Pergamon

Copyright

PII: SOO20-7519(96)00040-9

In~ermtiond Jownal/o~ Parmimiogy, Vol. 26, No. 5, pp. 499-508, 1996 I996 Australian Sc&ty for Parmtology. Published by Elsevier Science Ltd Printed in Great Britain 002&7519/96 Sl5.00+0.00

INVITED REVZEW

Why do some Nematode Parasites of the Alimentary Tract Secrete Acetylcholinesterase?

D. L. LEE

Department

of Biology, The University of Leeds, Leeds LS2 9JT, U.K.

(Received 2 January 1996; accepted 20 February 1996)

Abstract-Lee D.L. 19%. Why do some nematode parasites of the alimentary tract secrete acetylcholinesterase? ZnternatiomZ Journal fir ParasitoZogy 26~ 499-508. Many gastrointestinal nematodes secrete large amounts of acetylcholinesterases. Antibodia are produced against these secreted acetylcholinesterases and appear to give some protection against infection with some nematodes. The theory that acetylcholinesterax secreted by gastrointestinal nematodes may act as a biochemical holdfast by reducing contractions of the alimentary system has not been substantiatd, a vasoactive intestinal polypeptideJike protein is secreted hy some species and may be the biochemical holdfast. Secreted acetylcholinesterases may alter host cell permeability, have an anti-coagulant role, affect glycogenesis, and/or be important in certain aspects of acetate and choline metabolism. Probably the most important role for acetylcholinesterase secreted by nematodes is immune modulation and/or reduction of inflammation in the vicinity of the nematode. The reason why some species of gastrointestinal nematodes resistant to benzimidazoles contain elevated amounts of acetylcholinesterase is unclear. Copyright 0 19% Australian Society for Parasitology. Published by Elsevier Science Ltd. Key wor& Acetylcholine; acetylcholinesterase; E/S products; immunomodulation; nematode; secretions.

INTRODUCTION

In vertebrates acetylcholine (ACh) is a neurotransmitter that is found not only in the nervous system, in which it is involved in neuromuscular transmission and cardiac regulation, but also in other locations where it may have different functions. ACh receptors are divided into 2 main types, namely nicotinic and muscarinic. Nicotinic receptors are coupled to cation channels and mediate excitatory synaptic transmission at the neuromuscular junction, autonomic ganglia, and at various sites in the central nervous system. There are 5 types of muscarinic receptor. M,-receptors are found mainly in the central nervous system and the peripheral neurones; they are also involved in the secretion of gastric acid from parietal cells. Mz-receptors occur in the heart and on the pre-synaptic terminals of peripheral and central neurones; they cause a decrease in cardiac
Fax: +44 1 I3 233 2882; E-mail: pab6dll@leeds.ac.uk. 499

rate and force of contraction. M3-receptors stimulate glandular secretions, contraction of visceral smooth muscle and relaxation of vascular smooth muscle, with this latter resulting from the release of nitric oxide from neighbouring endothelial cells (Rang, Dale & Ritter, 1995); it is interesting to note that M3-receptors are present on mononuclear cells (Costa, Traver, Auger & Costa, 1994). Md-receptors occur in certain regions of the central nervous system. The pharmacological and physiological roles of the MS-receptors have yet to be characterized. All the muscarinic receptors are G-protein-coupled receptors. ACh is involved in the release of histamine through stimulation of receptors on the surface of histaminecontaining cells in the lungs (Kaliner, Orange & Austin, 1972; Kaliner & Austin, 1975) and in the alimentary tract; also in the release of mucus from goblet cells. It may also be associated with permeability of cells through the activation of phospholipase C.

