to Understand Compartmentalization and Vesicular Trafficking of Interferon-Gamma. Front Immunol 2018; 9:1542. [PMID: 30038615 PMCID: PMC6046373 DOI: 10.3389/fimmu.2018.01542] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2018] [Accepted: 06/21/2018] [Indexed: 12/16/2022] Open
Abstract
Human eosinophils release numerous cytokines that are pre-synthesized and stored within their cytoplasmic-specific (secretory) granules. For example, high levels of interferon-gamma (IFN-γ) are constitutively expressed in these cells, but the intracellular compartments involved in the transport and release of this cytokine remain to be established. In this work, we used a single-cell approach to investigate the subcellular localization of IFN-γ in human eosinophils stimulated or not with tumor necrosis factor alpha (TNF-α) or CC-chemokine ligand 11 CCL11 (eotaxin-1), inflammatory mediators that induce eosinophil activation and secretion. A pre-embedding immunonanogold transmission electron microscopy (TEM) technique that combines optimal epitope preservation and access to membrane microdomains was applied to detect precise localization of IFN-γ in combination with computational quantitative analyses. In parallel, degranulation processes and formation of eosinophil sombrero vesicles (EoSVs), large transport carriers involved in the transport of granule-derived cytokines, were investigated. Quantitative TEM revealed that both CCL11 and TNF-α-activated eosinophils significantly increased the total number of EoSVs compared to the unstimulated group, indicating that this vesicular system is actively formed in response to cell activation. Ultrastructural immunolabeling identified a robust pool of IFN-γ on secretory granules in both unstimulated and stimulated cells. Moreover, EoSVs carrying IFN-γ were seen around or/and in contact with secretory granules and also distributed in the cytoplasm. Labeling was clearly associated with EoSV membranes. The total number of IFN-γ-positive EoSVs was significantly higher in stimulated compared to unstimulated cells, and these labeled vesicles had a differential distribution in the cytoplasm of activated cells, being significantly higher in the cell periphery compared with the inner cell, thus revealing intracellular IFN-γ mobilization for release. IFN-γ extracellular labeling was found at the cell surface, including on extracellular vesicles. Our results provide direct evidence that human eosinophils compartmentalize IFN-γ within secretory granules and identify, for the first time, a vesicular trafficking of IFN-γ associated with large transport carriers. This is important to understand how IFN-γ is trafficked and secreted during inflammatory responses.
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Affiliation(s)
- Lívia A S Carmo
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil
| | - Kennedy Bonjour
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil
| | - Lisa A Spencer
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States
| | - Peter F Weller
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States
| | - Rossana C N Melo
- Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Brazil.,Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States
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11
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Melo RCN, Weller PF. Contemporary understanding of the secretory granules in human eosinophils. J Leukoc Biol 2018; 104:85-93. [PMID: 29749658 DOI: 10.1002/jlb.3mr1217-476r] [Citation(s) in RCA: 61] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2017] [Revised: 02/14/2018] [Accepted: 02/16/2018] [Indexed: 12/12/2022] Open
Abstract
Eosinophil secretory (specific) granules have a unique morphology and are both a morphologic hallmark of eosinophils and fundamental to eosinophil-mediated responses. Eosinophil mediators with multiple functional activities are presynthesized and stored within these granules, poised for very rapid, stimulus-induced secretion. The structural organization and changes of eosinophil specific granules are revealing in demonstrating the complex and diverse secretory activities of this cell. Here, we review our current knowledge on the architecture, composition, and function of eosinophil specific granules as highly elaborated organelles able to produce vesiculotubular carriers and to interplay with the intracellular vesicular trafficking. We reconsider prior identifications of eosinophil cytoplasmic granules, including "primary," "secondary," "microgranules," and "small granules"; and consonant with advances, we provide a contemporary recognition that human eosinophils contain a single population of specific granules and their developmental precursors and derived secretory vesicles.
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Affiliation(s)
- Rossana C N Melo
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Rua José Lourenço Kelmer, Juiz de Fora, Brazil.,Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
| | - Peter F Weller
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Rua José Lourenço Kelmer, Juiz de Fora, Brazil
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12
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Abstract
Eosinophils are a prominent cell type in particular host responses such as the response to helminth infection and allergic disease. Their effector functions have been attributed to their capacity to release cationic proteins stored in cytoplasmic granules by degranulation. However, eosinophils are now being recognized for more varied functions in previously underappreciated diverse tissue sites, based on the ability of eosinophils to release cytokines (often preformed) that mediate a broad range of activities into the local environment. In this Review, we consider evolving insights into the tissue distribution of eosinophils and their functional immunobiology, which enable eosinophils to secrete in a selective manner cytokines and other mediators that have diverse, 'non-effector' functions in health and disease.
