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Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from

Postgrad Med J (1994) 70, 536 552 PJ , The Fellowship of Postgraduate Medicine, 1994 Review Article

Eosinophils in the 1990s: New perspectives on their role in health and disease A.J. Wardlaw Department ofRespiratory Medicine, Leicester University Medical School, Glenfield Hospital, Groby Road, Leicester LE3 9QP, UK

Introduction have traditionally been somewhat imately 8 ytm in diameter. One of their most neglected over the years by clinicians and characteristic features are the membrane-bound pathologists alike, being seen as a sort of poor specific granules of which there are about 20 per man's , appearing in a supportive role as human . These are spherical or ovoid, part of 'acute ' with an occasional and contain a crystalline core surrounded by a less lead part in exotic diseases such as Churg-Strauss electron-dense matrix.6 The core is comprised of syndrome or hypereosinophilic syndrome. This major basic (MBP) and the matrix contains neglect is unwarranted, both because the the other three basic granule , eosinophil eosinophil is one of the most striking of cells, cationic protein (ECP), eosinophil peroxidase

readily identified in haematoxylin and eosin (H&E) (EPO) and eosinophil-derived neurotoxin (EDN orcopyright. stained sections with their bilobed nuclei and bright EPX).7 These basic proteins stain avidly with dyes red granules, and because the eosinophil is closely such as eosin from which the cell gained its name. associated with two of the most common and Eosinophils also contain lipid bodies which are universal of diseases, asthma and parasitic infec- non-membrane-bound organelles and the principal tion. The characteristic appearance of the store of arachidonic acid esterified into glycero- eosinophil resulted in its early identification by phospholipids.8 Eosinophil primary granules are a Ehrlich in 1879 and its association with asthma and third type of intracellular organelle which contain allergic disease was soon recognized.' Curiosity Charcot-Leyden crystal (CLC) protein. CLC pro- about its role in asthma and other diseases has tein is also found diffusely in the nucleus and http://pmj.bmj.com/ persisted ever since. In recent years there has been cytoplasm in activated eosinophils. Primary an explosion of interest in the eosinophil reflected granules are recognized by the absence of a core in an exponential increase in the number of pub- and are ofvariable size, being often larger than the lished papers and books devoted to the cell.2-5 This specific granules. They make up approximately 5% interest has been mainly the result of increasing of eosinophil granules.9 Tissue eosinophils also evidence that the eosinophil may be responsible for contain a number of small granules which stain