500 Acetylcholine reaction: is hydrolysed according to

D. L. Lee the

system. AChE is associated with nerve-muscle junctions, muscle membranes, certain sense organs, the nerve ring, and certain nerves (Lee I% Atkinson, ACh+HZO+acetate+choline 1976) and is presumably involved in hydrolysis of under the action of acetylcholinesterase. ACh at these sites. Large amounts of AChE are also Mammalian acetylcholinesterase is a very efficient present in the sub-ventral glands associated with the enzyme with a single molecule able to hydrolyse an excretory system and/or the oesophageal glands of estimated 300,000 molecules of acetylcholine every several nematodes, such as Ancylostoma ceylanicum, minute. Brugia malayi, Dictyocaulus viviparus, Necator Two types of mammalian cholinesterase are recogamericanus, Nematodirus battus, Nippostrongylus nized, namely acetylcholinesterase (AChE) (EC brasiliensis, Oesophagostomum radiatum, Stephanurus 3.1.1.7) and butyrylcholinesterase (BChE) (EC dentatus, Trichostrongylus colubrtformis, T axei. 3.1.18). These 2 types of enzyme are closely related T. retortaeformis, and the amphidial glands of N. in structure but differ in their distribution, substrate americanus (Blackburn & Selkirk, 1992a; Bremner, specificity, their functions and some inhibitors (see Ogilvie, Keith & Berrie, 1973; Griffiths & Pritchard, Chatonnet & Lockridge, 1989; Rang et aZ., 1995). A 1994; Lee, 1969, 1970; McKeand, Knox, Duncan & number of different molecular forms of acetylchoKennedy, 1994a, 1994b; McLaren, Burt & Ogilvie, linesterases, or true cholinesterases, can be found in 1974; Nakazawa, Yamada, Uchikawa & Arizono, animals. They occur as asymmetric (A) forms and 1995; Ogilvie et al., 1973; Pritchard, Brown & globular (G) forms. The A forms occur as Ad, Aa and Toutant, 1994; Rathaur, Robertson, Selkirk & Ai2 forms and are 1, 2 or 3 tetramers with a collagen Maizels, 1987; Rhoads, 1981; Rothwell, Ogilvie tail; the G forms occur as Gi, GZ and Gb forms & Love, 1973; Sanderson, 1969; Sanderson & Ogilvie, corresponding to monomers, dimers and tetramers of 1971; Tekwani, 1992). There has been much interest the catalytic subunits of the enzyme (Massoulie et al., in the secreted AChEs of nematodes since Lee (1969, 1993). AChE is bound to the basement membrane at 1970) first detected non-specific esterases and cholinergic synapses where it hydrolyses ACh recholinesterases in the oesophageal glands and the leased at the synapse. A soluble form of AChE occurs so-called excretory glands of adult N. brasihensis and in cholinergic nerve terminals, where it has a role in Sanderson (1972) quantified the amount of AChE regulating the free ACh concentration, and from secreted by this nematode. Lee (1969, 1970) suggested where it may be secreted. The function of this se- that the excretory glands were secretory rather than creted enzyme is not known. A soluble form of AChE excretory. Since then several authors have demonis found in cerebrospinal fluid. Membrane bound strated that a number of nematodes secrete AChE. AChE also occurs in erythrocytes and again its The amount secreted varies from species to species, function is not known. Mammalian AChE is most can vary from larvae to adults and between the sexes, active on ACh and closely related esters, such as and is immunogenic in the host (Jones & Ogilvie, acetyl B-methylcholine and acetylthiocholine (this 1972; Griffiths & Pritchard, 1994; Lawrence & latter substrate is used to detect the enzyme histoPritchard, 1993; McKeand et al., 1994a, 1994b; chemically and on gels), but most esters are not McKeand, Knox, Duncan & Kennedy, 1995a, 1995b; good substrates. Butyrylcholinesterase, or pseudoOgilvie et aZ., 1973; Pritchard et aZ., 1991, 1994; cholinesterase, has a widespread distribution in Pritchard, 1995; Rathaur et al., 1987; Rhoads, 1981, animals, being found in various organs such as the 1984; Rothwell & Merritt, 1974; Sanderson & liver, skin, brain and gastrointestinal smooth muscle Ogilvie, 1971). AChE is also secreted by some nemaand in the plasma. It has a broader substrate specifitodes, such as D. viviparus, Syngamus trachea and city than AChE but hydrolyses butyrylcholine more B. malayi, that do not inhabit the gastrointesrapidly than acetylcholine and is inactive on acetyl tinal tract (McKeand et al., 1994a, 1994b, 1995b; B-methylcholine. The function of this enzyme is Rathaur et al., 1987; Riga, Perry, Barrett & uncertain but the plasma enzyme is important in the Johnston, 1995). inactivation of certain drugs. In mammals AChE and BChE keep ACh in the plasma at undetectable low CHARACTERISTICS OF NEMATODE levels. AChE and BChE are both serine hydrolases. ACETYLCHOLINESTERASES For further reading see Rang et a/. (1995). Invertebrates appear to possess only globular forms ACETYLCHOLINESTERASE IN NEMATODES of AChE and tend to have less stringent substrate specificity than vertebrate AChEs (Chatonnet & Acetylcholine is an important neurotransmitter in Lockridge, 1989). The structure and characternematodes and is associated with the neuromuscular

