- Email: [email protected]
Eosinophils 1992
TRENDS
Eosinophils 1992 Christopher J.F. Spry, A. Barry Kay and Gerald J. Gleich
Four proteins make up the bulk of human eosinophil granules. Two of them, the eosinophil ribonucleases (eosinophil cationic protein (ECP) and eosinophil neurotoxin/protein X (EDN)) are closely related to the nonsecretory hepatic ribonucleases in structure and substrate specificity, and are distinct from pancreatic ribonuclease. ECP is more toxic than EDN, and the RNAase activity of these proteins is involved in their neurotoxicity (G. Gleich, Rochester). However, it is not known why there are two such closely related ribonucleases in eosinophil granules, or what role they play in host protection, although it is assumed that this relates to parasite immunity. Eosinophil major basic protein (MBP) can damage a variety of cells, and it has been thought to be the principal effector protein in many of the beneficial and harmful effects of eosinophils in disease. Guinea pigs have two different forms of MBP. Although there seems to be only one MBP gene in humans, MBP transcripts can undergo differential splicing (C. Spry, London). The kinetics and molecular basis of membrane damage by MBP has been studied recently using liposomes made up of acidic lipids, Eosinophil peroxidase (EPO) can injure cells and tissues directly, in addition to its capacity to produce reactive species with its preferred thiocyanate substrate (G. Gleich, Rochester). The Charcot Leyden crystal (CLC) protein, which is stored in the recently described 'primary' granules in eosinophils (Ann Dvorak, Boston), comprises up to 10% of the total protein in human eosinophils. CLC, which has been expressed in cos cells, belongs to the 'S-type' lectin superfamily, with homologies to rat immunoglobulin
This report from a recent meeting* highlights some of the key developments in eosinophil research and assesses their impact on current views of the role of eosinophils in inflammation. Considerable progress has been made in defining the structure, constituents and properties of eosinophils in vitro and these new insights into eosinophil biology are summarized. A new era is beginning in which the roles of eosinophils can be assessed and defined in vivo.
wise alterations in membrane capacitance as granules fuse with the plasma membrane or during endocytosis. A variety of pharmacological studies have suggested ways to prevent or reduce eosinophil activation and secretion (P. Barnes, London). Inhibitors have been found to reduce the responses of adrenoceptor agonists, pbosphodiesterase-mediated responses, phospholipase D and prostaglandin E2 (PGE2) production. Steroids seem to have few direct effects on (Ig)E-binding protein (S. Ackerman, eosinophils, although they have a Boston). Multicentre studies are in marked indirect effect by reducing progress to assess the possible roles cytokine production by lymphoof CLC protein in respiratory cytes. disease, on the basis that CLC Platelet activation factor (PAF) is protein may neutralize natural lung produced by stimulated eosinophils surfactants, and cause atelectasis. and has marked autocrine effects on them. It stimulates 02 uptake Secretion and signal transduction and superoxide release. It raises Regulation of the secretion of Ca 2+ and diacylglycerol levels and eosinophil granule proteins is increases the binding affinity of the complex, as shown by the range complement component iC3b. of responses of eosinophils to These findings show that PAF is stimulation: certain events take one of the most important sigonly a few seconds, while others nalling systems for converting peak several hours after stimu- blood eosinophils to their activated lation. The use of specific G- form (D. Roos, Amsterdam). protein inhibitors has shown that Endothelial cells are probably one protein (tyrosine) phosphorylation of the main producers of PAF to is an important regulatory step for activate eosinophils in inflamsome of these responses. O. mation (W. Busse, Madison). Cromwell (London) suggested that Granulocyte-macrophage colonyeosinophil 'activation' is the result stimulating factor (GM-CSF) inof enhanced or persistent phos- creases the synthesis of PAF by phorylation of signal-transducing eosinophils, but other factors are proteins. The process of exocytosis needed to induce PAF secretion of granule proteins, which is more from them (A. Lopez, Adelaide). pronounced in rodent than in The way in which secretion human eosinophils, has been occurs in vivo is still being investistudied by patch-clamp techniques; gated. One newly described type of these studies have revealed step- granule release, termed 'piecemeal' degranulation by A. Dvorak, is as*Eosinophils 1992 was organized by sociated with vesicular transport. She Barry Kay and Gerald Gleich. It was also described a spatial relationship held at Adare, Ireland on 1-3 June between eosinophils and nerve cells 1992. The proceedings will be in tissues, although the significance published by Marcel Dekker. of this has still to be discovered. 1992, Elsevier Science Publishers Ltd, UK.
