2016 Group 7 Project
- 1 Introduction
- 2 History
- 3 Birth, Life and Death in the Body
- 4 Structure
- 5 Signalling
- 6 Function
- 7 Role in Allergy and Disease
- 8 Glossary
- 9 References
Eosinophils are a type of leukocyte or white blood cell which has a role in the immune system in regulating an inflammatory response during infections, injuries, allergies and in the presence of tumours. In a healthy body, eosinophils normally account for 1-3% of all leukocytes in peripheral circulation, with approximately 30-350 cells per cubic mm of blood. This means most eosinophils can be found in tissues with only around 1/100 existing in the circulatory system. Maintaining this ratio via activation and cell death is important for maintaining a healthy immune system. In the case that there infections, allergies, etc. the level of eosinophils present in the blood stream increases can increase to be more than 4.5x108/L (450μl).
Eosinophils mature in the bone marrow via hematopoietic progenitor cells. They grow to be 10-12 µm in diameter and develop a bi-lobed or ‘kidney shaped’ nucleus which makes it easily distinguishable. They are also characterised by the presence of granules which can be classified into specific granuels, which occupies most of the cytoplasm, and primary granules. The eosinophil’s cytoplasm also contains lipid bodies, which generates eicosanoids, and sombrero vesicles which provide alternative secretory pathways to granules and have the appearance of a flattened tubule.
|1590||Compound microscope invented by Hans and Zacharias Janssen, making it possible to observe the components of blood.|
|1658||Jan Swammerdam (1637 -1680) first observed and identified red blood cells.|
|1843||French professor, Gabriel Andrai (1797–1878) and English practitioner, William Addison (1802–1881) simultaneously described leukocytes. They recognised that both red and white blood cells were altered during disease. Addison also postulated that pus cells were circulating leukocytes that had crossed the walls of blood vessels.
Gottlieb Gluge (1812–1898), a Belgium clinician and scientist published his major work, ‘Atlas der Pathologischen Anatomie’ describing ‘granule cells’ which he referred to as ‘compound inflammatory globules’. These cells were not only observed in inflammatory exudates (pus and serum) but also in colostrum and the ovaries.
German Pathologist Julius Vogel (1814–1880) published his revolutionary book. ‘Pathological Anatomy of the Human Body which contained various macroscopic and microscopic illustrations, including granular cells in inflammatory exudates. These depicted cells with the morphological characteristics of eosinophils, both intact and at different stages of degranulation.
|1846||British physiologist and ophthalmologist, Thomas Wharton Jones (1808–1891) described ‘finely granular and coarse granular blood cells’ in numerous species including the human, lamprey, fowl, frog, horse and elephant. The coarsely granular white blood cells demonstrated the distinctive appearance of eosinophils and Jones estimated the diameter of their granules to be approximately 1 micrometre.|
|1865||Max Johann Sigismund Schultze (1825–1874) first described four different types of leukocytes (now identified as monocytes, lymphocytes, neutrophils and eosinophils). He used warm stage microscopy to closely observe fine and coarse granular white blood cells and determined that these cells moved in an amoeboid like fashion and phagocytosed small particles.|
|1874||Heinrich Caro (1834–1910) discovered eosin, a fluorescent red dye formed by the addition of bromine to fluorescein. It is acidic and therefore stains basic proteins.|
|1879||Paul Ehrlich referred to the eosinophil for the first time. He developed a technique for staining blood films, which involved a simple heating and air-drying process to fix the samples and the addition of acidic (eosin) or basic coal tar dyes. This not only lead to the discovery of eosinophils, but neutrophils, lymphocytes and basophils as well. Ehrlich examined the distribution of eosinophils in tissues and described numerous features of granules (alpha granules). He noted their round or rod shaped appearance and correctly deduced that the granules contained secretory components. He also noted the variation in the number of granules and nuclear lobes from cell to cell. Ehrlich observed idulin (black) staining beta-granules in (eosinophil) myelocytes, likely to be immature crystalloid granules. He identified asthma, skin diseases, helminths and medication reactions as causes of eosinophilia.|
Birth, Life and Death in the Body
Differentiation of hematopoietic stem cells occurs in the bone marrow in response to growth factors produced by resident stromal cells and locally elevated cytokines. Hematopoietic progenitors are undifferentiated multipotent stem cells, meaning they can develop into all blood cell types and possess self-renewal capacities (by division) . These are identified by the presence of CD34 on their surface, a marker highly expressed on progenitor cells. These multipotent progenitor cells further to differentiate into either a common lymphoid progenitor or a common myeloid progenitor which then differentiate into downstream progenitors before terminal differentiation into a specific blood cell .
