The Role of the Eosinophil in Asthma'? s. T. HOLGATE, W. R. ROCHE, and M. K. CHURCH After the initial description of the histochemical characteristics of the eosinophil by Paul Erhlich in 1879 (1), recognition that this cell has associations with asthma was soon appreciated. Some of the earlier histopathologic descriptions of bronchial tissue from patients who had died from asthma contain vivid descriptions of the heavy eosinophil infiltrate of the airway walls, and for many years clinicians have also recognized the presence of free eosinophils in the sputum of patients with asthma, particularly during acute exacerbations (2). The characteristic Charcot-Leyden crystals frequently observed in eosinophilenriched sputum represent a crystallized form of the enzyme lysolecithinase derived from degenerated eosinophils (3). Finally, the presence of increased numbers of eosinophils in the peripheral blood of patients with both atopic and nonatopic (intrinsic) asthma (4) provides further evidence implicating these cells in the pathogenesis of this form of disordered airway function.
Ontogeny of Eosinophils Along with other polymorphonuclear leukocytes, eosinophils originate from bone marrow precursor cells under the influence of a number of identified growth or colony-stimulating factors. These include interleukin (IL)-3 (5), IL-5 (6), and granulocyte macrophage colony-stimulating factor (GM-CSF) (7). In humans, injections of IL-2 also stimulate a marked eosinophilia (8). Evidence for increased bone marrow eosinophilopoiesis participating in the inflammatory processesof asthma is largelyindirect. A relationship has been reported between the peripheral blood eosinophil count and clinical disease activity. More recently, bronchial provocation of the airwaysof atopic asthmatic subjects with inhaled allergen has been shown to produce an initial peripheral blood eosinopenia. This is followed by an eosinophilia occurring approximately 12 to 18 h after the challenge, which in magnitude correlates with the degree of nonspecific bronchial responsiveness measured before challenge (9). Preformed Mediators The eosinophil's characteristic uptake of red dyes such as eosin is the result of highly basic proteins stored in the secretory granules. Four have been isolated and studied in detail. Major Basic Protein Major basic protein (MBP) is localized specifically to the crystalloid core of the granules, and it received its name because it comprises approximately 55070 of the granule's protein content. MBP also has been localized to the secretory granules of basophils, but in smaller amounts. MBP is a highly cationic 566
protein with a pI of 10.9. Human eosinophil MBP has a molecular weight of 1"\.110 kDa and comprises 117 amino acids, with nine half cysteines and both hydrophilic and hydrophobic domains. The basicity of MBP resides in the large number of arginine residues. The large number of sulphydryl groups confers upon this molecule the propensity to stick to surfaces (10). The cDNA for human MBP has been isolated and sequenced to show that the molecule is translated as a 25,200-dalton preproprotein, which is cleaved as it passes from the endoplasmic reticulum to the granule stores (11). The preproprotein represents a nontoxic precursor of MBP. MBP is toxic to the schistosomula of Schistosoma mansoni (12) and several other parasites in vitro including the larvae of Trichinella spiralis and the amastigotes and epimastigotes of Trypanosoma cruzi. Using a rabbit polyclonal antibody directed to human MBP, large amounts of this eosinophil product have been found in the submucosa, epithelium, and intraluminal mucus of the airways of patients who have died from asthma (13). The observations that MBP is cytotoxic towards human (14)and guinea pig (15) bronchial epithelium has focused attention on this eosinophil product in the pathogenesis of epithelial disruption in asthma (16). Both sputum (17)and bronchoalveolar (BAL) fluid (18) from patients with asthma contain increased concentrations of MBP with levels correlating with eosinophil content and disease activity. In vitro MBP is a secretogogue for human basophils and rat mast cells (19) but probably not for human mast cells. MBP also can bind irreversiblyto C4b3b on the surface of erythrocytes and, by an interaction with the coagulation pathway, inhibit wholeblood clotting (20).
Eosinophil Cationic Protein Eosinophil cationic protein (ECP) is a basic protein with a molecular weight of 21,000kD and is localized to the matrix of eosinophil granules (21). Its N-terminal 59 amino acid residues show close homology with human pancreatic ribonuclease (22). ECP is synthesized for granule storage as a single-chain 22kD protein that is subsequently processed to 18to 20 kD prior to granule storage (23). ECP has a pI > 11.0and contains 2.5 mol of zinc per mole of protein. When secreted, ECP undergoes a structural change that can be demonstrated using a specific monoclonal antibody (24). The secreted form of ECP has been detected in the neighborhood of activated eosinophils in the skin, gastrointestinal tract, heart, and spleen and shares with MBP cytotoxicity towards parasites (25). Although ECP has ribonuclease activity, this function does not appear to account for its cytolytic action,
although it could contribute towards its neurotoxicity when injected into the cerebrospinal fluid. Other functions ofECP include inhibition oflymphocyte proliferation (26), induction of ion channels in artificialliposomes (27), anticoagulation through binding of factor XI, and inhibition of streptokinase and heparin functions (28).