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Affiliation(s)
- Peter F Weller
- Division of Allergy and Inflammation, Harvard Medical School, Beth Israel Deaconess Medical Center, CLS 943, 330 Brookline Avenue, Boston, Massachusetts 02215, USA
| | - Lisa A Spencer
- Division of Allergy and Inflammation, Harvard Medical School, Beth Israel Deaconess Medical Center, CLS 943, 330 Brookline Avenue, Boston, Massachusetts 02215, USA
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13
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Ethier C, Yu Y, Cameron L, Lacy P, Davoine F. Calcitriol Reduces Eosinophil Necrosis Which Leads to the Diminished Release of Cytotoxic Granules. Int Arch Allergy Immunol 2016; 171:119-129. [PMID: 27902981 DOI: 10.1159/000450951] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Accepted: 09/20/2016] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND Asthma severity and eosinophilia correlate with a deficiency in vitamin D and its active metabolite calcitriol. Calcitriol modulates numerous leukocyte functions, but its effect on eosinophils is not fully understood. We postulated that calcitriol exerts a direct effect on eosinophil biology by modulating cell survival. METHODS Purified peripheral blood eosinophils from atopic donors were incubated in the presence of calcitriol for up to 14 days with or without IL-5. The effect of calcitriol on eosinophil viability was measured using the annexin-V/propidium iodide flow cytometry assay. We also examined the release of eosinophil peroxidase (EPX) in media using a flow cytometry assay with anti-EPX antibodies, and the enzymatic activity of EPX was measured by an OPD-based colorimetric assay. RESULTS We observed that calcitriol sustained cell viability in eosinophils with a concurrent reduction of necrotic cells. This effect was amplified by the addition of IL-5. In parallel, we observed that a physiological dose of calcitriol (10 nM) significantly reduced eosinophil necrosis and cytolytic release of EPX in media when coincubated with IL-5. CONCLUSION These results suggest that calcitriol may exert a direct effect on eosinophils by reducing necrosis and the cytolytic release of inflammatory mediators like EPX.
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Affiliation(s)
- Caroline Ethier
- Pulmonary Research Group, University of Alberta, Edmonton, AB, Canada
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14
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Akuthota P, Carmo LAS, Bonjour K, Murphy RO, Silva TP, Gamalier JP, Capron KL, Tigges J, Toxavidis V, Camacho V, Ghiran I, Ueki S, Weller PF, Melo RCN. Extracellular Microvesicle Production by Human Eosinophils Activated by "Inflammatory" Stimuli. Front Cell Dev Biol 2016; 4:117. [PMID: 27833910 PMCID: PMC5081571 DOI: 10.3389/fcell.2016.00117] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2016] [Accepted: 10/07/2016] [Indexed: 01/08/2023] Open
Abstract
A key function of human eosinophils is to secrete cytokines, chemokines and cationic proteins, trafficking, and releasing these mediators for roles in inflammation and other immune responses. Eosinophil activation leads to secretion of pre-synthesized granule-stored mediators through different mechanisms, but the ability of eosinophils to secrete extracellular vesicles (EVs), very small vesicles with preserved membrane topology, is still poorly understood. In the present work, we sought to identify and characterize EVs released from human eosinophils during different conditions: after a culturing period or after isolation and stimulation with inflammatory stimuli, which are known to induce eosinophil activation and secretion: CCL11 (eotaxin-1) and tumor necrosis factor alpha (TNF-α). EV production was investigated by nanoscale flow cytometry, conventional transmission electron microscopy (TEM) and pre-embedding immunonanogold EM. The tetraspanins CD63 and CD9 were used as EV biomarkers for both flow cytometry and ultrastructural immunolabeling. Nanoscale flow cytometry showed that human eosinophils produce EVs in culture and that a population of EVs expressed detectable CD9, while CD63 was not consistently detected. When eosinophils were stimulated immediately after isolation and analyzed by TEM, EVs were clearly identified as microvesicles (MVs) outwardly budding off the plasma membrane. Both CCL11 and TNF-α induced significant increases of MVs compared to unstimulated cells. TNF-α induced amplified release of MVs more than CCL11. Eosinophil MV diameters varied from 20 to 1000 nm. Immunonanogold EM revealed clear immunolabeling for CD63 and CD9 on eosinophil MVs, although not all MVs were labeled. Altogether, we identified, for the first time, that human eosinophils secrete MVs and that this production increases in response to inflammatory stimuli. This is important to understand the complex secretory activities of eosinophils underlying immune responses. The contribution of the eosinophil-derived MVs to the regulation of immune responses awaits further investigation.