Immu,otogy today 3 8 4
vot. 13 No. I0 1992
TRENDS
Some eosinophils have receptors for IgA. It is now clear that crosslinked IgA, crosslinked secretory piece or secretory IgA (slgA) itself can stimulate eosinophils to secrete ECP and EPO (M. Capron, Lille). This may explain the presence of large numbers of eosinophils at mucosal surfaces where slgA is secreted. Capron's group, working with Po-Cbun Tai (who died in 1990, and whose many contributions to eosinophil research were remembered at the meeting), have shown a marked difference in the amounts of ECP and EPO secreted in response to IgE, IgG and IgA. This implies that there are separate transduction pathways for these signals, and/or distinct secretion pathways for the granule proteins. Some morphological evidence has been reported explaining this fine control of the functions of eosinophils.
and very late antigen (VLA-4)) on the loosely-bound eosinophils. Increased expression of adhesion molecules may take several hours to become fully effective, but the time course in vivo is unknown. Integrins are the: principal attachment sites on eosinophils for intracellular adhesion molecule 1 (ICAM-1), which is constitutively expressed on endothelial cells. IL-4 also induces the expression of vascular cell adhesion molecule 1 (VCAM-I) on endothelial cells, which binds to VLA-4 on eosinophils, providing specificity for eosinophil localization - neutrophils do not express VLA-4. This process may account for the accumulation of eosinophils in IL4-secreting tumours. Eosinophils also have VLA-6 adhesion receptors. The CD 11-CD 18 mCecules on eosinophils are required for them to emigrate into tissues.
Adhesion and emigration The localization of eosinophils in tissues is now known to involve families of adhesion molecules that are induced on circulating eosinophils and endothelial cells during inflammation (M. Vadas (Adelaide), A. Wardlaw (Leicester) and R. Schleimmer and B. Bochner (Baltimore). The process starts with a weak and reversible binding of eosinophil membrane sialyl Lewis X to selectins on postcapillary endothelial cells. Selectins in serum compete for this binding, so that eosinophils can 'roll' over the surface of endothelial cells. L-selectins can also be shed from the surface of eosinophils, and when this occurs in vitro eosinophils can no longer migrate across endothelial cell monolayers. Inflammatory mediators signal endothelial cells to transform this loose binding to a firm attachment; for example, endothelial-leucocyte adhesion molecule 1 (ELAM-1)is induced on endothelial cells, which then have stronger attachment sites for sialated Lewis X. lnterleukin 1 (IL-1) induces the release of PAF and other activating molecules from endothelial cells which, in turn, induce the expression of integrins (lymphocyte function associated molecule 1 (LFA-1), Mac-1
Activation CD25, the 13 chain of the IL-2 receptor, is expressed on eosinophils (Weller) but only following stimulation ((;apron). The expression of several other membrane antigens and receptors is also increased by stimulation: gammainterferon (IFN-y)-induced FcyRI and Fc~'RIII. Activated eosinophils also express CD69 (a membrane antigen found on activated T cells), and HLA-DR molecules (Wardlaw). Cultured eosinophils express HLA-DR molecules and can present antigens to T cells in an MHCrestricted manner as effectively as macrophages (Weller). Additional lipid bodies, five of which are normally found in each eosinophil, are induced by protein kinase C activating agents, but not by IL-5 or GM-CSF. These densely osmophilic bodies, which are not membrane bound, contain large amounts of arachidonic acid and enzymes involved in the prostaglandin and lipoxygenase synthesis pathways, suggesting that this is the principal site for their production in eosinophils (Weller). Lipid bodies also contain peroxidase (J. Abrams, Palo Alto). Mature eosinophils express C D l l b - C D 1 8 and receptors with C-type (FceRII, also known as
Immunology 7~day
385
CD23) and S-type (Mac2) lectin domains. Mac2 binds IgE and is involved in IgE-dependent eosinophil-mediated cytotoxicity, mRNA for Mac2 is present in human eosinophils, showing that the cells synthesize it themselves. Large amounts of soluble CD23 are found in the serum of patients with the idiopathic hypereosinophilic syndrome (HES).
