2016 Group 7 Project

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Eosinophil-pic.png

Introduction

Eosinophils are granulocytic effector cells, in healthy individuals they normally account for 1-3% of all leukocytes in peripheral circulation, with approximately 30-350 cells per cubic mm of blood [1].

History

Year Finding
1590 Compound microscope invented by Hans and Zacharias Janssen, making it possible to observe the components of blood[2].
1658 Jan Swammerdam (1637 -1680) first observed and identified red blood cells [3].
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 [3][2].

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[3].

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[3].

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 [3].
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[3].
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[3].
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)[3]. 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[3].

Birth, Life and Death in the Body

Histological images of an eosinophils. Note the well-defined, bi-lobed nucleus and heavily granulated cytoplasm.
Eosinophil Lineage

Hematopoietic progenitor cells give rise to mature eosinophils in bone marrow, these are identified primarily by their expression of CD34 as well as IL-5R and CCR3 proteins. They get produced in response to infections and diseases that cause an inflammatory responses which leads to an increase in amount of eosinophils present in the blood due to cytokine interleukin 5 (IL-5) [4], [5]. Eosinophils develop into their mature form via the influence of the regulatory molecules that control IL-5 [6], [7], [8]. Following stimulation by IL-5 and chemokines, such as eotaxin, eosinophils are mobilised, released from bone marrow into circulation and trafficked to tissues (for example during allergic inflammation or helminth infection). However, a large population of mature eosinophils will remain in bone marrow [9][10].


Once eosinophils enter circulation, they have a half-life of approximately 8–18 hours [9]. Under normal conditions, the vast majority of eosinophils are located in tissues (the tissue eosinophil/blood ratio is about 100:1) and once they enter, most do not recirculate [11]. 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) [9][10] [11]. 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 [12][9] [11]. IL-13 and IL-4 upregulate the expression of eotaxin 1 and VCAM-1, an endothelial adhesion molecule, to promote trafficking to these tissues [9]. Other important chemotactic molecules involved in this process include platelet activating factor, LTD2 and CCL5 [9] [11].


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[13]. Therefore, maintaining a certain level of eosinophils via activation and programmed cell death is important for the immune system[14]. This is because eosinophils can regulate antigens, tissues and they can also encourage the inflammatory process via release of cytokines and lipid mediators[14]. 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 [15].

Structure

Characteristics of Eosinophils [16]

Morphology

Eosinophils average 10-12 µm in diameter and possess a distinct bi-lobed nucleus, with highly condensed chromatin (nucleoli are not visible) [17]. The two nuclear lobes are of equal size and are usully 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) [9]. Specific granules occupy most of the cytoplasm; they are relatively large (~0.5-1 µm in diameter), round vesicles and contain basic cationic proteins that give eosinophils their distinctive staining properties (red or pink appearance when stained with eosin or other similar acidic dyes) [9][18][19].


Primary granules form early in eosinophil development and are enriched with Charcot-Leyden crystal proteins [9]. 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 [20][21].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 [20][21].

Smear showing hexagonal Charcot Leyden crystals in a background of inflammatory cells and necrotic material [22]


CLC proteins have been identified as members of the carbohydrate-binding family of galectins (Galectin-10) [20]. 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 [20][21][23]. (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 [9][24]. 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 [24].


Table here of products produced by lipid bodied and their functions- luekotrienes, prostaglandins etc


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 [25][26]. 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 [25]. 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 [25][27]. 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) [25][27].