Invited Review istics of AChEs have been studied in relatively few species of nematode. Biochemical studies on homogenates have revealed differences between species with regard to substrate specificity, sensitivity to inhibitors and molecular weight (Opperman & Chang, 1992). In the free-living nematode Cuenorhabditis elegans 3 different genes (ace-l, ace-2 and ace-3) code for AChE (Johnson et aZ., 1988) and this results in molecular polymorphism with 3 classes (A, B and C) of catalytic subunit which differ in their inhibitor and substrate specificities (Johnson & Russell, 1983). Somatic extracts and excretions/ secretions of the hookworm N. americanus appear to possess a class B AChE similar to that described for Caenorhabditis and to synthesize and secrete globular G* AChE with the basic catalytic subunit having a mass of 6670 kDa (Pritchard et ul., 1994). Earlier work by Yeates & Ogilvie (1976) had estimated the molecular weight of AChE in the excretions/secretions of Necator at 38(X400 kDa. Excretions/secretions from adult T. colubrtyormis possess 2 forms of hydrophilic AChE (80 kDa and 189 kDa), which are thought to be globular monomer (G,) and dimer (G*) forms of the enzyme (Griffiths & Pritchard, 1994). Three isoenzymes (A, B and C) are present in the supernatant of homogenates of normal adult N. brasihensis but these change during the course of an infection. There is an increase in the number of the B isoenzyme bands with the formation of extra bands B1 and B2 and an increase in staining intensity of the B and C bands with loss of the A band on acrylamide gels. However, in adapted worms from rats 7 days after a second or third infection the A band was greatly increased in size and staining intensity (Edwards, Burt & Ogilvie, 1971). Also, AChE levels in the homogenates of N. brasihensis increase as rats become immune (Sanderson & Ogilvie, 1971). AChE activity also increases in adult female N. battus up to 24 days after infection when young lambs begin to reject the nematodes (Lee & Martin, 1976). The reasons for these changes are not known. It is possible that by changing the form of the secreted AChEs the nematode is able to evade the action of antibodies directed against the earlier forms of secreted enzyme, and an increase in the amount of enzyme may also help the nematode to evade the immune response directed against the enzyme for a period of time. Hogarth Scott, Watt, Ogilvie & Rothwell (1973) showed that the molecular weight of AChE in the supernatant from homogenates of adult N. brasiliensis and T. colubrtyormis was between 65 and 75 kDa. Watts & Atkins (1981) detected a 61 kDa AChE in the supernatant fraction of homogenates of N. brasiliensis but also