Maturation Three related cytokines, GMCSF, IL-3 and IL-5, have pronounced effects on eosinophil precursors and mature cells. The source of these growth factors in most diseases has still to be defined. There is still controversy as to whether there is a human equivalent of the THI and TH2 phenotypes as described in mice, or whether any helper T cells can produce all of the cytokines characteristic of these two types of response. However it is clear that eosinopoietic events belong to the T~,2 class of response. IL-5 is an essential and sufficient growth factor for eosinophi]s in mice; Sanderson and colleagues have produced four lines of IL-5 transgenic mice bearing a T-cellspecific locus control region gene that drives IL-5 production throughout lift. When these mice were infected with Mesocestoides corti their eosinophil counts fell, suggesting that eosinophil production was still capable of being regulated. No specific pathCogy has been identified in any organs of these animals, although many are infiltrated with eosinophils. They have a small increase in serum IgM levels, although the levels are not as high as those seen in the IL-5 transgenic mice produced by K. Takatsu (Tokyo). IL-5 mRNA has been identified in human tissue eosinophils (Capron). This surprising observation, which implies an autocrine function for IL-5, has been corn firmed in several skin diseases and in eosinophilic endomyocardial fibrosis. IL-5, in the presence of IL-3 and GM-CSF, enhances secretion of eosinophil granule proteins by lgA and slgA but not by IgE or lgG.
v,,z. 17 No. 10 19~)2
TRENDS
IL-5 receptors have been defined on rodent and human eosinophils (Takatsu). They consist of two peptide chains, one (or possibly both) of which transduce signals. The IL-5 receptor has close structural homology with GM-CSF and IL-3 receptors, although the response of eosinophils to the three agonists can be clearly distinguished.
thesize IL-5 when stimulated in vitro. Current work centres on the development of in situ studies of cytokine synthesis in marrow and tissues to relate production to eosinophil production and activation. The three related cytokines, GM-CSF, IL-3 and IL-5, have pronounced effects on eosinophil precursors and mature cells. Alterations in the amino acid comCytokines position of the A chain of GM-CSF Eosinophils express mRNAs for and the fourth helix of IL-3 do not a wide range of peptide growth fac- inhibit the binding of these altered tors, including IL-10~, transforming proteins to their receptors, but do growth factor ~ (TGF-0t), TGF-~, block signal transduction, suggestGM-CSF, tumour necrosis factor ing that these engineered proteins (TNF-0~), IL-3, IL-5, IL-6 and IL-8. may be of therapeutic benefit Some of these findings are contro- (Vadas). Other potential ways to versial, as they depend on sensitive prevent eosinophil-mediated damage and complex assay systems that are through the inhibitory actions have a marked capacity to bind of P-selectins (Vadas) or by neutralnucleic acid probes non-specifically. izing MBP-induced injury with It is also not clear whether there is polyanions (Gleich). a correlation between the level of in There is no evidence to date that situ labelling and peptide secretion, IL-5 transgenic mice or mice lackor whether eosinophils secrete suf- ing the capacity to make IL-5 show ficient amounts of these growth a significantly altered response to factors to induce autocrine or parasite infections. This has come paracrine effects in tissues. S. Galli as a shock to many brought up on (Boston) noted that there were the view that eosinophils have a marked variations of unknown major role in parasite immunity. cause in the proportions of Although deletion experiments do eosinophils in nasal polyps that not eliminate accessory roles for contained TGF-0~mRNA. eosinophils, a reappraisal of the Antibodies to IL-5 suppress a essential roles of eosinophils in pararange of immune responses in sitic disease has become necessary antigen-stimulated guinea pigs, in view of these findings. It is possmice and monkeys (R. Egan, ible that the mucosal role of Bloomfield). This offers therapeutic eosinophils in worm expulsion has potential in humans; humanized been underestimated in the past monoclonal antibodies have been (Anthony Butterworth, Cambridge). produced to many of the peptide growth factors that affect Diseases eosinophils, offering one way to Several important new developreduce eosinophil-mediated tissue ments were reported on cellular injury (Abrams). aspects of asthma. (1) Mononuclear For measurement of cytokine cells from the lungs of patients with levels, bioassays are still more sen- asthma have been shown to contain sitive for serum IL-5 than immuno- IL-5 and IL-4 mRNA (B. Kay, assays. An increase in serum IL-5 is London). Such cells were present in seen following treatment of patients largest numbers where eosinophils with IL-2 who develop eosino- were also prominent. They were philia, and the periodicity of serum also seen in the bronchoalveolar IL-5 proceeds and mirrors the lavage fluid (Kay). It is therefore blood eosinophilia in patients with probable that the T,2 subset of cyclical eosinophilia syndromes. growth factors stimulate both the Surprisingly, there was no increase generation of eosinophils in the in serum IL-5 in patients with the marrow and their subsequent actieosinophilia myalgia syndrome. T vation in the tissues of patients cells from knockout mice lacking a with asthma. (2) In patients with functional IL-2 gene do not syn- nocturnal asthma, blood eosinophil