Eosinophil-derived Neurotoxin Eosinophil-derived neurotoxin (EDN) was named because it caused a characteristic neurologic lesion (the Gordon phenomenon) involving the cerebellum, pons, and spinal cord when injected into the cerebrospinal fluid or brain of rabbits or guinea pigs (29, 30). EDN has been purified to homogeneity and shown to have an estimated molecular weight of 17.4 kD. Structural homology with human ribonucleases indicate a common genetic origin (30),and it also has close homology with ECP. Although EDN is toxic to schistosomula it is less toxic than ECP in killing larvae of Trichinella (31). EDN has not been studied for its potentially damaging effect on bronchial epithelial cells. Eosinophil Peroxidase Eosinophil peroxidase (EPO) is a two-chained hemoprotein with a molecular weight of 71 to 77 kD (32). The enzyme is strongly basic with a pI> 11. The 49-kD heavy chain ofEPO has associated carbohydrate, whereas the 15kD light chain is slightly homologous with the light chain of myeloperoxidase (33). EPO is markedly inhibited by a 3-amino-l,2,4triazole, which means it can be assayed in the presence of myeloperoxidase (34). EPO in the presence of H 202 and iodide, chloride, or bromide will kill viruses, bacteria, parasites, fungi, and tumor cells, probably through the formation of hypohalous acid (35). Under similar circumstances it can also inactivate the leukotrienes. EPO is a secretogogue of mast cellsand basophils to which it binds with retention of enzymic activity (36). It can also prime macrophages for the more effectivekilling of microorganisms (37) and bind to the surface of neoplastic cells making them susceptible to macrophage-mediated cytolysis (38).
1 From the Immunopharmacology Group, Medicine I and Clinical Pharmacology, Southampton General Hospital, Southampton, United Kingdom. 2 Supported by a Program Grant from the Medical Research Council of Great Britain. 3 Correspondence and requests for reprints should be addressed to Professor S. T. Holgate, Immunopharmacology Group, Medicine I, Level D, Centre Block,- Southampton General Hospital, Southampton S09 4XY, UK.
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Other Preformed Mediators In addition to EPO and ribonucleases, the granules of eosinophils contain collagenase active against types 1 and 3 collagen. Acetylcholinesterase, catalase, arysulphatase, histaminase phospholipases B and D, alkaline phosphatase, and cathepsin G activities have also been described. These mediators along with the basic proteins are stored in association with chondroitin 4-sulphate (reviewed in 39).
superoxide. Eosinophil superoxide generation is tightly linked to the activation of the phosphatidylinositol cycle, thereby further emphasizing the importance of this biochemical pathway in the control of mediator functions in this cell (49).
Role of Eosinophils in Models of Asthma With the more widespread use of fiberoptic bronchoscopy as an investigative tool in Newly Generated Mediators asthma, a number of laboratories have reportHuman eosinophils contain microsomal cy- ed increased numbers of eosinophils recovclooxygenase along with terminal enzymes of ered by BAL (50-52) and in the aspirated muthis oxidative pathway leading to the generacus (53) in the absence of provocation. A step tion and release of prostaglandin (PG) E 2 and forward in understanding the cellular basis PGI 2 (prostacyclin). Human eosinophils also of asthma came when late-phase airway obpossess 5- and 15-lipoxygenase activity (40, struction after allergen challenge was shown 41). In contrast to human neutrophils, which to be accompanied by an increase in the numrelease the dihydroxy acid LTB4 as their ma- ber of eosinophils that could be recovered by jor 5-lipoxygenase product, human eosinoBAL and by showing increased concentrations phils preferentially generate and secrete LTC4 of ECP in the fluid phase indicative of secreand only a small amount of LTB4 (42). In ac- tion of eosinophil granule products (50). Durcordance with the concept of leukocyte priming or after allergen-induced late-phase obing, the ability of eosinophils to release LTC4 struction other investigators have shown eois greatly enhanced if the cells have had prior sinophil recruitment into the bronchoalveolar exposure to a chemotactic stimulus such as lumen together with a variable number of neuLTB4 or platelet-activating factor (PAF) or trophils (50,51). Wardlaw and coworkers (52) are preactivated by a variety of factors such report a positive correlation between the reas IL-5, IL-3, and GM-CSF (42). sponse of the airways to inhaled methachoHuman eosinophils possess the necessary line and both eosinophil number and MBP acetyl transferase enzyme required for the syn- content of BAL, supporting the view that thesis of PAF from the plasmalogen precur- these polymorphs participate actively in the sor,lyso-PAF (43). Primed eosinophils gener- mucosal inflammation of asthma. ate substantial quantities of PAF both with Further light on the potential role of eoan IgG and an IgE signal (44), although the sinophils in the pathogenesis of late-phase inamount of this mediator released from the flammatory events after allergen provocation cell constitutes only 5 to 10070 of that synthehas been gained from studies in animals. One sized. In addition to being released from ac- model that has provided interesting new intivated eosinophils, PAF is a potent secretoformation regarding the role of eosinophils gogue for this leukocyte, with cells obtained in late-phase inflammatory responses in the from the peripheral blood of asthmatic paairways is the guinea