The eosinophil lineage-committed progenitor (EoP) is derived from a common myeloid progenitor. These are distinguished by surface markers, CD34, IL-5 receptor alpha (IL-5Rα) and CD117 (lower levels) . EoPs mature into eosinophil precursors (preEos), like eosinophils, these contain granules however still possess the ability to proliferate. Upon stimulation with specific cytokines, a preEos will mature into an eosinophil (terminally differentiated and no proliferative capacity) . Cytokines IL-3, IL-5 and GM-CSF induce eosinophil maturation and formation from hematopoietic progenitors. IL-5 particularly directs differentiation encourages proliferation of EoPs and preEos cells 
Once eosinophils enter circulation, they have a half-life of approximately 8–18 hours. Under normal conditions, the vast majority of eosinophils are located in tissues (the tissue/blood eosinophil ratio is about 100:1) and once they enter tissue, most do not recirculate. They have a life span ranging from 2 to 5 days, however locally produced cytokines such as IL-5, IL-3, GM-CSF, IL-33, and interferon-γ may increase this survival time (up to 12 days). Eosinophils are predominantly trafficked to mucosal surfaces of the respiratory, lower genitourinary and gastrointestinal tracts where they reside within the lamina propria (excluding the oesophagus). They are also localised within the thymus (medulla and junction between the medulla and cortex), mammary glands, ovaries, uterus, spleen and lymph nodes as well as inflammation sites involving Th2 cells. During allergic reactions, they can be found in the lung, skin and oesophagus. IL-13 and IL-4 upregulate the expression of eotaxin 1 and VCAM-1, an endothelial adhesion molecule, to promote trafficking to these tissues . Other important chemotactic molecules involved in this process include platelet activating factor, LTD2 and CCL5.
Eosinophils are a type of leukocyte that has many functions in terms of inflammatory response: has a role in parasitic, bacterial and viral infection, allergies, tumours and injuries to tissues. Therefore, maintaining a certain level of eosinophils via activation and programmed cell death is important for the immune system. This is because eosinophils can regulate antigens, tissues and they can also encourage the inflammatory process via release of cytokines and lipid mediators. Eosinophil apoptosis is influenced by Natural Killer (NK) cells which was identified in Awad, Yassine, Barrier, et al. (2014) where they found that the rate of apoptosis of eosinophils was higher in a culture incubated with NK cells than the control.
Eosinophils average 10-12 µm in diameter and possess a distinct bi-lobed nucleus, with highly condensed chromatin (nucleoli are not visible). The two nuclear lobes are of equal size and are usually well demarcated, this has no obvious discernible function however is a useful indicator for observing Eosinophils in a blood smear.
Eosinophils are characterised by the presence of granules, there are two major types, specific granules (found only in eosinophils) and primary granules (these are similar to granules found in other granulocytes). Specific granules occupy most of the cytoplasm; they are relatively large (~0.5-1 µm in diameter), round vesicles and contain a multitude of cytokines and chemokines as well as basic cationic proteins that give eosinophils their distinctive staining properties (red or pink appearance when stained with eosin or other similar acidic dyes).
Primary granules form early in eosinophil development and are enriched with Charcot-Leyden crystal proteins. They are one of the most abundant proteins in eosinophils (7-10%) and form colourless crystals that are usually 2-4 µm in diameter and appear hexagonal or bi-pyramidal in shape.They have been found in body fluids, tissues and secretions thus have been considered a hallmark of eosinophil involvement in allergic reactions and other inflammatory reactions such as asthma or vasculitis. It has also been noted that CLC proteins persist long after eosinophil have died. However, their role in eosinophil function or inflammation is yet to be revealed.