Immunology Today
386
counts relate to the severity of illness (Busse). Although there was no circadian variation in the counts, blood eosinophils taken at 04:00 hrs had an increased capacity to survive in vitro. (3) Challenge of four segments of lung with different doses of antigens showed that epithelial shedding and free eosinophil granules in biopsy samples were characteristic of the higher antigen doses. Eosinophils in alveolar washings differed from blood eosinophils in response to F-Leu-Met-Phe (FLMP), producing a larger and longer lasting alteration in Ca 2+ mobilization. (4) Tight junctions between epithelial cells in the lungs may be damaged by light density eosinophils in asthma (S. Makino, Dokkyo University). (5) The expression of VCAM in the lungs is increased in asthma, and is related to the extent of MBP deposition in adjacent tissues (Cromwell). The late phase reaction in asthma has been studied for several years in bovine serum albumenimmunized guinea pigs (M. Church, Southampton). Recently, it has become possible to eliminate upper respiratory events which can dominate the response in this species, by direct endotracheal challenge of immunized animals. Using this method, a late rise in serum MBP after challenge was revealed, but this did not relate to the severity of bronchial narrowing. Other groups consider that monkeys provide a closer model of human asthma, and are now abandoning rodent models as they have proved to be so difficult to evaluate. Cynomologous monkeys immunized with Ascaris suum antigens have been of particular interest, first because many become dual (early and late phase) responders to challenge, and secondly because they can be studied in detail (R. Gundel, Richfield). These animals have severe respiratory impairment after challenge. Antibodies to ICAM-1 reduce the late phase response specifically; airways challenged with MBP, but not other eosinophil proteins, cause bronchial constriction, and this is inhibited with pro-MBP. Comparisons of the effects of neutrophils and
vot. 13 No. 10 1992
TRENDS eosinophils in this model have led to the conclusion that one of the main roles of eosinophils in the lungs is to prime them for subsequent allergic responses. The presence of eosinophil granule proteins has been demonstrated in several human skin diseases (K. Leiferman, Rochester). These include late phase reactions in the skin, solar urticaria and eczematoid reactions. Differences were found between atopic lesions and late phase reactions, suggesting that they have a different pathogenesis. In bullous pemphigoid, eosinophil granule protein deposition was especially prominent. The contribution of neutrophil-derived ECP and EDN to these lesions was suggested by equivalent deposits of neutrophil elastase. A new syndrome, eosinophil-mediated vasculitis, has been described in three patients in which there was necrotizing vasculitis with intense MBP deposition in the skin (Leiferman). The molecular basis of HES remains unknown. Some patients appear to have a disease driven by
overproduction of 1L-5. There may be an alteration in cytokine production in these patients from IL-5/IL-3 dependency to GM-CSF dependency (W. Owen, Boston t. An important new treatment for patients with severe forms of HES, IFN-¢~ therapy, has been introduced in the past two years. Five patients with HES in France have shown marked improvement with repeated courses of IFN-cz injections, 5 × 10" units/day for five days/week, and the improvements were sustained (Capron). A clinical trial is in progress in Rochester.
Conclusions Although there has been considerable progress in defining the properties of eosinophils in disease, little is known about their functions and interactions with other cells in tissues. New ideas about their possible roles as sources of peptide growth factors have been matched by observations casting doubt on the long-held view that they are necessary to destroy the tissue stages m nematode life cycles.
Decades of work on chemotactic factors have now been replaced by work showing that eosinophil localization in tissues derives from eosinophil-endothelial interactions through a complex series of adhesion molecules. Finally, there is now rear hope for the development of effective treatments for eosinophil-mediated diseases with drugs and reagents that block the pathways leading from T-cell responses to eosinophil proliferation, activation and secretion into tissues. With these compounds and reagents, and by developing transgenic and knockout mice, many of the current issues about the roles of eosinophils in disease should be resolved in the near future.
Christopher Spry is at St George's Hospital Medical School, London, UK SW17 ORE; Barry Kay is at the National Heart and Lung Institute, London, UK SW3 C~LY; and Gerald Gleich is at the Mayo Clinic and Foundation, Rochester, MN ~5905, USA.