501

found an AChE with a molecular weight of 980 kDa associated with the membrane fraction of the homogenate. Subsequently, Blackburn & Selkirk (1992a) showed that the excretions/secretions (E/S) of adult N. brusiliensis contain 3 major forms of AChE and these were equivalent to forms A, B and C identified by Edwards et aZ. (1971); 2 minor forms of AChE, B and B were also sometimes present. Blackburn & Selkirk (1992a) found isoforms of AChE of 74 kDa and 39 kDa and each isoform resolved as two species by isoelectric focusing. Both the 74 kDa and the 39 kDa AChEs were secreted by 4-day-old worms but there was a switch to predominant secretion of the 39 kDa form by day 8 of infection. They suggested that this change may be related to maturation of the nematodes. Nakazawa et ~2. (1995) detected only a 74 kDa form of the enzyme in the E/S of adult Nippostrongylus. Lawrence & Pritchard (1993) found that Heligmosomoides polygyrus (Nematospiroides dubius) secreted AChE with maximum production of the enzyme occurring in the secretions from the fourthstage larvae. The microfilariae and adults of the filarial nematode B. muluyi also secrete AChE with the adults secreting 100 kDa and 200 kDa forms of AChE while the microfilariae secrete 30, 40 and 200 kDa molecular forms of the enzyme (Rathaur et al, 1987). DeVos & Dick (1992) found 2 forms of AChE sedimenting at 5.3 and 13 S, which appear to be equivalent to class A forms of Caenorhabditis, in the supernatant of homogenates of the muscle stage larvae of Trichinella spiralis but did not detect class B or class C forms as found in Caenorhabditis; cholinesterase activity in the E/S of these larvae was low. The adult, gastrointestinal stage, of Trichinella was not investigated. Rhoads (198 1) characterized the cholinesterases of the tissue dwelling (kidney) nematode S. dentutus. The male released 27 times more cholinesterase than the female in vitro but the reason for this is not known. The molecular weight of the secreted enzyme is 100 kDa and there are 2 molecular forms of the enzyme. Adults of the cattle lungworm D. viviparus secrete AChE (over 200 times more activity per unit protein found in the E/S than in the homogenate supernatant of the nematodes) and there are 5 isoforms of the enzyme (McKeand et ul., 1994b). They found that relatively little AChE is produced by the larval stages. Blackburn & Selkirk (1992a) have drawn attention to the apparent requirement of AChEs of some invertebrates for proteolytic processing before full activity is achieved and suggested that the AChEs secreted by Nippostrongylus may require this sort of processing to become fully active.

502 ANTIBODIES AGAINST SECRETED AChE

D. L.

Lee the nematodes to stay in their preferred site and that protective antibodies inhibit this holdfast mechanism. Since then other possible roles for AChE secreted by nematodes have been proposed (see below) but the biochemical holdfast theory has remained one of the possible roles. Cholinesterase activity in whole worm homogenates and in secretions from some species varies enormously, sometimes within the same genus. Adult NQpostrongylus release large quantities of AChE with up to 11% of the total enzyme being released per hour into culture fluid (Sanderson, 1972); presumably the enzyme is also secreted by the nematodes in vivo. In contrast, adult H. poZygyrus released about 1% of their total cholinesterase per hour in vitro (Sanderson, 1972). This leads to difficulties in assigning a biochemical holdfast function to secreted AChE. For example, it is dilhcult to assign a holdfast function to the AChE secreted by adult S. dentutus, which inhabit the kidney, and to adult D. vivipurus, which live in the bronchi and trachea of cattle. Surprisingly, although the biochemical holdfast theory is now well established in the literature, until recently no attempt had been made to test this hypothesis. Foster, Dean & Lee (1994) have shown that the supernatant of homogenates and the excretions/secretions from adult Nippostrongylus significantly decreased the amplitude of contractions when injected into the lumen of segments of uninfected rat intestine maintained in vitro. They also showed that AChE from the electric eel did not affect the contraction of the preparation, so it is unlikely that AChE secreted by the nematode caused the inhibition of contractions of the intestinal wall. This suggests that the EIS of adult Nippostrongylus contains a substance, or substances, other than AChE, which is responsible for the decrease in amplitude of contractions of rat intestine in vitro and that AChE is not the biochemical holdfast. Lee & Foster (1995) and Foster & Lee (1996) have shown that a 3% 50 kDa fraction of the EIS of adult Nippostrongylus significantly reduced the amplitude of contractions of rat intestine in vitro. Gel electrophoresis of this 30-50 kDa fraction revealed a 30 kDa band and this band was recognized by rabbit anti-porcine vasoactive intestinal polypeptide (VIP) antiserum in western blots. Rabbit anti-porcine VIP anti-serum also recognized supernatant of homogenate and E/S of Nippostrongyfus in dot blots. The inhibitory effect of E/S from the nematode on contractions of the rat intestine was significantly reduced when the EIS were mixed with rabbit anti-porcine VIP anti-serum. It would appear, therefore, that the E/S of this nematode contains a VIP-like protein. Mammalian VIP directly inhibits intestinal smooth