pig. In the version of tients being more susceptible than those from this model that we have described (54) guinea normal control subjects (45). Recent work pigs are exposed on two occasions, 1 wk apart, suggests that PAF-induced mediator secretion to aerosolized ovalbumin, which is a suffirequires an interaction with specific receptors cient stimulus to sensitize the airways and renon eosinophils, which may be competitively der then hyperresponsive to inhaled histamine antagonized with the selective antagonist and methacholine (55). When nonanestheWEB-2086 (46). tized animals are challenged 1 wk later with Human eosinophils share with dispersed inhaled ovalbumin under the protection of tracheal epithelial cells the capacity to gener- an antihistamine, both early and late phases ate monohydroxy and dihydroxy acids of ar- of airway obstruction occur that can be measured by whole-body plethysmography. In this achidonic acid using the 15-lipoxygenasepathway (47). Although there is some doubt as model the late-phase changes in airway resisto whether 15-lipoxygenase is activated in eo- tance are accompanied by an influx of granusinophils under physiologic conditions, the lar leukocytes into the airways (54). BAL unpresence of detectable concentrations of 15- dertaken at the onset of the first late reaction hydroxyeicosatetranoeic acid (15-HETE) in has demonstrated predominantly neutrophils, BAL fluid from patients with atopic asthma with numbers reaching maximum at 17hand declining to baseline by 72 h. The neutrophilia and as much as a 3-fold increase in the concentration of this mediator occurring after is followed by an eosinophilia that progresallergen challenge (48) indicates its in vivo sivelyincreases between 17and 72 h after chalgeneration in the inflamed asthmatic airway. lenge, at which time eosinophils constitute In common with other granulocytes, eo- almost half of the total nucleated cells recovsinophils have an active pentose monophos- ered by BAL. Light microscopy of the lung phate shunt for the generation of reactive spe- confirms the presence of extensive granulocies of oxygen. Almost any stimulus that cyte infiltration of the submucosal and epireleases granule-derived and newly generated thelial region. By transmission of electron milipid mediators from this cell also generate croscopy these changes are accompanied by
degranulation of goblet cells and the formation of intraluminal mucus plugs. A cytotoxic serum specific for guinea pig neutrophils that produces a peripheral blood neutropenia has been shown to ablate the influx of neutrophils but not eosinophils into the airways, but it has no significant effect either on the earlyor late-phase airway events after challenge (56). Thus, in this model, it is likely that eosinophils rather than neutrophils serve as important mediator-secreting cells, but final proof of this will have to await the use of a specificantieosinophil antiserum. Eosinophils have also been reported as components of the leukocyte infiltrate of the airways during latephase responses in sheep (57) and in rabbits (58), but their functional significance has not been fully evaluated. In a number of animal models of late-phase reactions, the PAF antagonists BN 52063 or WEB 2086 administered prior to challenge have been shown to attenuate both the latephase pulmonary mechanical response and the eosinophil influx into the airways (59, 60). In our guinea-pig model administration of WEB 2086 prior to allergen challenge foreshortened the first late reaction and reduced eosinophil influx at 17 h postchallenge (60). If the drug is administered between the earlyand late-phase reactions during the active phase of leukocyte recruitment into the airways, then both the late-phase changes in airway mechanics and the delayed influx of eosinophil leukocytes at 72 h are suppressed. Although WEB 2086 has a short duration of action in this animal species, these phamacologic studies provide some evidence that PAF is a candidate as a chemotactic mediator for the recruitment and activation of eosinophils. Eosinophils in Day-to-Day Asthma Although there is overwhelming evidence demonstrating massive influx of eosinophils into the inflamed airways of patients who have died from asthma (2, 3), evidence incriminating these cellsin day-to-day asthma is limited. In an attempt to address this deficiency we have undertaken fiberoptic bronchoscopy in a group of eight subjects with mild atopic asthma and three nonasthmatic control subjects to compare the BAL with endobronchial biopsy findings (61). The asthmatic subjects were young, atopic, and had hyperresponsive airways measured by histamine provocation (geometric mean PC2 0 FEV h 0.9 mg/rnl), Their baseline airway caliber measured as FEV 1 ranged from 70 to 130% of predicted, and their treatment requirements wereinhaled ~radrenoreceptor agonists alone when needed (n = 6) or no treatment (n = 2). The control subjects also were young males with a geometric mean PC 20 histamine of > 32 mg/ml and normal baseline values of FEV r- When the BAL between the control and the asthmatic subjects were compared, the latter contained more eosinophils and a 5-fold greater number of bronchial epithelial cells. The epithelial cells appeared in clumps, possessed functional cilia, and had intact intercellular
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HOLGATE, ROCHE, AND CHURCH
Fig. 1. Transmission electron micrograph of the basement membrane region of the bronchial epithelium of a normal subject (left panel) and a subject with mild asthma (right panel) (uranyl acetate-lead citrate; x 5,000).