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~.,m.ndog~, ~,d,~,
387
V,,t. /~ No. zo ~9~,2
Eosinophils 1992 Christopher J.F. Spry, A. Barry Kay and Gerald J. Gleich
Four proteins make up the bulk of human eosinophil granules. Two of them, the eosinophil ribonucleases (eosinophil cationic protein (ECP) and eosinophil neurotoxin/protein X (EDN)) are closely related to the nonsecretory hepatic ribonucleases in structure and substrate specificity, and are distinct from pancreatic ribonuclease. ECP is more toxic than EDN, and the RNAase activity of these proteins is involved in their neurotoxicity (G. Gleich, Rochester). However, it is not known why there are two such closely related ribonucleases in eosinophil granules, or what role they play in host protection, although it is assumed that this relates to parasite immunity. Eosinophil major basic protein (MBP) can damage a variety of cells, and it has been thought to be the principal effector protein in many of the beneficial and harmful effects of eosinophils in disease. Guinea pigs have two different forms of MBP. Although there seems to be only one MBP gene in humans, MBP transcripts can undergo differential splicing (C. Spry, London). The kinetics and molecular basis of membrane damage by MBP has been studied recently using liposomes made up of acidic lipids, Eosinophil peroxidase (EPO) can injure cells and tissues directly, in addition to its capacity to produce reactive species with its preferred thiocyanate substrate (G. Gleich, Rochester). The Charcot Leyden crystal (CLC) protein, which is stored in the recently described 'primary' granules in eosinophils (Ann Dvorak, Boston), comprises up to 10% of the total protein in human eosinophils. CLC, which has been expressed in cos cells, belongs to the 'S-type' lectin superfamily, with homologies to rat immunoglobulin
This report from a recent meeting* highlights some of the key developments in eosinophil research and assesses their impact on current views of the role of eosinophils in inflammation. Considerable progress has been made in defining the structure, constituents and properties of eosinophils in vitro and these new insights into eosinophil biology are summarized. A new era is beginning in which the roles of eosinophils can be assessed and defined in vivo.
wise alterations in membrane capacitance as granules fuse with the plasma membrane or during endocytosis. A variety of pharmacological studies have suggested ways to prevent or reduce eosinophil activation and secretion (P. Barnes, London). Inhibitors have been found to reduce the responses of adrenoceptor agonists, pbosphodiesterase-mediated responses, phospholipase D and prostaglandin E2 (PGE2) production. Steroids seem to have few direct effects on (Ig)E-binding protein (S. Ackerman, eosinophils, although they have a Boston). Multicentre studies are in marked indirect effect by reducing progress to assess the possible roles cytokine production by lymphoof CLC protein in respiratory cytes. disease, on the basis that CLC Platelet activation factor (PAF) is protein may neutralize natural lung produced by stimulated eosinophils surfactants, and cause atelectasis. and has marked autocrine effects on them. It stimulates 02 uptake Secretion and signal transduction and superoxide release. It raises Regulation of the secretion of Ca 2+ and diacylglycerol levels and eosinophil granule proteins is increases the binding affinity of the complex, as shown by the range complement component iC3b. of responses of eosinophils to These findings show that PAF is stimulation: certain events take one of the most important sigonly a few seconds, while others nalling systems for converting peak several hours after stimu- blood eosinophils to their activated lation. The use of specific G- form (D. Roos, Amsterdam). protein inhibitors has shown that Endothelial cells are probably one protein (tyrosine) phosphorylation of the main producers of PAF to is an important regulatory step for activate eosinophils in inflamsome of these responses. O. mation (W. Busse, Madison). Cromwell (London) suggested that Granulocyte-macrophage colonyeosinophil 'activation' is the result stimulating factor (GM-CSF) inof enhanced or persistent phos- creases the synthesis of PAF by phorylation of signal-transducing eosinophils, but other factors are proteins. The process of exocytosis needed to induce PAF secretion of granule proteins, which is more from them (A. Lopez, Adelaide). pronounced in rodent than in The way in which secretion human eosinophils, has been occurs in vivo is still being investistudied by patch-clamp techniques; gated. One newly described type of these studies have revealed step- granule release, termed 'piecemeal' degranulation by A. Dvorak, is as*Eosinophils 1992 was organized by sociated with vesicular transport. She Barry Kay and Gerald Gleich. It was also described a spatial relationship held at Adare, Ireland on 1-3 June between eosinophils and nerve cells 1992. The proceedings will be in tissues, although the significance published by Marcel Dekker. of this has still to be discovered. 1992, Elsevier Science Publishers Ltd, UK.