Granules

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. [28] [29] [30]

Transmission Electron Microscope Image of an Eosinophil and its Granules

Contents

Granula Contents Function
Major Basic Protein (MBP)
  • Forms crystalloid structure of granules
  • Toxic to helminthic worms [31]
  • Cytotoxic to airways (partialy responsible for tissue damage in asthema) [32]
Eosinophil Cationic Protein (ECP)
  • Creates voltage-insensitive, ion-selective toxic pores in the membranes of target cells (to allow other cytotoxins to enter) [33]
  • Ribonuclease activity (but not as strong as EDN)[34]
  • Suppression of T cell proliferative responses [35]
  • Immunoglobulin synthesis by B cells [35]
  • Mast cell degranulation stimulation of airway mucus secretion [35]
  • Glycosaminoglycan production by human fibroblasts [35]
Eosinophil Perocidase (EPO)
  • Catalyze the oxidation of halides, pseudohalides, and nitric oxide to form highly reactive oxygen species which go on to oxidise nucleophil species on proteins causing oxidative stress on the host cell, leading to apoptosis and cell death [36]
Eosinophil-Derived Neurotoxins (EDN)
  • Ribonuclease activity
  • Anti-viral activity [37]
  • Induce migration and maturation of dendritic cells [38]
Mechanisms of granule secretion [39]

Mechanisms for contents release

  • Classic Exocytosis
    Eosinophil in PMD. A: Full granule B: Partial granule release C: Full granule release

When a single granule fuses with the plasma membrane of the cell, releasing its contents extracellularly. [40]

  • 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. [28]

  • 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. [41] [42]

  • Eosinophil Cytolysis
    Eosinophil undergoing cytolysis. Note the free intact extracellular granules

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. [43] [44].

Surface Markers

Eosinophils migrating to different tissues in the body are part of its function[45]. Eosinophils that are part of the circulatory system remain inactive until they reach the tissue[46]. When eosinophils migrate to endothelial cells, interleukin (IL)-4 or IL-Beta encouragse further migration[46]. The rate of this process further increases if a chemoattractant is used[46]. 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[46]. CD69 is an early marker and CD35 is a receptor[47]. Both of these are controlled by endothelial cells and thus their expression increased when the eosinophils migrated to the endothelial cells[47].

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 [48]. 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 [48].

Signalling

Function

Inflammation

Infection

Helminth

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 [49], however, other IL-5 receptor cells such as B-cells and basophils have not been ruled out in that model [50]. 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 [51]. 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 [52].

Time Lapse Video of Eosinophils Attacking a Helminth:

YouTube Link


Viral

Eosinophil granular proteins (ECP and EDN) have been shown to degrade single stranded RNA containing viruses, such as the respiratory syncytial virus [53]. 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 [54]).

Fungal

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 [37]. 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 [55].

Bacterial

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. [56]
Skin lesions on the inside of the elbow of an Atopic Dermatitis sufferer

Role in Allergy and Disease

Atopic Dermatitis (Eczema)

An elevated level of eosinophil granule protein 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 [37]. Even in the absence of eosinophils, Davis et al, (2003) showed high levels of positive staining for MBP in the skin lesions [57]. These findings suggest a role for eosinophils in the pathogenesis of AD.






Asthma

IL-5 and eotaxin-induced eosinophil recruitment in allergic asthma
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 [58] [59]. 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 [60]. 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 [61]. The disease is mainly controlled by T-helper cells with the assistance of eosinophils[62]. The disease can evolve to slowly change the structure of the respiratory tract[62]. Eosinophilic asthma can be monitored to prevent the worsening of the condidtion via:

1. Monitoring levels of sputum eosinophils[63]

2. Looking into the blood eosinophil count[64]

Sputum eosinophils are directly related to eosinophilic asthma as they control inflammation[61].



Eosinophilic Esophagitis, Gastritis, and Gastroenteritis

Inflammation of the eosophagus 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[37]. 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 eosophogus eosinophilic [65].

Eosinophils and Vasculitis

Schematic representation of eosinophil trafficking.jpg

Schematic representation of eosinophil trafficking [65].

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 [66]. Eosinophils have been implicated in systemic vasculitis, with a significant linkage demonstrated between eosinophilia and asthma [67] as well as other allergic conditions. Khoury et al. [65]. discusses the connection between eosinophilic granular chemokines and EGPA, additionally showing linkages between these biomarkers, eosinophilia and asthma.

Eosinophilia

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 (idopathic). 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.[68]

  • 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


Glossary

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