tight junctions. An inversecorrelation was observed between the number of BAL epithelial cells and the log PC 20 histamine (rho = 0.64, p = 0.03), supporting the view that in asthma there is defective adherence of epithelium to its basement membrane (16) and that this abnormality may have a bearing on the pathogenesis of bronchial hyperresponsiveness. The ultrastructural appearance of the endobronchial biopsy obtained from the nonasthmatic control subjects showed the characteristic pseudostratified ciliated epithelium attached on a thin basement membrane beneath which there was a narrow submucosal space containing a rich sinusoidal vascular network (figure 1). Although the submucosa contained stromal cells and a small amount of supporting collagen, polymorphonuclear leukocytes were notably absent. All of the bronchial biopsies of the subjects with mild asthma wereabnormal. Although we wereunable to comment about the integrity of the epithelium because of the small size of the biopsies obtained, areas of normal or even hyperplastic epithelium were frequently seen. Beneath the epithelium all the biopsies showed extensive thickening of the basement membrane region (figure 1).Using monoclonal and polyclonal antibodies as immunohistochem-
ical reagents, the true basement membrane wasseen to be of normal thickness and stained positively for type 4 collagen and laminin. The apparent "thickening" of the subbasement membrane region was due to the deposition of types 3 and 5 collagen together with fibronectin, suggesting a fibroblast origin. Types 3 and 5 collagen also were seen to be extensively deposited throughout the submucosal region and extending into the smooth muscle, a somewhat surprising finding considering the apparent mild state of the asthma being investigated. In the biopsies from the normal subjects, mast cells wereseen mostly in relation to basement membrane and submucosal region where they exhibited characteristic granule structures in the form of scrolls. In the asthma biopsies, mast cells were also seen in the subepithelial region in abundance, but they invariably showed evidence of degranulation. One of the most striking features of the asthma biopsies was the large number of eosinophils seen to infiltrate the submucosal and sub-basement membrane region. Transmission electron microscopy demonstrated granule heterogeneity, some showing selective loss of the major basic protein containing crystalloid core, whereas others showing preferential loss of granule matrix. These ultrastructural changes
are compatible with the eosinophil being in an "activated" form. Eosinophils were observed in clumps beneath the basement membrane, in association with strands of collagen, in which some appeared to be entombed, and between epithelial cells. They also were observed in increased numbers within postcapillary venules where they had a close relationship with the endothelial cells suggestive of integrin-mediated adherence (62). Thus, the evidence from these endobronchial biopsies in mild atopic asthma supports the view that eosinophils are selectively recruited into the airways from the bloodstream. No correlation could be established between the number of eosinophils in the submucosa and the measured level of bronchial responsiveness. Infiltration with neutrophils was not a prominent feature of the biopsies.
Conclusions In asthma associated with allergen exposure, there is strong evidence for eosinophils playing an active role in pathogenesis. Although the eosinophil has been linked to the pathogenesis of airway hyperresponsiveness in asthma, possibly through disrupting the epithelium, final proof of this as a pathogenetic mechanism is still lacking. Nevertheless, this unique granulocyte, with its capacity to selec-
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THE ROLE OF THE EOSINOPHIL IN ASTHMA
tively infiltrate the airways in asthma and to show evidence of proinflammatory mediator release, is likely to playa key role in the pathogenesis of certain forms of this disease, and with increased understanding of its functions, it may provide an important therapeutic target for antiasthmatic drugs.
Acknowledgment The writers would like to acknowledge Mr. Christopher Inman for preparing the transmission electron microscopy figures and M. Dowling for typing the manuscript.