Immu,otogy today 3 8 4
vot. 13 No. I0 1992
TRENDS
Some eosinophils have receptors for IgA. It is now clear that crosslinked IgA, crosslinked secretory piece or secretory IgA (slgA) itself can stimulate eosinophils to secrete ECP and EPO (M. Capron, Lille). This may explain the presence of large numbers of eosinophils at mucosal surfaces where slgA is secreted. Capron's group, working with Po-Cbun Tai (who died in 1990, and whose many contributions to eosinophil research were remembered at the meeting), have shown a marked difference in the amounts of ECP and EPO secreted in response to IgE, IgG and IgA. This implies that there are separate transduction pathways for these signals, and/or distinct secretion pathways for the granule proteins. Some morphological evidence has been reported explaining this fine control of the functions of eosinophils.
and very late antigen (VLA-4)) on the loosely-bound eosinophils. Increased expression of adhesion molecules may take several hours to become fully effective, but the time course in vivo is unknown. Integrins are the: principal attachment sites on eosinophils for intracellular adhesion molecule 1 (ICAM-1), which is constitutively expressed on endothelial cells. IL-4 also induces the expression of vascular cell adhesion molecule 1 (VCAM-I) on endothelial cells, which binds to VLA-4 on eosinophils, providing specificity for eosinophil localization - neutrophils do not express VLA-4. This process may account for the accumulation of eosinophils in IL4-secreting tumours. Eosinophils also have VLA-6 adhesion receptors. The CD 11-CD 18 mCecules on eosinophils are required for them to emigrate into tissues.
Adhesion and emigration The localization of eosinophils in tissues is now known to involve families of adhesion molecules that are induced on circulating eosinophils and endothelial cells during inflammation (M. Vadas (Adelaide), A. Wardlaw (Leicester) and R. Schleimmer and B. Bochner (Baltimore). The process starts with a weak and reversible binding of eosinophil membrane sialyl Lewis X to selectins on postcapillary endothelial cells. Selectins in serum compete for this binding, so that eosinophils can 'roll' over the surface of endothelial cells. L-selectins can also be shed from the surface of eosinophils, and when this occurs in vitro eosinophils can no longer migrate across endothelial cell monolayers. Inflammatory mediators signal endothelial cells to transform this loose binding to a firm attachment; for example, endothelial-leucocyte adhesion molecule 1 (ELAM-1)is induced on endothelial cells, which then have stronger attachment sites for sialated Lewis X. lnterleukin 1 (IL-1) induces the release of PAF and other activating molecules from endothelial cells which, in turn, induce the expression of integrins (lymphocyte function associated molecule 1 (LFA-1), Mac-1
Activation CD25, the 13 chain of the IL-2 receptor, is expressed on eosinophils (Weller) but only following stimulation ((;apron). The expression of several other membrane antigens and receptors is also increased by stimulation: gammainterferon (IFN-y)-induced FcyRI and Fc~'RIII. Activated eosinophils also express CD69 (a membrane antigen found on activated T cells), and HLA-DR molecules (Wardlaw). Cultured eosinophils express HLA-DR molecules and can present antigens to T cells in an MHCrestricted manner as effectively as macrophages (Weller). Additional lipid bodies, five of which are normally found in each eosinophil, are induced by protein kinase C activating agents, but not by IL-5 or GM-CSF. These densely osmophilic bodies, which are not membrane bound, contain large amounts of arachidonic acid and enzymes involved in the prostaglandin and lipoxygenase synthesis pathways, suggesting that this is the principal site for their production in eosinophils (Weller). Lipid bodies also contain peroxidase (J. Abrams, Palo Alto). Mature eosinophils express C D l l b - C D 1 8 and receptors with C-type (FceRII, also known as
Immunology 7~day
385
CD23) and S-type (Mac2) lectin domains. Mac2 binds IgE and is involved in IgE-dependent eosinophil-mediated cytotoxicity, mRNA for Mac2 is present in human eosinophils, showing that the cells synthesize it themselves. Large amounts of soluble CD23 are found in the serum of patients with the idiopathic hypereosinophilic syndrome (HES).