Evidence that AChE is secreted by certain species of nematode in vivo comes from the detection of antibodies against nematode AChE in the serum of infected humans and animals (Griffiths & Pritchard, 1994; Jones & Ogilvie, 1972; McKeand et ul., 1995a, 1995b; Pritchard er ul., 1989, 1991, 1994; Rathaur er ul., 1987; Rathaur, Muller, Maizels & Walter, 1992; Rhoads, 1981, 198q RothweIl & Merritt, 197q Sanderson & Ogilvie, 1971). Some authors have attempted to vaccinate animals using excretory/ secretory products of nematodes (Dopheide et ul., 1991; McKeand et uZ., 1995a, 1995b; ODonnell et uZ., 1989a, 1989b; Rothwell & Love, 1974) and some have used nematode AChE for this purpose. For example, Griffiths & Pritchard (1994) obtained up to 59% protection of sheep against mixed infections of T. colubrz~ormis, Huemonchus contortus and Cooperiu oncophoru using purified secretory AChE from T colubrljkmis. Similarly, McKeand et ul. (1995b) have shown that excretory/secretory products of D. vivipurus, enriched with the nematode AChE, stimulated high levels of enzyme-specific antibody in immunized guinea pigs, and guinea pigs which received the AChE-enriched fraction of the excretory/secretory products had significantly lower worm burdens than control animals. This indicates that the secreted AChE plays an important role in the maintenance of the infection in these animals.

EFFECT OF NEMATODES ON GASTROINTESTINAL MOTILITY

Certain species of nematode that inhabit the alimentary system of animals can affect contractions of the wall of the alimentary tract and alter the movement of gut contents along the tract. Some species increase the motility of certain regions of the hosts alimentary tract and decrease transit time, whereas others appear to inhibit contractions resulting in an increase in transit time through the alimentary tract (Holmes, 1986; Lee & Foster, 1995). Lee (1969, 1970, 1972) suggested that the AChE produced by the so-called excretory glands of adult Nippostrongylus may inhibit local spasm in the ulcerated region of the intestinal wall inhabited by the nematodes, making a calmer environment for them. He also suggested that the enzyme may be the factor which reduces peristalsis in the infected rat intestine (Symons, 1966) thus enhancing the survival of the nematode in the inflamed intestine. Ogilvie & Jones (1971) and Edwards et uZ. (1971) subsequently developed this concept by suggesting that the parasites AChE could act as a biochemical holdfast enabling