much of the tissue damage seen in asthma and the intensely for acid phosphatase and aryl sul- on September 25, 2021 by guest. Protected hope that modulation of eosinophil function may phatase.'°" be an effective therapy for the disease. The purpose of this paper is firstly to review recent findings about the biology of the eosinophil, emphasizing Eosinophil production the features that are distinctive about the Eosinophils like other leucocytes differentiate from eosinophil compared to other leucocytes and stem cell precursors in the bone marrow. They then especially . Secondly, I will briefly sum- migrate into the peripheral blood where they marize the possible role of the eosinophil in the circulate with a half life of about 18 hours before diseases with which it is associated. migrating into tissue. Eosinophils are primarily tissue-dwelling cells with about one blood Morphology and ultrastructure eosinophil for every 100 tissue eosinophils.'2 Nor- mal human adult bone marrow contains about 3% Eosinophils are non-dividing, bone marrow- eosinophils of which a third are mature and derived, granule-containing cells. They are approx- two-thirds are myelocytic precursors. Eosinophilic myelocytes are large cells with a single-lobed Correspondence: A.J. Wardlaw, F.R.C.P., Ph.D. nucleus, expanded Golgi and extensive dilated Received: 7 January 1994 cisterns of rough endoplasmic reticulum. They Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from EOSINOPHILS 537 become identifiable when they develop the core detected in mast cells and eosinophils,36 it is likely containing specific granules which initially are that T- are the principal source of this interspersed with large numbers of homogenous . T cells, as well as being divided into dense granules.'3 subsets on the basis of their receptor phenotype, There is now substantial evidence that the mas- can be distinguished by their cytokine profile. sive increase in eosinophils associated with helmin- T-helper type 1 (Thl) cells produce IL-2 and thic parasitic infection is T-cell dependent.'4"15 interferon-y (IFN-'), Th2 cells IL-4 and IL-5, Three T-cell-derived have been shown to whereas GM-CSF and IL-3 are elaborated by both promote eosinophil growth and differentiation, cell types.37 T-cells with a Th2 profile of cytokine interleukin-3 (IL-3), IL-5 and -macro- production are found in allergic and eosinophilic phage colony-stimulating factor (GM-CSF). parasitic disease. Eosinophilia in many diseases Eosinophil and colonies appear together therefore appears to be due to a specific type of in colony-forming assays.'6 Mouse IL-5, but not T-cell response to certain types of . For human IL-5, is a growth factor for B cells. IL-5 is a example, in allergic disease and parasitic disulphide-linked homodimeric glycoprotein with in helminthic infections. Drug-induced a molecular weight of 40-45 kDa.'7"8 The dimers eosinophilia may be due to the drug acting as a are aligned in a head to tail fashion and dimeriza- hapten for a Th2 response. tion is essential for function.'9 Culture of mouse bone marrow suggested that IL-5 was a late differentiation factor and could not support eosinophil growth from early precursors. These Eosinophil heterogeneity steps appeared to require other cytokines such as IL-3 and GM-CSF.20 However, IL-5 transgenic Peripheral blood eosinophils from normal individ- mice had a marked peripheral blood and tissue uals are relatively dense cells which separate out eosinophilia with increased numbers of eosinophil from other leucocytes in the lower bands ofPercoll precursors in their bone marrow.2"22 Despite a or Metrizamide discontinuous density gradients. marked eosinophilia these mice had no obvious For many years these differences were the basis for copyright. pathological defect. The observation that IL-5 the standard method ofpurifying eosinophils. This alone was sufficient to generate an eosinophilia is has now been largely superseded by immuno- consistent with the fact that increases in numbers of magnetic selection based on differences in expres- eosinophils are often seen without expansion ofthe sion of the low-affinity (immunoglobulin receptor. other myeloid lineages. Both IL-3 and GM-CSF (Fc)TyRIII, cluster of differentiation (CD)16) IgG induce eosinophil production in vitro in human receptor by eosinophils and neutrophils.38 A pro- cord blood culture23 and after in vivo administra- portion of eosinophils from individuals with a tion, although the increase in the number of raised eosinophil count are less dense than eosinophils was modest compared to other eosinophils from normal subjects.39 The http://pmj.bmj.com/ lineages.24 In humans the genes for IL-3, IL-4, IL-5 mechanism for this heterogeneity is unclear. and GM-CSF are clustered on the long arm of Hypodense eosinophils appear to be vacuolated chromosome 5.2S The receptors for IL-3, IL-5 and and contain smaller-sized granules, although of GM-CSF are structurally similar.26 They consist of equal numbers to normal density eosinophils.4 It is unique though homologous a chains, which bind generally considered that hypodense eosinophils with low affinity to their respective cytokines with a represent an activated phenotype.4' Thus hypo- Kd in the region of 10 nmol/l. There is a common P dense eosinophils have increased oxygen consump- on September 25, 2021 by guest. Protected chain which is non-covalently associated with the a tion,41 increased cytotoxicity towards helminths43 chains at the cell surface and transforms the and increased leukotrine C4 (LTC4) production." receptor into one ofhigh affinity (Kd 150 picomoles They release less platelet-activating factor (PAF) (pm)). The P chain is required for signal transduc- after stimulation with IgG sepharose beads but this tion. Unlike the a chains of IL-3 and GM-CSF, the appears to be the result of increased acetyl hydro- a chain of the IL-SR can bind IL-5 with relatively lase activity.45 Stimulation of eosinophils either in high affinity (Kd 250-590 pm). 27'28 the short term with PAF or in long-term culture In many conditions associated with increases in with cytokines results in a hypodense phenotype eosinophils, including asthma,29 parasitic disease,30 and enhanced effector function.' In contrast, IL-2 therapy,3' hypereosinophilic syndrome hypodense eosinophils have a similar profile of (HES)32 and eosinophilia/myalgia syndrome,33 leucocyte integrin and Fc'y receptor expression to evidence of increased IL-5 production has been normal density cells47 and normal density cells from obtained. IL-5 messenger RNA (mRNA) has been individuals with an eosinophilia are also primed. detected in eosinophilic Hodgkins disease.34 Nonetheless, the weight of evidence suggests that against IL-5 abolishes the eosinophilia hypodensity represents a primed or partially in parasitized animals.35 Although IL-S has been activated phenotype. Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from 538 A.J. WARDLAW

Eosinophil receptors interaction is relatively weak and the leucocyte rolls along the surface of the endothelium until it comes Like all leucocytes, eosinophils express various into contact with a priming stimulus such as a membrane receptors through which they com- chemotactic mediator. This allows the leucocyte municate with the extracellular environment. integrin receptor to bind to its corresponding These include adhesion receptors, Fc receptors and immunoglobulin-like ligand. The resultant bond is receptors for the recognition of soluble mediators much firmer than the selectin carbohydrate bond (Figure 1). and results in the leucocyte flattening and trans- migrating between endothelial cells.5' Three events Adhesion receptors are therefore required for migration to occur: (1) engagement of a selectin and its receptor; (2) A critical aspect of leucocyte function is migration leucocyte activation; and (3) engagement of the from the vascular space into extracellular tissue. intergrin/immunoglobulin receptor bond. Having The initial step in this process is adherence to transmigrated through the endothelium, the postcapillary venular endothelium. This is leucocyte interacts with the extracellular matrix mediated by binding of adhesion receptors on the proteins through its integrin and other adhesion surface of leucocytes to their ligands or counter- receptors. Adhesion receptors and their ligands structures on endothelium. Adhesion receptors are potentially involved in eosinophil function are grouped into several gene superfamilies, and in- summarized in Table I. As with neutrophils, L- clude the integrin superfamily, members of the selectin is shed when eosinophils are stimulated and immunoglobulin superfamily, and the selectins.48'49 BAL eosinophils that have migrated into the Integrins bind to members of the immunoglobulin airways express very little L-selectin.52 receptor family and selectins bind, via their lectin One potential mechanism for preferential domain, to carbohydrate counterstructures that localization of eosinophils (as opposed to neut- include the moiety sialyl Lewis X'5° Transmigration rophils) at inflammatory foci is a selective adhesion through vascular endothelium is a staged process in pathway. IL-5 and IL-3 increase eosinophil, but which the leucocyte is first tethered to the not neutrophil, adhesion to unstimulated human endothelial cell by binding of a selectin receptor to umbilical vein endothelial cells (HUVEC).53copyright. its carbohydrate ligand. The binding affinity ofthis Eosinophils but not neutrophils can utilize the very