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59. Stevenson lS, Tallant M, Blinder L, Abraham WM. Modification of antigen-induced late response with an antagonist of platelet-activating factor (WEB 2086). Fed Proc 1987; 46:6683. 60. Hutson PA, Holgate ST, Church MK. Effect of WEB 2086 on early and late airways response to ovalbumin challenge in conscious guinea-pigs. Br 1 Pharmacol 1988; 95(Suppl:770). 61. Beasley R, Roche WR, Roberts lA, Holgate ST. Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis 1989; 139:806-17. 62. Kimani G, Tonnesen MG, Henson PM. Stimulation of eosinophil adherence to human vascular endothelial cells in vitro by platelet activating factor. 1 Immunol 1988; 140:3161-6.
Ontogeny of Eosinophils Along with other polymorphonuclear leukocytes, eosinophils originate from bone marrow precursor cells under the influence of a number of identified growth or colony-stimulating factors. These include interleukin (IL)-3 (5), IL-5 (6), and granulocyte macrophage colony-stimulating factor (GM-CSF) (7). In humans, injections of IL-2 also stimulate a marked eosinophilia (8). Evidence for increased bone marrow eosinophilopoiesis participating in the inflammatory processesof asthma is largelyindirect. A relationship has been reported between the peripheral blood eosinophil count and clinical disease activity. More recently, bronchial provocation of the airwaysof atopic asthmatic subjects with inhaled allergen has been shown to produce an initial peripheral blood eosinopenia. This is followed by an eosinophilia occurring approximately 12 to 18 h after the challenge, which in magnitude correlates with the degree of nonspecific bronchial responsiveness measured before challenge (9). Preformed Mediators The eosinophil's characteristic uptake of red dyes such as eosin is the result of highly basic proteins stored in the secretory granules. Four have been isolated and studied in detail. Major Basic Protein Major basic protein (MBP) is localized specifically to the crystalloid core of the granules, and it received its name because it comprises approximately 55070 of the granule's protein content. MBP also has been localized to the secretory granules of basophils, but in smaller amounts. MBP is a highly cationic 566
protein with a pI of 10.9. Human eosinophil MBP has a molecular weight of 1"\.110 kDa and comprises 117 amino acids, with nine half cysteines and both hydrophilic and hydrophobic domains. The basicity of MBP resides in the large number of arginine residues. The large number of sulphydryl groups confers upon this molecule the propensity to stick to surfaces (10). The cDNA for human MBP has been isolated and sequenced to show that the molecule is translated as a 25,200-dalton preproprotein, which is cleaved as it passes from the endoplasmic reticulum to the granule stores (11). The preproprotein represents a nontoxic precursor of MBP. MBP is toxic to the schistosomula of Schistosoma mansoni (12) and several other parasites in vitro including the larvae of Trichinella spiralis and the amastigotes and epimastigotes of Trypanosoma cruzi. Using a rabbit polyclonal antibody directed to human MBP, large amounts of this eosinophil product have been found in the submucosa, epithelium, and intraluminal mucus of the airways of patients who have died from asthma (13). The observations that MBP is cytotoxic towards human (14)and guinea pig (15) bronchial epithelium has focused attention on this eosinophil product in the pathogenesis of epithelial disruption in asthma (16). Both sputum (17)and bronchoalveolar (BAL) fluid (18) from patients with asthma contain increased concentrations of MBP with levels correlating with eosinophil content and disease activity. In vitro MBP is a secretogogue for human basophils and rat mast cells (19) but probably not for human mast cells. MBP also can bind irreversiblyto C4b3b on the surface of erythrocytes and, by an interaction with the coagulation pathway, inhibit wholeblood clotting (20).
Eosinophil Cationic Protein Eosinophil cationic protein (ECP) is a basic protein with a molecular weight of 21,000kD and is localized to the matrix of eosinophil granules (21). Its N-terminal 59 amino acid residues show close homology with human pancreatic ribonuclease (22). ECP is synthesized for granule storage as a single-chain 22kD protein that is subsequently processed to 18to 20 kD prior to granule storage (23). ECP has a pI > 11.0and contains 2.5 mol of zinc per mole of protein. When secreted, ECP undergoes a structural change that can be demonstrated using a specific monoclonal antibody (24). The secreted form of ECP has been detected in the neighborhood of activated eosinophils in the skin, gastrointestinal tract, heart, and spleen and shares with MBP cytotoxicity towards parasites (25). Although ECP has ribonuclease activity, this function does not appear to account for its cytolytic action,
although it could contribute towards its neurotoxicity when injected into the cerebrospinal fluid. Other functions ofECP include inhibition oflymphocyte proliferation (26), induction of ion channels in artificialliposomes (27), anticoagulation through binding of factor XI, and inhibition of streptokinase and heparin functions (28).