Maturation Three related cytokines, GMCSF, IL-3 and IL-5, have pronounced effects on eosinophil precursors and mature cells. The source of these growth factors in most diseases has still to be defined. There is still controversy as to whether there is a human equivalent of the THI and TH2 phenotypes as described in mice, or whether any helper T cells can produce all of the cytokines characteristic of these two types of response. However it is clear that eosinopoietic events belong to the T~,2 class of response. IL-5 is an essential and sufficient growth factor for eosinophi]s in mice; Sanderson and colleagues have produced four lines of IL-5 transgenic mice bearing a T-cellspecific locus control region gene that drives IL-5 production throughout lift. When these mice were infected with Mesocestoides corti their eosinophil counts fell, suggesting that eosinophil production was still capable of being regulated. No specific pathCogy has been identified in any organs of these animals, although many are infiltrated with eosinophils. They have a small increase in serum IgM levels, although the levels are not as high as those seen in the IL-5 transgenic mice produced by K. Takatsu (Tokyo). IL-5 mRNA has been identified in human tissue eosinophils (Capron). This surprising observation, which implies an autocrine function for IL-5, has been corn firmed in several skin diseases and in eosinophilic endomyocardial fibrosis. IL-5, in the presence of IL-3 and GM-CSF, enhances secretion of eosinophil granule proteins by lgA and slgA but not by IgE or lgG.
v,,z. 17 No. 10 19~)2
TRENDS
IL-5 receptors have been defined on rodent and human eosinophils (Takatsu). They consist of two peptide chains, one (or possibly both) of which transduce signals. The IL-5 receptor has close structural homology with GM-CSF and IL-3 receptors, although the response of eosinophils to the three agonists can be clearly distinguished.
thesize IL-5 when stimulated in vitro. Current work centres on the development of in situ studies of cytokine synthesis in marrow and tissues to relate production to eosinophil production and activation. The three related cytokines, GM-CSF, IL-3 and IL-5, have pronounced effects on eosinophil precursors and mature cells. Alterations in the amino acid comCytokines position of the A chain of GM-CSF Eosinophils express mRNAs for and the fourth helix of IL-3 do not a wide range of peptide growth fac- inhibit the binding of these altered tors, including IL-10~, transforming proteins to their receptors, but do growth factor ~ (TGF-0t), TGF-~, block signal transduction, suggestGM-CSF, tumour necrosis factor ing that these engineered proteins (TNF-0~), IL-3, IL-5, IL-6 and IL-8. may be of therapeutic benefit Some of these findings are contro- (Vadas). Other potential ways to versial, as they depend on sensitive prevent eosinophil-mediated damage and complex assay systems that are through the inhibitory actions have a marked capacity to bind of P-selectins (Vadas) or by neutralnucleic acid probes non-specifically. izing MBP-induced injury with It is also not clear whether there is polyanions (Gleich). a correlation between the level of in There is no evidence to date that situ labelling and peptide secretion, IL-5 transgenic mice or mice lackor whether eosinophils secrete suf- ing the capacity to make IL-5 show ficient amounts of these growth a significantly altered response to factors to induce autocrine or parasite infections. This has come paracrine effects in tissues. S. Galli as a shock to many brought up on (Boston) noted that there were the view that eosinophils have a marked variations of unknown major role in parasite immunity. cause in the proportions of Although deletion experiments do eosinophils in nasal polyps that not eliminate accessory roles for contained TGF-0~mRNA. eosinophils, a reappraisal of the Antibodies to IL-5 suppress a essential roles of eosinophils in pararange of immune responses in sitic disease has become necessary antigen-stimulated guinea pigs, in view of these findings. It is possmice and monkeys (R. Egan, ible that the mucosal role of Bloomfield). This offers therapeutic eosinophils in worm expulsion has potential in humans; humanized been underestimated in the past monoclonal antibodies have been (Anthony Butterworth, Cambridge). produced to many of the peptide growth factors that affect Diseases eosinophils, offering one way to Several important new developreduce eosinophil-mediated tissue ments were reported on cellular injury (Abrams). aspects of asthma. (1) Mononuclear For measurement of cytokine cells from the lungs of patients with levels, bioassays are still more sen- asthma have been shown to contain sitive for serum IL-5 than immuno- IL-5 and IL-4 mRNA (B. Kay, assays. An increase in serum IL-5 is London). Such cells were present in seen following treatment of patients largest numbers where eosinophils with IL-2 who develop eosino- were also prominent. They were philia, and the periodicity of serum also seen in the bronchoalveolar IL-5 proceeds and mirrors the lavage fluid (Kay). It is therefore blood eosinophilia in patients with probable that the T,2 subset of cyclical eosinophilia syndromes. growth factors stimulate both the Surprisingly, there was no increase generation of eosinophils in the in serum IL-5 in patients with the marrow and their subsequent actieosinophilia myalgia syndrome. T vation in the tissues of patients cells from knockout mice lacking a with asthma. (2) In patients with functional IL-2 gene do not syn- nocturnal asthma, blood eosinophil