Immunoglobulin FcR Adhesion receptors Mac-1 (CR3) receptors i3 / \ ~~~~~~~LFA-1 http://pmj.bmj.com/ FcRp1,9

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W CD 45 C5 I / \ / ~~~ ~ ~~~~~PAF Not expressed f-MLP on neutrophils LTB CD / ~~CD 25 ICAM-1 IL-5 CD 16 HLA-DR | I Mediator RANTES. receptors Newly expressed receptors

Figure 1 Schematic representation ofan eosinophil with its bibbed nucleus and specific granules illustrating the major eosinophil membrane receptors. Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from EOSINOPHILS 539

Table I Eosinophil adhesion receptors and their eosinophils can undertake a number of IgE- counterstructures dependent functions, including killing ofschistom- somes opsonized with specific IgE.65 It was thought Eosinophil Endothelial Matrix that the eosinophil IgE receptor was related to the receptor receptor protein low-affinity IgE receptor found on B-lymphocytes, Integrin platelets and - FcsRIII (CD23).' VLA-4 (Q4PI) VCAM-1 Fibronectin However, peripheral blood eosinophils incon- m4P7 MadCAM-1 Fibronectin sistently express messenger RNA (mRNA) for VLA-6 (m6l1) Laminin CD23 and do not stain with a panel ofmonoclonal LFA-1 ICAM-1, ICAM-2 antibodies (mAbs) directed against this receptor. Mac-I ICAM-1 Fibrinogen Eosinophils express the IgE-binding protein Mac-2 Immunoglobulin-like but so do neutrophils that lack IgE-dependent PECAM PECAM functions.67 The nature of eosinophil IgE binding Selectin therefore remains to be clarified. There are three L-selectin GlyCam-1 receptors for IgG: the high-affinity receptor, FcyRI (CD64), and two low-affinity receptors, FcyRII Carbohydrate (CDw32) and FcyRIII (CD16).' Only CD32 is P-selectin ligand P-selectin constitutively expressed by eosinophils to any E-selectin ligand E-selectin significant degree.42 A number of eosinophil func- tions are mediated via this receptor, including schistosomula killing, phagocytosis, the secretion late antigen (VLA)-4/vascular cell adhesion mole- of granule proteins, and the generation of newly cule (VCAM)-1 pathway.54 VCAM-1 expression is formed, membrane-derived lipid mediators such as selectively upregulated by IL-4" and IL-4 is PAF and LTC4. After stimulation for 2 days in vitro generated at sites of allergic inflammation.56 IL-4 with IFN-y, eosinophils express CD16 and CD64 enhances eosinophil transmigration through endo- as well as CD32.69 The eosinophil also expresses thelium in a man- IgA receptors that, when engaged by IgA-coated