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Affiliation(s)
- Praveen Akuthota
- Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical SchoolBoston, MA, USA; Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California San DiegoLa Jolla, CA, USA
| | - Lívia A S Carmo
- Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences (ICB), Federal University of Juiz de Fora Juiz de Fora, Brazil
| | - Kennedy Bonjour
- Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences (ICB), Federal University of Juiz de Fora Juiz de Fora, Brazil
| | - Ryann O Murphy
- Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School Boston, MA, USA
| | - Thiago P Silva
- Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences (ICB), Federal University of Juiz de Fora Juiz de Fora, Brazil
| | - Juliana P Gamalier
- Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences (ICB), Federal University of Juiz de Fora Juiz de Fora, Brazil
| | - Kelsey L Capron
- Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School Boston, MA, USA
| | - John Tigges
- Flow Cytometry Core, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School Boston, MA, USA
| | - Vasilis Toxavidis
- Flow Cytometry Core, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School Boston, MA, USA
| | - Virginia Camacho
- Flow Cytometry Core, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School Boston, MA, USA
| | - Ionita Ghiran
- Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School Boston, MA, USA
| | - Shigeharu Ueki
- Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine Akita, Japan
| | - Peter F Weller
- Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School Boston, MA, USA
| | - Rossana C N Melo
- Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical SchoolBoston, MA, USA; Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences (ICB), Federal University of Juiz de ForaJuiz de Fora, Brazil
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15
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Mesnil C, Raulier S, Paulissen G, Xiao X, Birrell MA, Pirottin D, Janss T, Starkl P, Ramery E, Henket M, Schleich FN, Radermecker M, Thielemans K, Gillet L, Thiry M, Belvisi MG, Louis R, Desmet C, Marichal T, Bureau F. Lung-resident eosinophils represent a distinct regulatory eosinophil subset. J Clin Invest 2016; 126:3279-95. [PMID: 27548519 DOI: 10.1172/jci85664] [Citation(s) in RCA: 402] [Impact Index Per Article: 44.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2015] [Accepted: 06/09/2016] [Indexed: 12/21/2022] Open
Abstract
Increases in eosinophil numbers are associated with infection and allergic diseases, including asthma, but there is also evidence that eosinophils contribute to homeostatic immune processes. In mice, the normal lung contains resident eosinophils (rEos), but their function has not been characterized. Here, we have reported that steady-state pulmonary rEos are IL-5-independent parenchymal Siglec-FintCD62L+CD101lo cells with a ring-shaped nucleus. During house dust mite-induced airway allergy, rEos features remained unchanged, and rEos were accompanied by recruited inflammatory eosinophils (iEos), which were defined as IL-5-dependent peribronchial Siglec-FhiCD62L-CD101hi cells with a segmented nucleus. Gene expression analyses revealed a more regulatory profile for rEos than for iEos, and correspondingly, mice lacking lung rEos showed an increase in Th2 cell responses to inhaled allergens. Such elevation of Th2 responses was linked to the ability of rEos, but not iEos, to inhibit the maturation, and therefore the pro-Th2 function, of allergen-loaded DCs. Finally, we determined that the parenchymal rEos found in nonasthmatic human lungs (Siglec-8+CD62L+IL-3Rlo cells) were phenotypically distinct from the iEos isolated from the sputa of eosinophilic asthmatic patients (Siglec-8+CD62LloIL-3Rhi cells), suggesting that our findings in mice are relevant to humans. In conclusion, our data define lung rEos as a distinct eosinophil subset with key homeostatic functions.
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16
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Melo RCN, Weller PF. Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy. Exp Cell Res 2016; 347:385-90. [PMID: 27562864 DOI: 10.1016/j.yexcr.2016.08.016] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2016] [Revised: 08/19/2016] [Accepted: 08/20/2016] [Indexed: 01/21/2023]
Abstract
Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles - EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses.