Eosinophil-derived Neurotoxin Eosinophil-derived neurotoxin (EDN) was named because it caused a characteristic neurologic lesion (the Gordon phenomenon) involving the cerebellum, pons, and spinal cord when injected into the cerebrospinal fluid or brain of rabbits or guinea pigs (29, 30). EDN has been purified to homogeneity and shown to have an estimated molecular weight of 17.4 kD. Structural homology with human ribonucleases indicate a common genetic origin (30),and it also has close homology with ECP. Although EDN is toxic to schistosomula it is less toxic than ECP in killing larvae of Trichinella (31). EDN has not been studied for its potentially damaging effect on bronchial epithelial cells. Eosinophil Peroxidase Eosinophil peroxidase (EPO) is a two-chained hemoprotein with a molecular weight of 71 to 77 kD (32). The enzyme is strongly basic with a pI> 11. The 49-kD heavy chain ofEPO has associated carbohydrate, whereas the 15kD light chain is slightly homologous with the light chain of myeloperoxidase (33). EPO is markedly inhibited by a 3-amino-l,2,4triazole, which means it can be assayed in the presence of myeloperoxidase (34). EPO in the presence of H 202 and iodide, chloride, or bromide will kill viruses, bacteria, parasites, fungi, and tumor cells, probably through the formation of hypohalous acid (35). Under similar circumstances it can also inactivate the leukotrienes. EPO is a secretogogue of mast cellsand basophils to which it binds with retention of enzymic activity (36). It can also prime macrophages for the more effectivekilling of microorganisms (37) and bind to the surface of neoplastic cells making them susceptible to macrophage-mediated cytolysis (38).
1 From the Immunopharmacology Group, Medicine I and Clinical Pharmacology, Southampton General Hospital, Southampton, United Kingdom. 2 Supported by a Program Grant from the Medical Research Council of Great Britain. 3 Correspondence and requests for reprints should be addressed to Professor S. T. Holgate, Immunopharmacology Group, Medicine I, Level D, Centre Block,- Southampton General Hospital, Southampton S09 4XY, UK.
AM REV RE5PIR DI5 1991; 143:566-570
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THE ROLE OF THE EOSINOPHIL IN ASTHMA
Other Preformed Mediators In addition to EPO and ribonucleases, the granules of eosinophils contain collagenase active against types 1 and 3 collagen. Acetylcholinesterase, catalase, arysulphatase, histaminase phospholipases B and D, alkaline phosphatase, and cathepsin G activities have also been described. These mediators along with the basic proteins are stored in association with chondroitin 4-sulphate (reviewed in 39).
superoxide. Eosinophil superoxide generation is tightly linked to the activation of the phosphatidylinositol cycle, thereby further emphasizing the importance of this biochemical pathway in the control of mediator functions in this cell (49).
Role of Eosinophils in Models of Asthma With the more widespread use of fiberoptic bronchoscopy as an investigative tool in Newly Generated Mediators asthma, a number of laboratories have reportHuman eosinophils contain microsomal cy- ed increased numbers of eosinophils recovclooxygenase along with terminal enzymes of ered by BAL (50-52) and in the aspirated muthis oxidative pathway leading to the generacus (53) in the absence of provocation. A step tion and release of prostaglandin (PG) E 2 and forward in understanding the cellular basis PGI 2 (prostacyclin). Human eosinophils also of asthma came when late-phase airway obpossess 5- and 15-lipoxygenase activity (40, struction after allergen challenge was shown 41). In contrast to human neutrophils, which to be accompanied by an increase in the numrelease the dihydroxy acid LTB4 as their ma- ber of eosinophils that could be recovered by jor 5-lipoxygenase product, human eosinoBAL and by showing increased concentrations phils preferentially generate and secrete LTC4 of ECP in the fluid phase indicative of secreand only a small amount of LTB4 (42). In ac- tion of eosinophil granule products (50). Durcordance with the concept of leukocyte priming or after allergen-induced late-phase obing, the ability of eosinophils to release LTC4 struction other investigators have shown eois greatly enhanced if the cells have had prior sinophil recruitment into the bronchoalveolar exposure to a chemotactic stimulus such as lumen together with a variable number of neuLTB4 or platelet-activating factor (PAF) or trophils (50,51). Wardlaw and coworkers (52) are preactivated by a variety of factors such report a positive correlation between the reas IL-5, IL-3, and GM-CSF (42). sponse of the airways to inhaled methachoHuman eosinophils possess the necessary line and both eosinophil number and MBP acetyl transferase enzyme required for the syn- content of BAL, supporting the view that thesis of PAF from the plasmalogen precur- these polymorphs participate actively in the sor,lyso-PAF (43). Primed eosinophils gener- mucosal inflammation of asthma. ate substantial quantities of PAF both with Further light on the potential role of eoan IgG and an IgE signal (44), although the sinophils in the pathogenesis of late-phase inamount of this mediator released from the flammatory events after allergen provocation cell constitutes only 5 to 10070 of that synthehas been gained from studies in animals. One sized. In addition to being released from ac- model that has provided interesting new intivated eosinophils, PAF is a potent secretoformation regarding the role of eosinophils gogue for this leukocyte, with cells obtained in late-phase inflammatory responses in the