VLA-4/VCAM-l-dependent copyright. ner.57 IL-4 transgenic mice have a tissue Sepharose beads, trigger substantial release of eosinophilia and manifest an inflammatory condi- eosinophil granule proteins.70 tion in the eye similar to allergic conjunctivitis.58 Anti-VLA-4 mAb's inhibit eosinophil migration Other receptors into tissue in guinea pigs.59 In contrast, VCAM-1 expression in eosinophilic tissue, such as nasal An interesting feature ofthe eosinophil is its ability biopsies, nasal polyps, and endobronchial biopsies to express receptors de novo after prolonged from asthmatic individuals and in the skin of (> 48 hour) culture in a number of cytokines. For allergic individuals after challenge, is very example, after culture in GM-CSF, eosinophils http://pmj.bmj.com/ weak or non-existent despite strong expression of express human leucocyte antigen (HLA-DR) other endothelial adhesion molecules.W61 The antigens and increased amounts ofICAM-1, which evidence that selective expression of adhesion are associated with an in vitro capacity to present molecules is responsible for eosinophil localization antigen to T cells.7' Peripheral blood eosinophils in tissues in humans is therefore at best contradic- express the early activation antigen CD69 after tory. cytokine stimulation in vitro as do bronchoalveolar Eosinophils adhere to a number of extracellular lavage (BAL), eosinophils from patients with on September 25, 2021 by guest. Protected matrix proteins. For example, fibronectin binds to asthma and pulmonary eosinophilia.72 Eosinophils eosinophils through VLA-4, laminin through also respond to a number ofsoluble mediators such VLA-6 and hyaluronate through CD44. Adhesion as f-MLP, C5a, C3a and RANTES,73 as well as to to matrix proteins results in priming for increased lipid mediators such as PAF, by means of specific LTC4 and hydrogen peroxide release.62'63 receptors, many of which belong to the rhodopsin Eosinophils also survive for prolonged periods family of seven transmembrane region G-protein- when cultured on fibronectin, as a result of auto- linked receptors. crine stimulation of IL-3 and GM-CSF produc- tion.' This is a possible mechanism for prolonged survival of tissue eosinophils in both health and Eosinophil chemotaxis disease. A number of eosinophil chemotaxins have been Immunoglobulin receptors described, although few are both effective and specific. PAF and C5a are highly active on human The eosinophil expresses receptors for IgG, IgA eosinophils but are equally active on neutrophils.74 and IgD. The eosinophil also binds IgE and LTB4 and 5,15,diHETE act as guinea pig eosino- Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from 540 A.J. WARDLAW phil and neutrophil chemoattractants." IL-5; IL-3 Eosinophil mediators and GM-CSF have variable and generally weak activity in the Boyden chamber assay, although Eosinophils have the capacity to secrete a number they are active at very low concentrations."6 They ofpotent mediators (Figure 2). These include basic are only active on eosinophils from individuals proteins stored in eosinophil granules, lipid with normal eosinophil counts. However, they can mediators newly formed ater eosinophil activation, effectively prime eosinophils for enhanced cytokines, various eosinophil proteases, and com- chemotactic responsiveness to suboptimal concen- ponents of the oxygen burst, including superoxide trations ofPAF and LTB4, as well as enhancing the and hydrogen peroxide. generally negligible activity that f-MLP and IL-8 have for unactivated eosinophils." Recently, it has Lipidmediators been reported that the C-C chemokine RANTES is an effective and selective (in the sense of having no Eosinophils generate an array of lipid mediators activity for neutrophils) chemoattractant for principally eicosanoids and PAF.8' Eosinophils can eosinophils." In vivo PAF injected into the skin generate relatively large amounts (up to 70 ng/106 caused the accumulation of eosinophils in atopic cells) of the sulphidopeptide leukotriene, LTC4, individuals, but neutrophils were prominent in after stimulation with the calcium ionophore, but non-atopic subjects.78 Increased numbers of neut- only negligible amounts of LTB4.82 This is in rophils, but not eosinophils, appeared in BAL fluid contrast to neutrophils which can produce large 4-6 hours after the inhalation ofPAF in a group of amounts of LTB4, but little, if any LTC4. LTC4 eight normal subjects, three ofwhom were atopic.79 generation by human eosinophils was also A highly effective but non-specific mediator such as observed after stimulation with both opsonized PAF could combine with a selective but weakly zymosan and via an FcyII-dependent mechanism chemotactic agent such as IL-5 to promote the using Sepharose beads coated with IgG.4 specific accumulation of eosinophils in allergic Eosinophils can also generate substantial quan- disease. Inhalation of LTE4 produced eosinophil tities of 1 5-HETE via 1 5-lipoxygenase. Eosinophils migration into the airways.80 The mechanism of generated 25 and 2 ng/106 cells of PAF after this effect is not clear. stimulation with calcium ionophore and IgG-copyright.

Granule-derived mediators Lysophospholipase Phospholipase D Arylsulphatase http://pmj.bmj.com/ Newly synthesized Histaminase membrane-derived 14A Catalase mediators Acid phosphatase LTC4 Non-specific esterases PAF Glucosaminoglycans 15-HETE Hexoseaminicdase PGE1 and 2