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Affiliation(s)
- Rossana C N Melo
- Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Rua José Lourenço Kelmer, Juiz de Fora, MG 36036-900, Brazil; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215, USA.
| | - Peter F Weller
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215, USA
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17
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Melo RCN, Morgan E, Monahan-Earley R, Dvorak AM, Weller PF. Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes. Nat Protoc 2014; 9:2382-94. [PMID: 25211515 DOI: 10.1038/nprot.2014.163] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Precise immunolocalization of proteins within a cell is central to understanding cell processes and functions such as intracellular trafficking and secretion of molecules during immune responses. Here we describe a protocol for ultrastructural detection of proteins in leukocytes. The method uses a pre-embedding approach (immunolabeling before standard processing for transmission electron microscopy (TEM)). This protocol combines several strategies for ultrastructure and antigen preservation, robust blocking of nonspecific binding sites, as well as superior antibody penetration for detecting molecules at subcellular compartments and membrane microdomains. A further advantage of this technique is that electron microscopy (EM) processing is quick. This method has been used to study leukocyte biology, and it has helped demonstrate how activated leukocytes deliver specific cargos. It may also potentially be applied to a variety of different cell types. Excluding the initial time required for sample preparation (15 h) and the final resin polymerization step (16 h), the protocol (immunolabeling and EM procedures) can be completed in 8 h.
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Affiliation(s)
- Rossana C N Melo
- 1] Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil. [2] Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
| | - Ellen Morgan
- Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
| | - Rita Monahan-Earley
- Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
| | - Ann M Dvorak
- Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
| | - Peter F Weller
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
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18
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Melo RCN, Weller PF. Unraveling the complexity of lipid body organelles in human eosinophils. J Leukoc Biol 2014; 96:703-12. [PMID: 25210147 DOI: 10.1189/jlb.3ru0214-110r] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Lipid-rich organelles are common in many cell types. In cells, such as adipocytes, these organelles are termed LDs, whereas in other cells, such as leukocytes, they are called LBs. The study of leukocyte LBs has attracted attention as a result of their association with human diseases. In leukocytes, such as eosinophils, LB accumulation has been documented extensively during inflammatory conditions. In these cells, LBs are linked to the regulation of immune responses by compartmentalization of several proteins and lipids involved in the control and biosynthesis of inflammatory mediators (eicosanoids). However, it has been unclear how diverse proteins, including membrane-associated enzymes involved in eicosanoid formation, incorporate into LBs, especially if the internal content of LBs is assumed to consist solely of stores of neutral lipids, as present within adipocyte LDs. Studies of the formation, function, and ultrastructure of LBs in eosinophils have been providing insights pertinent to LBs in other leukocytes. Here, we review current knowledge of the composition and function of leukocyte LBs as provided by studies of human eosinophil LBs, including recognitions of the internal architecture of eosinophil LBs based on 3D electron tomographic analyses.
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Affiliation(s)
- Rossana C N Melo
- Laboratory of Cellular Biology, Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Brazil; and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
| | - Peter F Weller
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
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19
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An extragranular compartment of blood eosinophils contains eosinophil protein X/eosinophil-derived neurotoxin (EPX/EDN). Inflammation 2013; 36:320-9. [PMID: 23053729 DOI: 10.1007/s10753-012-9549-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Serum and plasma profiles of eosinophil protein X (EPX/EDN) and those of other eosinophil proteins differ in various conditions, suggesting a different mobilisation from storage granules. This work studied the subcellular localisation of EPX/EDN in non-primed and in vivo primed blood eosinophils from healthy and allergic subjects, during and out of the pollen season. Primed eosinophils contain easily mobilisable secretory proteins. By fractionation on sucrose density gradients, EPX/EDN localised in the specific granules as well as in a cytoplasmic extra-granular compartment of low equilibrium density that partially overlapped with vesicular structures, cytosolic proteins and plasma membranes. This compartment was clearly separate from the low density peak of ECP that increases during the pollen season. There were no significant differences in the amounts of EPX/EDN present in the low density peak of healthy and allergic subjects. Immuno-gold labelling electron microscopy showed EPX/EDN in specific granules, cytoplasm and associated to plasma membranes. In conclusion, substantial amounts of EPX/EDN localise in an extra-granular, low equilibrium density compartment of human eosinophils.