Invited

Review

503

muscle (Bitar & Makhlouf, 1982) and VIP-ergic neurones are important inhibitory neurones in the gastrointestinal tract of mammals (Costa & Fumess, 1983; DAmato, Beurme & Lefebvre, 1988). Injection of porcine VIP at the same concentration as that used in the nematode EIS experiments significantly reduced the amplitude of contractions of rat intestine in vitro (Foster & Lee, 1996). Savin et ul. (1990) have shown that secretions from adult T. colubrt@rmis contain a homologue of porcine VIP and that it has a molecular weight of 30 kDa. The molecular weight of the VIP-like substance from Trichostrongyks and from N@postrongylus is much higher than that of porcine VIP (3 kDa) but Savin et ul. (1990) have cloned the VIP-like protein from Trichostrongylus and this has indicated that it is a 15 kDa protein with 4 N-linked carbohydrate chains and internal disulphide bonds, which may explain the differences in molecular weight. It seems, therefore, that AChE in the E/S of adult Nippostrongylus is not responsible for the reduction in motility of the host intestine but that a VIP-like protein is wholly or partially responsible for this effect and may be the biochemical holdfast (Foster & Lee, 1996; Lee & Foster, 1995). As a VIP-like protein is produced by adult Trichostrongylus (Savin et al., 1990) and is present in the E/S of adult N. buttus (which inhabit the intestine of lambs) (unpublished results) it is possible that it is present in the E/S of many parasitic nematodes that cause a reduction in motility of the gastrointestinal tract of other animals. It is interesting to note that in rats infected for 6 days with T spiralis there was suppression of release of acetylcholine from the myenteric plexus in the jejunum and also in the worm-free ileum (Collins et ul., 1992). These authors concluded that suppression of acetylcholine release occurs as a result of the inflammatory response to the presence of the nematode and this has an effect on smooth muscle function.
AChE AND CELL MEMBRANE PERMEABILITY

scoleces and brood capsules of the cestode Echinococcus granulosus and found that AChE inhibitors resulted in more rapid outward passage of glucose through the hydatid cyst wall and suggested that AChE could be involved in permeability control across the cyst wall. Lee (1970) suggested that the AChE in the excretions/secretions of Nippostrongylus may alter the permeability of the host cells thus allowing nutrients to leak into areas where the nematodes were clustered together and feeding. The AChE secreted by nematodes might, therefore, have a role in making nutrients more freely available to the nematodes.
AChE AND AN ANTI-COAGULANT ROLE

Blackburn & Selkirk (1992b) have shown that the excretions/secretions of adult Nippostrongylus contain a substance, possibly AChE, which inhibits platelet-activating factor. This could reduce or abolish the ability of platelets to aggregate. Riga et aZ. (1995) found that adult S. truchea release AChE and suggested that one of the functions of the enzyme may be as an anti-coagulant to assist in feeding by this blood-feeding nematode. However, it is more likely that proteases secreted by blood-feeding nematodes act as the anti-coagulant rather than AChE (Pritchard, 1995).

AChE

AND

GLYCOGENESIS

Acetylcholinesterases block the uptake of Na+ and K+ by the gills of the crustacean Eriochier and sodium transport across frog skin and the anal papillae of Chironomus (Prosser & Brown, 1961). This suggests that ACh may be important in maintaining ion gradients across cell membranes in these structures. It may also be associated with permeability of cells through the activation of phospholipase C which is involved in metabolism of phosphatidylinositol in cell membranes, thus triggering release of Ca2+ into the cytoplasm of cells. Schwabe, Koussa & Acra (1961) detected AChE in homogenates of the

A known function of ACh is the stimulation of glycogen synthase activity in mammalian tissues (Akpan, Gardner & Wagle, 1974). Yeates & Ogilvie (1976) suggested that secreted AChE may bring about the breakdown of glycogen to glucose by neutralizing the action of ACh as ACh stimulates glycogen synthesis. This could possibly aiso result in the blocking of the conversion of glucose to glycogen. Either of these possibilities may make glucose more readily available in the environment of the nematodes, especially if the cells have become leaky (see above).

AChE,

ACETATE FOR

AND CHOLINE METABOLISM

PRECURSORS

Secreted nematode AChE may provide acetate and choline precursors for metabolism (Pritchard, 1993) or for use in the neuromuscular system when it hydrolyses any ACh in the environment. Choline in particular may be made available to the nematodes this way. It is probable, however, that this is of minor or no importance for nematodes.