Immunology Today
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counts relate to the severity of illness (Busse). Although there was no circadian variation in the counts, blood eosinophils taken at 04:00 hrs had an increased capacity to survive in vitro. (3) Challenge of four segments of lung with different doses of antigens showed that epithelial shedding and free eosinophil granules in biopsy samples were characteristic of the higher antigen doses. Eosinophils in alveolar washings differed from blood eosinophils in response to F-Leu-Met-Phe (FLMP), producing a larger and longer lasting alteration in Ca 2+ mobilization. (4) Tight junctions between epithelial cells in the lungs may be damaged by light density eosinophils in asthma (S. Makino, Dokkyo University). (5) The expression of VCAM in the lungs is increased in asthma, and is related to the extent of MBP deposition in adjacent tissues (Cromwell). The late phase reaction in asthma has been studied for several years in bovine serum albumenimmunized guinea pigs (M. Church, Southampton). Recently, it has become possible to eliminate upper respiratory events which can dominate the response in this species, by direct endotracheal challenge of immunized animals. Using this method, a late rise in serum MBP after challenge was revealed, but this did not relate to the severity of bronchial narrowing. Other groups consider that monkeys provide a closer model of human asthma, and are now abandoning rodent models as they have proved to be so difficult to evaluate. Cynomologous monkeys immunized with Ascaris suum antigens have been of particular interest, first because many become dual (early and late phase) responders to challenge, and secondly because they can be studied in detail (R. Gundel, Richfield). These animals have severe respiratory impairment after challenge. Antibodies to ICAM-1 reduce the late phase response specifically; airways challenged with MBP, but not other eosinophil proteins, cause bronchial constriction, and this is inhibited with pro-MBP. Comparisons of the effects of neutrophils and
vot. 13 No. 10 1992
TRENDS eosinophils in this model have led to the conclusion that one of the main roles of eosinophils in the lungs is to prime them for subsequent allergic responses. The presence of eosinophil granule proteins has been demonstrated in several human skin diseases (K. Leiferman, Rochester). These include late phase reactions in the skin, solar urticaria and eczematoid reactions. Differences were found between atopic lesions and late phase reactions, suggesting that they have a different pathogenesis. In bullous pemphigoid, eosinophil granule protein deposition was especially prominent. The contribution of neutrophil-derived ECP and EDN to these lesions was suggested by equivalent deposits of neutrophil elastase. A new syndrome, eosinophil-mediated vasculitis, has been described in three patients in which there was necrotizing vasculitis with intense MBP deposition in the skin (Leiferman). The molecular basis of HES remains unknown. Some patients appear to have a disease driven by
overproduction of 1L-5. There may be an alteration in cytokine production in these patients from IL-5/IL-3 dependency to GM-CSF dependency (W. Owen, Boston t. An important new treatment for patients with severe forms of HES, IFN-¢~ therapy, has been introduced in the past two years. Five patients with HES in France have shown marked improvement with repeated courses of IFN-cz injections, 5 × 10" units/day for five days/week, and the improvements were sustained (Capron). A clinical trial is in progress in Rochester.
Conclusions Although there has been considerable progress in defining the properties of eosinophils in disease, little is known about their functions and interactions with other cells in tissues. New ideas about their possible roles as sources of peptide growth factors have been matched by observations casting doubt on the long-held view that they are necessary to destroy the tissue stages m nematode life cycles.
Decades of work on chemotactic factors have now been replaced by work showing that eosinophil localization in tissues derives from eosinophil-endothelial interactions through a complex series of adhesion molecules. Finally, there is now rear hope for the development of effective treatments for eosinophil-mediated diseases with drugs and reagents that block the pathways leading from T-cell responses to eosinophil proliferation, activation and secretion into tissues. With these compounds and reagents, and by developing transgenic and knockout mice, many of the current issues about the roles of eosinophils in disease should be resolved in the near future.
Christopher Spry is at St George's Hospital Medical School, London, UK SW17 ORE; Barry Kay is at the National Heart and Lung Institute, London, UK SW3 C~LY; and Gerald Gleich is at the Mayo Clinic and Foundation, Rochester, MN ~5905, USA.
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