CLC proteins have been identified as members of the carbohydrate-binding family of galectins (Galectin-10). They lack secretory signal peptides, suggesting they primarily function intracellularly, however their exportation and secretion also provides evidence for an extracellular function as well. Studies have also noted their interaction with cationic proteins and involvement in the secretion of EDN and ECP specifically. It is known that CLC proteins have a role in immunoregulation and immunomodulation in regulatory T cells (t regs). Eosinophils also possess these abilities, hence it has been stipulated that the protein may play a part in regulating the proliferation and function of CD4+ cells. (can move this paragraph elsewhere if need be)
Eosinophils also possess lipid bodies, these are lipid-rich cytoplasmic inclusions that form rapidly following eosinophil activation. These cytoplasmic structures are not membrane bound, they contain eicosanoid synthetic enzymes (5-LO, LTC4 synthase, COX) which act to generate important eicosanoids such as Leukotrienes, Prostaglandins and lipoxins. Eicosanoids are biologically active, oxygenated fatty acids that behave as paracrine mediators of inflammation (act on nearby cells to stimulate the inflammatory response) in addition to intracellular signals. They have been identified as key molecules in the pathogenesis of many inflammatory diseases such as psoriasis and rheumatoid arthritis. (Table here of products produced by lipid bodied and their functions- luekotrienes, prostaglandins etc)
Eosinophil Sombrero Vesicles
Large vesiculotubular carriers called eosinophil sombrero vesicles (EoSVs) exist within the cytoplasm of eosinophils. These are flattened, folded and elongated tubules with relatively large membrane surface areas and a diameter of 150–300 nm. EoSVs offer a different secretory pathway in which they rapidly transport products (cytokines and cationic proteins) from granules to the plasma membrane for extracellular release during the absence of granule-granule or granule-plasma membrane fusion. This process occurs upon stimulation of eosinophils with the typical agonists (e.g. MBP has been found in vesicles of eosinophils stimulated by eotaxin) and is characteristic of eosinophils undergoing piecemeal degranulation. EoSVs arise from specific granules and have been observed budding from emptying granules. They are also released from eosinophils undergoing cytolysis where they are deposited within target tissues together with free granules. Furthermore, EoSVs are present in non-stimulated eosinophils (with or without granule products).
What are they?
Granules are trilaminar membrane bound organelles with a matrix surrounding an electron dense crystalline core composed of MBP-1 and MBP-2. The granules contain highly structured internal membranes that serve to compartmentalize the granule. It has been shown with TEM and electron tomography studies that following agonist activation these compartments rearranged themselves. Suggesting that different stimuli can lead to the contents of the granules to be segregated, sorted and selectively secreted from the organelle.
|Major Basic Protein (MBP)|
|Eosinophil Cationic Protein (ECP)||
|Eosinophil Perocidase (EPO)||
|Eosinophil-Derived Neurotoxins (EDN)|
Mechanisms for contents release
- Classic Exocytosis
When a single granule fuses with the plasma membrane of the cell, releasing its contents extracellularly.
- Compound Exocytosis
Where more than one granule is released extracellularly when the granules first fuse with each other then the plasma membrane. Mainly used to combat big targets such as helminthic (worm) parasites.
- Piecemeal Degranulation (Predominant Method)
Process where the contents of the granules are selectively extracted into tubular or spherical vesicles which then travel through the cytoplasm to the plasma membrane where they fuse and release their contents.
- Eosinophil Cytolysis
Following lysis of the cell and loss of the plasma membrane, membrane bound granules are released freely into the surrounding tissue. This is seen mostly in eopsinophilic disorders, such as being found in the sputum of asthma sufferers.
Eosinophils migrating to different tissues in the body are part of its function. Eosinophils that are part of the circulatory system remain inactive until they reach the tissue. When eosinophils migrate to endothelial cells, interleukin (IL)-4 or IL-Beta encouragse further migration. The rate of this process further increases if a chemoattractant is used. In an experiment where a culture is used, the endothelial cells that were treated to prevent this chemotactic event lead to a decrease in the expression of CD68. CD69 is an early marker and CD35 is a receptor. Both of these are controlled by endothelial cells and thus their expression increased when the eosinophils migrated to the endothelial cells.