TXB2 on September 25, 2021 by guest. Protected

Cytokines IL-1 GM-CSF IL-3 IL-5 IL-6 TGF-a TGF-f IL-8

Figure 2 Schematic representation of eosinophil-derived mediators. Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from EOSINOPHILS 541 coated Sepharose beads, respectively.45 Much of EDN, also called EPX, is a 16 kDa glycosylated the PAF remained cell associated. Eosinophils can protein possessing marked ribonuclease activity. also generate mediators of the cycloxygenase path- Like ECP, it is a member ofribonuclease multigene way, including prostaglandins El and E2 and family.9' EDN expression is not restricted to thromboxane B2 (TXB2). eosinophils, as it is found in mononuclear cells and possibly neutrophils. It is also probably secreted by Eosinophilgranuleproteins the liver. It does not appear to be toxic to parasites or mammalian cells and its only known function, MBP has a molecular weight of 13.801 and a pl of other than its ribonuclease activity, is the neurotox- 10.9. It contains 17 arginine residues, which icity exhibited in the Gordon phenomenon. accounts for its basicity. It is initially synthesized as A major constituent ofeosinophil is CLC protein an acidic proprotein, which may neutralize MBP's which has been shown to be lysophospholipase. It toxicity while it is stored in the eosinophil granule.83 constitutes up to 10% of eosinophil protein and is Purified MBP was shown to be cytotoxic for the also found in large quantities in , thus schistosomula of S. mansoni, and adherence of highlighting the similarities between these two cell eosinophils to IgG-coated schistosomula resulted types. Its function is unclear.92 in the secretion ofMBP on to the integument ofthe larvae.84 MBP at concentrations as low as 10 gtg/ml has also been shown to be toxic for both guinea pig Cytokines and human respiratory epithelial cells.85 The inhalation of MBP, albeit at high concentrations Eosinophils can synthesize an array of cytokines. (1 mg/ml), produced increased bronchial hyperres- Activated eosinophils have been shown to secrete ponsiveness in monkeys.86 MBP and EPO were significant amounts oftransforming growth factor- shown to be strong agonists for platelet activation alpha (TGF-a).9" After stimulation with calcium as well as inducing the non-cytolytic activation of ionophore, eosinophils can also generate GM-CSF mast cells, basophils and neutrophils.87 The and IL-3, which prolong eosinophil survival as well mechanism of as IL-8.9496 Eosinophils in allergic tissue expressed action of MBP is likely to be related copyright. to its hydrophobicity and strong negative charge. mRNA for IL-5 and eosinophils have been shown EPO is a heme-containing protein composed ofa to generate significant quantities of TGFP and 14,000 Da (light) and a 58,000 Da (heavy) subunit IL-6.9799 IL-1 has also been detected in human derived from the same strand of mRNA and eosinophils. subsequently cleaved. The cDNA also demon- strates the presence ofa prosequence.88 EPO shares Other mediators a 68% amino-acid identity with human neutrophil myeloperoxidase as well as other peroxidase The eosinophil contains a number of granule- enzymes. EPO is toxic for parasites, respiratory stored enzymes whose role in eosinophil function is http://pmj.bmj.com/ epithelium, and pneumocytes, either alone, or not clear (reviewed by Spry"'). They include acid (more potently) when combined with H202 and phosphatase (large amounts of which have been halide, the preferred ion in vivo being bromide. isolated from eosinophils), collagenase, arylsul- ECP is an arginine-rich protein with a pI of 10.8 phatase B, histaminase, phospholipase D, catalase, of 133 amino acids with a molecular mass of non-specific esterases, vitamin B12-binding proteins 15.6 kDa ECP shows 66% amino-acid homology and glycosaminoglycans. Eosinophils can undergo with EDN and 31% homology with human pan- a respiratory burst with release of superoxide ion on September 25, 2021 by guest. Protected creatic ribonuclease.89 It has low ribonuclease and H202 in response to stimulation. activity compared to EDN. It appears to be expressed only in eosinophils or eosinophilic cell lines. ECP is toxic for helminthic parasites, isolated myocardial cells, and guinea pig tracheal epithelium. ECP also inhibits pro- Eosinophil secretion and activation liferation in vitro. Both ECP and EDN produce neurotoxicity (the Gordon phenomenon) when A striking feature of eosinophil-rich inflammatory injected into the cerebrospinal fluid of experimen- reactions is the marked deposition of granule tal animals. The secreted form of ECP differs proteins often in the presence of relatively small structurally and antigenically from the stored form. numbers of intact eosinophils. The mechanism of This difference has been used to differentiate eosinophil secretion in vivo is still poorly under- between resting eosinophils and activated stood. Eosinophils are cytotoxic for the larvae of eosinophils in which active secretion is occurring helminthic parasites such as schistosomulae of S. with the mAb EGl recognizing the stored form and mansoni but only when the larvae have been the mAb EG2, the activated state.90 opsonized with either complement or immuno- Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from 542 A.J. WARDLAW suggesting that triggering of eosinophil misleading picture of eosinophil involvement in a secretion is dependent on perturbation of Fcy or particular disease. The blood eosinophil count complement receptors, particularly Mac-1. represents the balance between the rate of Eosinophils preferentially secrete their mediators eosinophil migration from the bone marrow and on to a large surface, a process described as entry into the tissues. Once in the tissue, frustrated phagocytosis. Opsonized zymosan eosinophils can survive for many days under the interacts with eosinophils, triggering generation of influence of locally generated cytokines. Their hydrogen peroxide and PAF through Mac-I."' removal appears to be largely the result of pro- The ability ofeosinophils to secrete their mediators grammed cell death (apoptosis) and subsequent is markedly enhanced by priming with soluble phagocytosis of the senescent cell by macro- mediators such as chemotactic factors and phages.'08 Despite the usually modest peripheral cytokines. Chemotactic agents can also elicit the blood eosinophil counts, there are large numbers of direct secretion of both granule proteins and lipid eosinophils and their precursors in the normal bone mediators, although soluble mediators are marrow. generally ineffective secretogogues except with Eosinophils can be enumerated in the peripheral highly activated eosinophils or when used in con- blood either by 'wet counts' in modified Neubauer junction with cytochalasin B, which inhibits chambers, differential counts on dried smears or by cytoskeletal assembly.'02 Differential secretion of automated cell counting. The automated counting granule proteins depending on the stimulus has that uses detection of eosinophil peroxidase is the been reported. (IgG) com- most accurate method followed by counting in a plexes induced the secretion of ECP but not EPO, cell chamber. Counting on smears is least accurate whereas IgE complexes induced secretion of EPO because of the tendency for eosinophils to congre- but not ECP.'03 However, secretion was low in both gate at the margins of the smear. It is preferable to instances. Eosinophils release their granule com- record the eosinophil count in absolute numbers ponents by exocytosis, with individual granules rather than as a percentage, as the latter will fusing with the plasma membrane. This process depend on the total cell count. The normal count is involves a guanosine 5'-triphosphate (GTP)- eosinophil (generally taken as) less thancopyright. binding protein and is modulated by the intracel- 0.4 x 109/l, although a study of 765 medical lular calcium concentration.'"4 Priming of students in the USA measured counts ranging from eosinophils involves a rise in intracellular calcium 0.015-0.65 x 109/l.'" It is higher in neonates."0 and triggering of PI turnover. As is the case with The eosinophil count varies with age, time of day, degranulation, the signal transduction pathways exercise and environmental stimuli, particularly involved in priming appear broadly similar to those allergen exposure. Blood eosinophil counts described for mast cells and neutrophils.'05 undergo diurnal variation, being lowest in the Several cytokines have a marked effect on morning and highest at night. This effect resulted in eosinophil function. IL-5, besides being a growth a greater than 40% variation in one study."' This http://pmj.bmj.com/ maturation factor for eosinophils, also selectively may be related to the reciprocal diurnal variation in stimulates a number of mature human eosinophil cortisol levels which are highest in the morning. functions, including prolonged survival, cytotox- The causes of an eosinophilia can be usefully icity toward helminth targets, and increased classified according to the degree and frequency of adhesion to vascular endothelium.'" IL-3 and occurrence (Table II). Division of eosinophil GM-CSF have similar though less selective counts into degree is arbitary but a mild activities. stimulates eosinophilia could be regarded as 0.4-1.5 x IFN-'y eosinophil cytotoxicity, 109/l, on September 25, 2021 by guest. Protected prolongs eosinophil survival and results in expres- a moderate count as 1.5-5 x 109/l and a highcount sion of mRNA for GM-CSF. TNF-a stimulated as greater than 5 x 109/l. The commonest cause of eosinophil cytotoxicity toward endothelium.'07 IL- an eosinophilia worldwide is infection with helmin- 3, IL-5 and GM-CSF have both short-term prim- thic parasites, which can often result in a very high ing effects on eosinophils, which are maximal count. The commonest causes ofan eosinophilia in within an hour and more long-term effects, which industrialized countries are the atopic allergic include increased receptor expression and depend diseases, seasonal and perennial rhinitis, atopic on protein synthesis. dermatitis and asthma. Allergic disease generally results in only a mild increase in eosinophil counts. A moderate or high eosinophil count in asthma raises the possibility of a complication such as Churg-Strauss syndrome or allergic bronchopul- Eosinophilia monary aspergillosis (ABPA). Apart from allergic disease and helminthic parasites, a raised An isolated count of eosinophil numbers in the eosinophil count, especially a moderate or high blood offers only a limited and sometimes count, is unusual. Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from EOSINOPHILS 543