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20
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Melo RCN, Liu L, Xenakis JJ, Spencer LA. Eosinophil-derived cytokines in health and disease: unraveling novel mechanisms of selective secretion. Allergy 2013; 68:274-84. [PMID: 23347072 DOI: 10.1111/all.12103] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/30/2012] [Indexed: 12/13/2022]
Abstract
Over the past two decades, our understanding of eosinophils has evolved from that of categorically destructive effector cells to include active participation in immune modulation, tissue repair processes, and normal organ development, in both health and disease. At the core of their newly appreciated functions is the capacity of eosinophils to synthesize, store within intracellular granules, and very rapidly secrete a highly diverse repertoire of cytokines. Mechanisms governing the selective secretion of preformed cytokines from eosinophils are attractive therapeutic targets and may well be more broadly applicable to other immune cells. Here, we discuss recent advances in deciphering pathways of cytokine secretion, both from intact eosinophils and from tissue-deposited cell-free eosinophil granules, extruded from eosinophils undergoing a lytic cell death.
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Affiliation(s)
| | - L. Liu
- Division of Allergy and Inflammation; Department of Medicine; Beth Israel Deaconess Medical Center; Harvard Medical School; Boston; MA; USA
| | - J. J. Xenakis
- Division of Allergy and Inflammation; Department of Medicine; Beth Israel Deaconess Medical Center; Harvard Medical School; Boston; MA; USA
| | - L. A. Spencer
- Division of Allergy and Inflammation; Department of Medicine; Beth Israel Deaconess Medical Center; Harvard Medical School; Boston; MA; USA
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21
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Muniz VS, Weller PF, Neves JS. Eosinophil crystalloid granules: structure, function, and beyond. J Leukoc Biol 2012; 92:281-8. [PMID: 22672875 DOI: 10.1189/jlb.0212067] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Eosinophils are granulocytes associated with host defense against parasitic helminths with allergic conditions and more recently, with immunoregulatory responses. Eosinophils are distinguished from leukocytes by their dominant population of cytoplasmic crystalloid (also termed secretory, specific, or secondary) granules that contain robust stores of diverse, preformed cationic proteins. Here, we provide an update on our knowledge about the unique and complex structure of human eosinophil crystalloid granules. We discuss their significance as rich sites of a variety of receptors and review our own recent research findings and those of others that highlight discoveries concerning the function of intracellular receptors and their potential implications in cell signaling. Special focus is provided on how eosinophils might use these intracellular receptors as mechanisms to secrete, selectively and rapidly, cytokines or chemokines and enable cell-free extracellular eosinophil granules to function as independent secretory structures. Potential roles of cell-free eosinophil granules as immune players in the absence of intact eosinophils will also be discussed.
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Affiliation(s)
- Valdirene S Muniz
- Institute of Biomedical Sciences, Federal University of Rio de Janeiro, UFRJ, Rio de Janeiro, Brazil
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22
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Caruso RA, Fedele F, Parisi A, Paparo D, Bonanno A, Finocchiaro G, Branca G, Scardigno M, Rigoli L. Chronic allergic-like inflammation in the tumor stroma of human gastric carcinomas: an ultrastructural study. Ultrastruct Pathol 2012; 36:139-144. [PMID: 22455876 DOI: 10.3109/01913123.2012.656883] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Inflammatory cell infiltration around the sites of carcinoma invasion is believed to play important roles in tumor biological behavior. The status of inflammatory cell infiltration at the sites of frank invasion in 92 cases of gastric carcinomas was examined, with special emphasis on tumor-associated tissue eosinophilia (TATE). TATE was found in 7 out of 92 (7.6%) gastric carcinomas (6 of intestinal-type and 1 of diffuse-type). Electron microscopy, selectively performed in the 7 cases of gastric carcinomas with TATE, showed that eosinophils participated in the stromal reaction by interacting with tumor cells, mast cells, and each other. Most of the tumor-infiltrating mast cells exhibited anaphylactic or piecemeal degranulation, indicating that the mast cells had been activated in situ. Some mast cells were noted in close contact to viable tumor cells, suggesting the existence of direct cell-to-cell interactions. There was also extracellular deposition of free eosinophil granules and Charcot-Leyden crystals. These morphologic findings are similar to that described in late/chronic-phase allergic reaction in both human and experimental animals, where angiogenesis and fibrosis/tissue repair are also present. In conclusion, TATE may indicate a chronic allergic-like Th2 host-tumor reaction, and understanding these pathways should create tools to enhance defence and contrast neoplastic disease.
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23
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Shamri R, Melo RCN, Young KM, Bivas-Benita M, Xenakis JJ, Spencer LA, Weller PF. CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules. FASEB J 2012; 26:2084-93. [PMID: 22294786 DOI: 10.1096/fj.11-200246] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC(50) 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.
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Affiliation(s)
- Revital Shamri
- Division of Allergy and Inflammation, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
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