Granules express receptors for cytokines and G protein coupled receptors (CCR3) for chemokines. These are located on their surface membranes and respond to external cytokines and chemokines by activating a signal-transduction pathway within. IFN-γ (cytokine) and eotaxin (chemokine) are primarily responsible for stimulating secretion of the cationic proteins, enzymes and cytokines originating from granules. Granules essentially function as individual secretory vessels outside of eosinophils in diseased tissue sites, this reveals how they may contribute to inflammation mediated by eosinophils as well as immunoregulation and immunomodulation.
Large influxes of eosinophils from circulation occur mostly in sites during an infection or an inflammatory response. These cells are released into the blood stream before migrating to the part of the tissue that is inflammed. This action is dependent two main factors. One being the relationship between the adhesion molecules, that are found on eosinophils, and the endothelium and the second being the chemotactic signals generated by cytokines, chemokines and chemoattractant receptors. Some of the chemotactic molecules include FMLP, platelet-activating factor, complement 5a, and Chemokines. Human chemokines can be classified into CXC, CC, CX3C, and C subfamilies. Chemokines bind to G-protein-coupled seven-transmembrane receptors on different leukocytes which results in different cellular events. Humans express four different types of G-protein-coupled chemokine receptors (CCR):
Initially, the cytokines Interleukin (IL) -3 , IL-5 and GM-CSF prime the eosinophil whilst they are still in the bone marrow, before migrating to blood vessels. The transduction pathways of eosinophils then involve CCR1 and CCR3, where CCR3 is utilised for eotaxin signals. From here eosinophils migrate according to the chemotactic gradient caused by chemokines which promotes the adhesion and transmigration process from the peripheral blood vessel to the tissue after chemokine receptors aggregate with the anterior pole of the eosinophil and cause it to polarise.
In the context of response to allergic challenges in the airways of mice, eosinophils have demonstrated the ability to activate naïve T cells by acting as antigen presenting cells (APCs) in contrast to their normal role as end-stage effector cells that release granular cationic proteins. With the increasing prevalence of asthma, research into the role of eosinophils in the bronchial inflammatory response has increased, with the release of IL-5, eotaxin and other eosinophilic cytokines and chemokines being seen as the target for novel chemotherapeutic agents due to the damage that they cause to the bronchial epithelium. Allergic challenge stimulates the eosinophils to release IL-5 and granulocyte macrophage-colony-stimulating factor (GM-CSF), both of which are implicated in the inflammatory response characteristic of asthma. The release of toxic eosinophilic cationic proteins (ECP) allows the detection of inflammation in rhinitis by correlating serum ECP with that in nasal fluid, differentiating between systemic and local pathology.
- Studying the role eosinophils play in helminth infection is quite difficult, mainly due to the fact that humans and rodents don’t share many common helminthic worms making interpretations from rat models difficult. They is some evidence that they have a role for IL-5 (eosinophil activator) in protective immunity, however, other IL-5 receptor cells such as B-cells and basophils have not been ruled out in that model. However it has been shown that during parasitic infection, the granules such as eosinophil peroxides (EPO) and major basic protein-1 (MBP-1) eosinophil-derived neurotoxin and eosinophil catatonic protein can deposit its contents onto the helminth to kill it. Thus eosinophils are needed for eliminating the the parasite as without it, the parasite would be able to survive in the body for a longer period of time.
Time Lapse Video of Eosinophils Attacking a Helminth:
- Eosinophil granular proteins (ECP and EDN) have been shown to degrade single stranded RNA containing viruses, such as the respiratory syncytial virus. Paradoxically, they have also been shown to be susceptible to HIV-1 infection in vitro so may be a reservoir for the virus in vivo).
- Through release of their cytotoxic granule proteins (EDN and MBP) in the extracellular matrix, eosinophils have demonstrated a role in fungal infection fighting, as the cytotoxins, upon contact, can kill fungal organisms. Yoon et al. (2008) showed that through β2 integrin protein, the eosinophils were able to adhere to and react to the β-glucan found in the cells walls of the fungus Alternaria alternata, prompting the Eosinophil to release EDN, compromising the integrity of the fungus’ cell wall.
- Upon exposure to bacteria, eosinophils readily release mitochondrial DNA and both ECP and MBP, together they form extracellular structures like traps. These traps then bind to and kill the bacteria.