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Eosinophils and disease Eosinophils and asthma The role ofeosinophils It is well established that large numbers of Views on the role of eosinophils in health and eosinophils together with mononuclear cells are disease have changed with time. For years they frequently found in and around the bronchi with were thought to ameliorate inflammatory res- patients who have died of asthma. The immuno- ponses, now they are believed to have a tissue- staining of bronchial tissue from such patients has damaging role."2"'3 Even more recently it has revealed the existence of large amounts of MBP become apparent that eosinophils are the source of deposited in the airways."8 The presence of in- a range ofcytokines several ofwhich are thought to creased numbers ofperipheral blood eosinophils in have a homeostatic, rather than pro-inflammatory both atopic and non-atopic chronic asthma is well function. For example, the observation that known, although this elevation is not as great as eosinophils secrete TGFa together with studies that seen in other eosinophil-associated diseases showing increased numbers of eosinophils at the and the peripheral blood eosinophil count is often edges of healing wounds suggests that they may be normal. important in wound healing."4 Cytokine- Full appreciation of the extent of eosinophil stimulated eosinophils secrete IL-1, express HLA involvement in asthma has come with the use of class II receptors and present antigen to T cells in fibreoptic bronchoscopy to obtain BAL fluid and vitro, suggesting they may be important as acces- endobronchial biopsies from the airways in sory cells in T-cell-mediated reactions. There is patients with mild to moderate asthma. evidence that eosinophils slow the rate of progres- Aerosolized challenge ofsensitized asthmatics with sion of solid tumours, presumably by being allergen results in an influx of inflammatory cells cytotoxic against tumour cells."5 Nonetheless, consisting of eosinophils, neutrophils and there is also little doubt that eosinophils can cause mononuclear cells into the airways and an increase severe tissue damage under certain circumstances. in the amount of eosinophil granule proteins in Persistently high eosinophil counts from many lavage fluid."9 A similar picture has been observed causes including drug reactions, parasitic infec- after challenge with agents that cause occupational tions, eosinophilic leukaemia and hypereosinoph- asthma.'20"2' The eosinophilia associated with copyright. lic syndrome are associated with endomyocardial segmental challenge down the bronchoscope is fibrosis, a condition that presents with heart failure even more dramatic. Twenty-four hours after and signs consistent with a restrictive car- segmental challenge up to 50% of the lavage cells diomyopathy. The ventricle is thickened and his- were eosinophils.'22 Similar findings have been tologically there are areas of fibrosis, thrombus found after allergen challenge to the skin and formation and inflammation in the endomyocar- nose 123,124 dium with large numbers of both intact and An almost invariable increase in the number of degranulating eosinophils. Eosinophil granule pro- eosinophils, in association with increased numbers http://pmj.bmj.com/ ducts are deposited adjacent to myocytes and in of mast cells and epithelial cells has been observed vitro have been shown to be toxic for cardiac in BAL fluid and endobronchial biopsies from myocytes. HES is a condition in which there is a clinical asthmatics compared with normal con- high eosinophil count ofunknown aetiology and it trols.'25'2' A lesser, but often significant, increase in is associated with a number of features, several of airway eosinophils is seen in atopic non-asthmatics which could be ascribed to the toxic properties of or seasonal asthmatics out of season. Airway eosinophils."16 eosinophils in asthma are activated as determined Much ofthe work undertaken in recent years on by staining with mAb EG2 and expression of the on September 25, 2021 by guest. Protected eosinophils has been in association with allergic activation receptor CD69.'29"30 Eosinophil infil- disease and parasitic infection. The observation in tration is accompanied by increased numbers of the mid-1970s that eosinophils could kill parasite activated CD25-positive T-lymphocytes, which targets led to the hypothesis that the teleological have a Th2-like profile ofcytokine secretion'3' and role of eosinophils was to counter parasitic infec- evidence ofepithelial desquamation with increased tion."7 The realization that eosinophils could numbers ofepithelial cells in BAL fluid and signs of release pro-inflammatory mediators such as PAF epithelial fragility in bronchial biopsies.'32 The and eiconsanoids, and the observation that increase in eosinophils has been noted in intrinsic eosinophil basic proteins were toxic for airway and occupational asthma as well as atopic epithelium has led to a consensus that eosinophils asthma.'33"34 A BAL eosinophilia is relatively are a major effector cell for tissue damage in asthma specific to asthma, although it is also seen in and could cause many of the pathological features pulmonary eosinophilia and some patients with of the disease. These conditions provide a useful fibrosing alveolitis.'35 The numbers of eosinophils model for eosinophil involvement in disease pro- in BAL fluid in asthma are generally only modestly cesses. raised ranging from 1% to 5% (normals, <1%), Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from 546 A.J. WARDLAW although occasionally eosinophil counts can be in reinfection. Moreover, treatment of mice infected the range of30- 50%. There is a general correlation with N. brasiliensis or S. mansoni with neutralizing between the numbers ofairway eosinophils and the anti-IL-5 mAbs abolished the eosinophilia without severity ofasthma. Inhibition ofan airway eosino- modulating the disease process."5 philia by disodium chromoglycate (DSCG),'37 or more effectively corticosteroids,'38"39 is associated Other eosinophilic disorders with an improvement in bronchial hyperrespon- siveness, symptoms and lung function. Inhibition More unusual eosinophilic disorders include pul- of migration of eosinophils into the airways of monary eosinophilia, idiopathic hypereosinophilic allergen-challenged non-human primates, using an syndrome, eosinophil leukaemia and Churg- monoclonal directed against the adhesion Strauss syndrome, which is a life-threatening con- molecule ICAM-1, also inhibited the development dition characterized by eosinophilic vasculitis, of airway hyperresponsiveness.'40 However, none asthma and a peripheral blood eosinophilia.'5' of these treatments is specific to the eosinophil. These conditions are of unknown aetiology. Man- Glucocorticoids, for example, probably act to a agement generally consists of treatment with high- large extent through inhibition of the release of dose oral glucocorticoids supplemented by chemo- eosinophil active cytokines from T-cells and therapy, if the condition is only partially respon- .'4' Airway eosinophilia can also occur sive, as is generally the case in HES and without asthma or airways hyperresponsiveness. 141 Churg-Strauss syndrome. More recently therapy For eosinophils to cause tissue damage in the with interferon a and y has been used with anec- airways, they need to be actively secreting their dotal success in HES. IL-5 antagonists, when they mediators. Measurements of eosinophilic basic become available, may also be effective. Two proteins may therefore be a better guide to the interesting and related conditions that have been degree of eosinophilic inflammation than described recently are toxic oil syndrome (TOS) eosinophil numbers. For example, Adelroth and and eosinophilia-myalgia syndrome.'52 Eosino- coworkers found that, whereas inhaled cor- philia-myalgia syndrome was first described in ticosteroids had no effect on the number of October 1989 in New Mexico and 1,500 cases were eosinophils in BAL fluid from asthmatics, they reported by mid-1990 with 27 deaths. It was causedcopyright. markedly reduced the amounts of ECP in lavage by ingestion of a batch of tryptophan and thought fluid.'43 to be due to a contaminant possibly 1, '-ethyl- idenebis (tryptophan). It was characterized by Eosinophils andparasitic disease severe myalgia in association with an eosinophil count ofgreater than 1 x 109/l with a median count Although infection with helminths is by far the of4-6 x I09/l. Patients also complained offatigue, commonest cause of a moderate to high shortness of breath, cough, rash and headache.