from the peripheral blood of asthmatic paairways is the guinea pig. In the version of tients being more susceptible than those from this model that we have described (54) guinea normal control subjects (45). Recent work pigs are exposed on two occasions, 1 wk apart, suggests that PAF-induced mediator secretion to aerosolized ovalbumin, which is a suffirequires an interaction with specific receptors cient stimulus to sensitize the airways and renon eosinophils, which may be competitively der then hyperresponsive to inhaled histamine antagonized with the selective antagonist and methacholine (55). When nonanestheWEB-2086 (46). tized animals are challenged 1 wk later with Human eosinophils share with dispersed inhaled ovalbumin under the protection of tracheal epithelial cells the capacity to gener- an antihistamine, both early and late phases ate monohydroxy and dihydroxy acids of ar- of airway obstruction occur that can be measured by whole-body plethysmography. In this achidonic acid using the 15-lipoxygenasepathway (47). Although there is some doubt as model the late-phase changes in airway resisto whether 15-lipoxygenase is activated in eo- tance are accompanied by an influx of granusinophils under physiologic conditions, the lar leukocytes into the airways (54). BAL unpresence of detectable concentrations of 15- dertaken at the onset of the first late reaction hydroxyeicosatetranoeic acid (15-HETE) in has demonstrated predominantly neutrophils, BAL fluid from patients with atopic asthma with numbers reaching maximum at 17hand declining to baseline by 72 h. The neutrophilia and as much as a 3-fold increase in the concentration of this mediator occurring after is followed by an eosinophilia that progresallergen challenge (48) indicates its in vivo sivelyincreases between 17and 72 h after chalgeneration in the inflamed asthmatic airway. lenge, at which time eosinophils constitute In common with other granulocytes, eo- almost half of the total nucleated cells recovsinophils have an active pentose monophos- ered by BAL. Light microscopy of the lung phate shunt for the generation of reactive spe- confirms the presence of extensive granulocies of oxygen. Almost any stimulus that cyte infiltration of the submucosal and epireleases granule-derived and newly generated thelial region. By transmission of electron milipid mediators from this cell also generate croscopy these changes are accompanied by
degranulation of goblet cells and the formation of intraluminal mucus plugs. A cytotoxic serum specific for guinea pig neutrophils that produces a peripheral blood neutropenia has been shown to ablate the influx of neutrophils but not eosinophils into the airways, but it has no significant effect either on the earlyor late-phase airway events after challenge (56). Thus, in this model, it is likely that eosinophils rather than neutrophils serve as important mediator-secreting cells, but final proof of this will have to await the use of a specificantieosinophil antiserum. Eosinophils have also been reported as components of the leukocyte infiltrate of the airways during latephase responses in sheep (57) and in rabbits (58), but their functional significance has not been fully evaluated. In a number of animal models of late-phase reactions, the PAF antagonists BN 52063 or WEB 2086 administered prior to challenge have been shown to attenuate both the latephase pulmonary mechanical response and the eosinophil influx into the airways (59, 60). In our guinea-pig model administration of WEB 2086 prior to allergen challenge foreshortened the first late reaction and reduced eosinophil influx at 17 h postchallenge (60). If the drug is administered between the earlyand late-phase reactions during the active phase of leukocyte recruitment into the airways, then both the late-phase changes in airway mechanics and the delayed influx of eosinophil leukocytes at 72 h are suppressed. Although WEB 2086 has a short duration of action in this animal species, these phamacologic studies provide some evidence that PAF is a candidate as a chemotactic mediator for the recruitment and activation of eosinophils. Eosinophils in Day-to-Day Asthma Although there is overwhelming evidence demonstrating massive influx of eosinophils into the inflamed airways of patients who have died from asthma (2, 3), evidence incriminating these cellsin day-to-day asthma is limited. In an attempt to address this deficiency we have undertaken fiberoptic bronchoscopy in a group of eight subjects with mild atopic asthma and three nonasthmatic control subjects to compare the BAL with endobronchial biopsy findings (61). The asthmatic subjects were young, atopic, and had hyperresponsive airways measured by histamine provocation (geometric mean PC2 0 FEV h 0.9 mg/rnl), Their baseline airway caliber measured as FEV 1 ranged from 70 to 130% of predicted, and their treatment requirements wereinhaled ~radrenoreceptor agonists alone when needed (n = 6) or no treatment (n = 2). The control subjects also were young males with a geometric mean PC 20 histamine of > 32 mg/ml and normal baseline values of FEV r- When the BAL between the control and the asthmatic subjects were compared, the latter contained more eosinophils and a 5-fold greater number of bronchial epithelial cells. The epithelial cells appeared in clumps, possessed functional cilia, and had intact intercellular
568
HOLGATE, ROCHE, AND CHURCH
Fig. 1. Transmission electron micrograph of the basement membrane region of the bronchial epithelium of a normal subject (left panel) and a subject with mild asthma (right panel) (uranyl acetate-lead citrate; x 5,000).