Role in Allergy and Disease
Atopic Dermatitis (Eczema)
An elevated level of ECP in the peripheral blood seems to correlate with the level of disease activity in Atopic Dermatitis (AD) patients. More specifically, the cytotoxic granule proteins from eosinophils are deposited within the skin lesions. Even in the absence of eosinophils, Davis et al, (2003) showed high levels of positive staining for MBP in the skin lesions. Eosinophilia has been demonstrated as being highly predictive of food allergies in AD sufferers, with food allergy being shown as causative of AD symptom aggravation in greater than 70% of the patients studied. Additional linkages between AD and food allergies are discussed by Majamaa et al., with faecal ECP and Tumour Necrosis Factor alpha (TNF) concentrations increasing after ingestion of cow's milk in children with AD ezcema. These findings are highly suggestive that eosinophils play a significant role in the pathogenesis of AD.
- Significant amounts of MBP have been found in the bronchoalveolar lavage fluid of patients with asthma, enough to induce cytotoxicity in host tissues such as epithelial cells as well as increase smooth muscle activity, therefore indirectly causing airway hyperactivity. In addition, the presence of eosinophils in respiratory tissue may lead to increased vascular permeability, potent smooth muscle constrictions and mucus secretion due to their generation of cysteinyl leukotrienes. Therapeutically, it has been found to be beneficial to treat allergic airway disease through cysteinyl leukotriene inhibitors.
Half of all asthma patients are affected by eosinophilic asthma. The disease is mainly controlled by T-helper cells with the assistance of eosinophils. The disease can evolve to slowly change the structure of the respiratory tract. Eosinophilic asthma can be monitored to prevent the worsening of the condidtion via:
1. Monitoring levels of sputum eosinophils
2. Looking into the blood eosinophil count
Sputum eosinophils are directly related to eosinophilic asthma as they control inflammation.
Eosinophilic Esophagitis, Gastritis, and Gastroenteritis
- Inflammation of the esophagus and gastrointestinal tract due to mass migration of eosinophils into these tissues in response to an allergen, leading to difficulty swallowing and discomfort in digestion. Interestingly, it has been found by Akei et al. (2006) that in rat models, rats with AD showed eosinophilic esophagitis in response to an allergen with migration of eosinophils to the tissue, where as rats without AD did not. This suggests a co-pathogenesis of eosinophilic inflammation in the skin and eosinophilic esophagitis.
Eosinophils and Vasculitis
The role played by eosinophils in vasculitis has mainly been examined in conjunction with eosinophilic granulomatosis with polyangiitis (EGPA), which was renamed from Churg–Strauss Syndrome in 2012 to better reflect the disease pathology. Eosinophils have been implicated in systemic vasculitis, with a significant linkage demonstrated between eosinophilia and asthma as well as other allergic conditions. Eosinophilic vasculitis appears to be connected to connective tissue disease, with a similar pathogenesis to vasculitis being demonstrated by the presence of ECP in the biopsied connective tissue lesions. Khoury et al. discusses the connection between eosinophilic granular chemokines and EGPA, additionally showing robust linkages between these biomarkers, eosinophilia and asthma.
Within all circulating leukocytes, less than 7% are eosinophils. In a normal patient, the eosinophil blood count would be under 4.5x108/L (450μl), however if the eosinophil blood count is greater then 4.5x108/L (450μl) in the peripheral blood. Diagnosis of eosinophilia is completed by a complete blood count (CBC) while in certain circumstances an absolute eosinophil count is conducted. The exceeding eosinophils can be found either in the bloodstream or directly found in tissues. An increase in eosinophils is commonly just a secondary side effect of another disease or in rare cases it by may be via a primary disease (idiopathic). Once the complete and absolute blood counts are completed, the underlying cause can be identified and is targeted in variety of treatments used to lower the eosinophil levels.