eosinophilia in association with parasites, Histopathologically there was a perivascular lym- http://pmj.bmj.com/ eosinophilia in association with protozoan infec- phocyte and eosinophilic infiltrate in the dermis, tions has been described and ectoparasites such as fascia and skeletal muscle with a pulmonary vas- head lice and scabies can produce a local culitis and alveolitis. The histological appearances eosinophilic reaction. The commoner helminthic were similar to eosinophilic fascilitis. Symptoms causes of an eosinophilia are summarized in Table were persistent and not very responsive to treat- III. '"6 Eosinophils have been shown to be able to ment with glucocorticoids and immunosuppres- kill a number of opsonized parasites including sants. TOS was an epidemic in Spain in 1981 caused newborn larvae of T. spiralis, larvae of Nippostro- by the ingestion of industrial rapeseed oil. A total on September 25, 2021 by guest. Protected gylus brasiliensis, a gut parasite in the rat, and F. of20,000 people were affected with 300 deaths. The hepatica as well as schistosomulae of S. mansoni.'47 acute phase of TOS was characterized by an adult In vivo parasite larvae become coated with specific respiratory distress syndrome-(ARDS) likc picture IgG and IgE antibodies and can activate comple- with a profound eosinophilia but the chronic phase ment. Dead larvae of S. haematobium and other resembled eosinophilia-myalgia syndrome both parasites have been detected surrounded by clinically and histopathologically. Fifty per cent of eosinophils and eosinophil granule products in the patients had complete resolution after 8 years. The skin.'48 Adult worms both in vitro and in vivo precise aetiological agent is unknown. appear resistant to eosinophil-mediated damage. Despite this circumstantial evidence for eosinophils being involved in host defence against parasites, there remains some doubt about their Eosinopenia role. Except for one study in the Gambia'" there is no obvious correlation between the degree of The normal eosinophil count is often low, although eosinophilia and protection against infection or in one study of over 20,000 patients only 24 had Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from EOSINOPHILS 547