tight junctions. An inversecorrelation was observed between the number of BAL epithelial cells and the log PC 20 histamine (rho = 0.64, p = 0.03), supporting the view that in asthma there is defective adherence of epithelium to its basement membrane (16) and that this abnormality may have a bearing on the pathogenesis of bronchial hyperresponsiveness. The ultrastructural appearance of the endobronchial biopsy obtained from the nonasthmatic control subjects showed the characteristic pseudostratified ciliated epithelium attached on a thin basement membrane beneath which there was a narrow submucosal space containing a rich sinusoidal vascular network (figure 1). Although the submucosa contained stromal cells and a small amount of supporting collagen, polymorphonuclear leukocytes were notably absent. All of the bronchial biopsies of the subjects with mild asthma wereabnormal. Although we wereunable to comment about the integrity of the epithelium because of the small size of the biopsies obtained, areas of normal or even hyperplastic epithelium were frequently seen. Beneath the epithelium all the biopsies showed extensive thickening of the basement membrane region (figure 1).Using monoclonal and polyclonal antibodies as immunohistochem-
ical reagents, the true basement membrane wasseen to be of normal thickness and stained positively for type 4 collagen and laminin. The apparent "thickening" of the subbasement membrane region was due to the deposition of types 3 and 5 collagen together with fibronectin, suggesting a fibroblast origin. Types 3 and 5 collagen also were seen to be extensively deposited throughout the submucosal region and extending into the smooth muscle, a somewhat surprising finding considering the apparent mild state of the asthma being investigated. In the biopsies from the normal subjects, mast cells wereseen mostly in relation to basement membrane and submucosal region where they exhibited characteristic granule structures in the form of scrolls. In the asthma biopsies, mast cells were also seen in the subepithelial region in abundance, but they invariably showed evidence of degranulation. One of the most striking features of the asthma biopsies was the large number of eosinophils seen to infiltrate the submucosal and sub-basement membrane region. Transmission electron microscopy demonstrated granule heterogeneity, some showing selective loss of the major basic protein containing crystalloid core, whereas others showing preferential loss of granule matrix. These ultrastructural changes
are compatible with the eosinophil being in an "activated" form. Eosinophils were observed in clumps beneath the basement membrane, in association with strands of collagen, in which some appeared to be entombed, and between epithelial cells. They also were observed in increased numbers within postcapillary venules where they had a close relationship with the endothelial cells suggestive of integrin-mediated adherence (62). Thus, the evidence from these endobronchial biopsies in mild atopic asthma supports the view that eosinophils are selectively recruited into the airways from the bloodstream. No correlation could be established between the number of eosinophils in the submucosa and the measured level of bronchial responsiveness. Infiltration with neutrophils was not a prominent feature of the biopsies.
Conclusions In asthma associated with allergen exposure, there is strong evidence for eosinophils playing an active role in pathogenesis. Although the eosinophil has been linked to the pathogenesis of airway hyperresponsiveness in asthma, possibly through disrupting the epithelium, final proof of this as a pathogenetic mechanism is still lacking. Nevertheless, this unique granulocyte, with its capacity to selec-
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THE ROLE OF THE EOSINOPHIL IN ASTHMA
tively infiltrate the airways in asthma and to show evidence of proinflammatory mediator release, is likely to playa key role in the pathogenesis of certain forms of this disease, and with increased understanding of its functions, it may provide an important therapeutic target for antiasthmatic drugs.
Acknowledgment The writers would like to acknowledge Mr. Christopher Inman for preparing the transmission electron microscopy figures and M. Dowling for typing the manuscript.
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