- Corticosteroids - Inhibits eosinophil survival and stimulates eosinophil clearance from tissues
- Imatinib - Inhibits receptor tyrosine kinase activity
- Hydroxyurea - Cytotoxic agent
- Interferon-α - Inhibits eosinophil growth and functional responses
|Eosinophil||A white blood cell that is accountable for combating multi cellular parasites and infections.|
|Granulocytic Effector Cells||Are cells that act in defence against helminth infections.|
|Leukocytes||Circulating cells in the blood which act as a defence against foreign material and diseases, e.g. monocytes, granulocytes and lymphocytes|
|Inflammatory Exudates||Is a fluid that filters via the circulatory system into lesion or regions of inflammation, can either be transparent fluids or pus filled.|
|Amoeboid||Is a common form of locomotion in eukaryotic cells|
|Hematopoietic Cell||Are stem cells in which give rise to all other known blood cells via haematopoiesis.|
|CD34||Is a cluster of diversity in a cell surface glycoprotein and its roles is to act as a cell to cell adhesion factor.|
|IL-5R||Interleukin-5 receptor is a type 1 cytokine receptor.|
|CCR3||C to C chemokine type 3 receptor.|
|Chemokines||A group of small cytokines/signalling proteins.|
|IL-5||Interleukin 5 is produced by Type 2 T helper cells/mast cells.|
|IL-3||Improves the body’s natural response to a disease.|
|GM-CSF||Granulocyte macrophage colony-stimulator factor is a monomeric glycoprotein that is secreted by a variety of cells such as mast cells, T cells, Macrophages, NK cells, Endothelial cells and Fibroblasts|
|IL-33||Increases the production of T helper-2 cytokines.|
|Interferon-γ||A dimerised soluble cytokine, that is important for innate and adaptive immunity against viral infections and also some protozoal and bacterial infections.|
|IL-13||Induces differentiation of helper T cells to T Helper 2 cells.|
|IL-4||A class of proteins which have carbohydrate groups attached to the polypeptide chain.|
|Eotaxin 1||Known as CCL11 (C-C chemokine 11) – Recruits eosinophils by preventing their chemotaxis in order to be implicated in allergic responses.|
|VCAM-1||Vascular Cell Adhesion Molecule 1 mediates the adhesion of lymphocytes, monocytes, basophils and eosinophils to vascular endothelium.|
|CCL5||Chemokine ligand 5 is chemotactic for eosinophils, basophils and T cells while it also recruites leukocytes into inflammatory areas.|
|Apoptosis||Is a process of cell death.|
|Charcot-Leyden Crystal Proteins||Are microscopic crystals that are found in patients with allergic disease such as asthma, pneumonia or ascariasis.|
|Vasculitis||Inflammation of a blood vessels or multiple blood vessels|
|Galectins||A group of proteins that bind specifically to β-galactoside sugars.|
|Immunoregulation||The control of immune responses and interactions between B and T lymphocytes and macrophages.|
|Immunomodulation||A process in which an immune response is altered to a preferred level.|
|ECP||Eosinophil Cationic Protein is a released during degranulation in eosinophils.|
|CD4+||Is a glycoprotein found on the surface of T helper cells, Monocytes, Macrophages and Dendritic cells.|
|Eicosanoids||Are signalling molecules.|
|Leukotrienes||Are a group of biologically active compounds, originally isolated from leucocytes (metabolites of arachidonic acid).|
|Prostaglandins||Are a group of compounds that have different hormone like effects (cyclic fatty acids)|
|Lipoxins||Are a group of compounds generated form arachidonic acid.|
|MBP-1||Mlul-box binding protein is a transcription factor involved in the regulation of cell cycle progression from G1 to S phase.|
|TEM||Transmission elector microscopy is a microscopy technique used to capture images at higher resolutions than light microscopes .|
|Chemoattractant||Are substances that possess chemotaxis inducers in motile cells|
|CD68||Used as a marker for monocytes and macrophages.|
|CD69||Is induced by the activation of T lymphocytes and NK cells.|
|Respiratory Syncytial Virus||Is a syncytial virus that causes respiratory tract infections.|
|β2 integrin protein||Is a transmembrane receptor that acts as a cell to cell and cell to extracellular matrix interactions.|
|Polyangiitis||Is an autoimmune disease that is characterised by a systemic, pauci-immune, necrotising, small vessel vasculitis.|
|Idiopathic||A disease or condition which arises spontaneously or has an unknown cause.|
|Corticosteroids||A class of steroid hormones that are produced in the adrenal cortex.|
|Imatinib||Is a tyrosine – kinase inhibitor that is used in the treatment of multiple cancers.|
|Hydroxyurea||Antineoplastic drug used in myeloproliferative disorders.|
|Interferon-α||Is a group of interferon proteins that assist in regulating the activity of the immune system.|
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