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rA Ad C13 0 4- t3 cn 0 0 &.. .4 0 (U A Ad Q la rA 'a 0 C, 0 C, CIS 0 4... 0 Q ;,4 4- cd Q 4., .3t cis U C's E rA Q E cis 0 (U z Postgrad Med J: first published as 10.1136/pgmj.70.826.536 on 1 August 1994. Downloaded from 548 A.J. WARDLAW counts of less than 0.01 x l09/l. In each case this cell type, eosinophils utilize VLA-4/VCAM-l as an could be ascribed to the disease or treatment.3 adherence pathway and have a number of other Acute infections, treatment with glucocorticoids receptors not shared by neutrophils. These include and adrenaline decrease eosinophil counts. In recognition units for IgE (distinct from CD23), and contrast, P blockers inhibit adrenaline-induced receptors for IL-5, IL-3 and RANTES. Following eosinopenia and can cause a rise in the eosinophil stimulation with a variety of agents, eosinophils count. The mechanism by which eosinopenia preferentially elaborate LTC4 as the major 5- occurs in these circumstances is not fully under- lipoxygenase product of arachidonic acid and stood. Beeson and Bass found that acute infection produce substantial amounts ofPAF. Ofparticular in mice resulted in a rapid fall in eosinophil counts interest is the ability of eosinophils to synthesize a due to either margination or migration into tissue number of cytokines. Thus eosinophils have and a more prolonged eosinophilia due to inhibi- marked pro-inflammatory potential. tion of bone marrow production. A soluble There is now convincing evidence that eosino- mediator of greater than 30,000 kDa appeared to philia is T-cell dependent. The Th2-type cell, which be involved.'"3 There have been several isolated selectively secretes IL-5 and IL-4, seems particularly case reports of patients with absent eosinophils in involved. IL-5, IL-3 and GM-CSF are required for the blood and bone marrow.'54 Often the cause of eosinophil maturation, and cause activation and the eosinopenia was related to some prolonged survival of the mature cell. IL-5 is immunological problem. For example, in one case unique in that it promotes terminal differentiation it occurred after drug-induced agranulocytosis'l" of the committed eosinophil precursor and in vivo and in another there was a serum inhibitor of in mice appears to be sufficient on its own for eosinophil colony formation.'56 A rare disorder is eosinophil growth from uncommited stem cells. EPO deficiency, cases of which may be brought to IL-4 selectively upregulates VCAM-1 expression light by automatic counting that uses detection of on endothelial cells thus augmenting VLA-4- EPO to count eosinophils. EPO deficiency does not dependent eosinophil adhesion. The role of appear to have any adverse clinical conse- eosinophils in disease is complex but in general quences."'" their numbers are increased in helminthic parasitic disease and atopic and asthma. Eosinophilcopyright. products can produce many of the pathological Summary and conclusions features of asthma, and helminthic larvae coated with immunoglobulin or complement are part- Eosinophils are characterized by their unique cry- icularly susceptible to eosinophil-mediated stalloid granules that contain four basic proteins - cytotoxicity. Eosinopenia is often related to acute MBP, ECP, EDN and EPO. The cell has many inflammation or stress. common features with neutrophils but, unlike that http://pmj.bmj.com/

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