504
EFFECT OF ANTHELMINTICS AChE ON SECRETION

D. L. Lee OF

Benzimidazole anthelmintics, such as mebendazole or oxfendazole, bring about a marked reduction in the secretion of AChE by Nippostrongylus and by A. ceylanicum, with a corresponding rise in the amount of the enzyme within the nematode (Tekwani, 1992; Watts, Rapson, Atkins & Lee, 1982). As benzimidazoles are known to have an effect on microtubule function it was suggested that inhibition of secretion was caused by the loss of microtubular function in glands that secrete the enzyme, and that the anthelmintics did not interfere with production of the enzyme. Whereas the reduction in secretion of the AChE coincided with the expulsion of the nematodes from the intestine it is probable that expulsion is related to a number of factors, including interference with the neuromuscular system through disruption of neurotubules and other functions of cells which depend on microtubular functions, rather than solely because AChE is no longer secreted. AChE secretion is also inhibited in H. polygyrus treated with levamisole, the dose required to inhibit 50% of secretion of the enzyme by males being twice that for females (Mallet & Kerboeuf, 1993). This is apparently because significantly more of the enzyme is secreted by males than by females. There is also a fall in AChE activity of adult T. colubri$ormis in vivo after treatment of the host with levamisole. This occurs within a short period after administration of the levamisole. It is possible that there is reduced metabolism of the nematodes following treatment with levamisole and this may have an effect on production of AChE by the nematodes (Sharpe & Lee, 1981).

levels has been correlated to the levels of resistance in some arthropods (Iwata & Hama, 1972). It has also been suggested that the esterases may prevent activity of the insecticides by non-specific binding of enzyme to the insecticide (Devonshire, 1975). It is therefore possible that non-specific binding of AChE to some anthelmintics may occur, resulting in their inactivation in resistant strains of nematode. Benzimidazoles have no ester group and are therefore unlikely to be broken down by the esterases in resistant nematodes.
IMMUNE MODULATION/ANTI-INFLAMMATORY ROLE FOR NEMATODE AChE

AChE

AND

ANTHELMINTIC

RESISTANCE

An intriguing finding, and one that still requires a satisfactory explanation, is that the third-stage larvae of strains of H. contortus, Ostertagia circumcincta and T. colubrz~ormis that are resistant to thiabendazole contain significantly greater amounts of AChE than susceptible strains (Sutherland, Lee & Lewis, 1988, 1989). Sutherland (1987) also showed that the third-stage larvae of strains of 0. circumcincta and T. colubr$ormis, known to be resistant to both morantel tartrate and levamisole, had significantly higher amounts of AChE than susceptible strains. There have been many studies on the role of elevated levels of esterases in insecticide-resistant arthropods. It is thought that the enzyme may be responsible for hydrolysis of organophosphate insecticides (Sawicki et al., 1978). The rise in enzyme

It has been suggested that one of the functions of AChE secreted by nematodes is to modulate the immune system of the host (Pritchard, 1993; Rhoads, 1984). The cholinergic system plays a role in the neuro-immune network of mammals with a high degree of integration between cells of the immune system and the chohnergic system (Rinner et aZ., 1995). Mezey & Palkovits (1992) have shown that immunocytes, including plasmocytes and macrophages, in the lamina propria of the alimentary tract possess muscarinic receptors and that they play a role in certain inflammatory diseases, such as gastric and duodenal ulcers, by stimulating gastric acid secretion. AChE produced by nematodes, such as Haemonchus that inhabit the stomach or and Ostertagia, abomasum might reduce inflammation and local ulceration by hydrolysing ACh which stimulates this gastric acid secretion. It is interesting to note that these 2 nematodes decrease pH in the abomasum. It is known that muscarinic receptors occur on the surface of white blood cells (Costa et al., 1994) and plasma cells can respond to ACh by increasing secretion of immunoglobulins (Brink et al, 1994). Acetylcholine enhances release of lysosomal enzymes by polymorphonuclear leukocytes and neutrophils during uptake of immune complexes (Ignarro, 1974; Ignarro & Colombo, 1973; Ignarro & George, 1974; Zurier et al., 1974). Ignarro & George (1974) and Zurier et al. (1974) have suggested that this release of lysosomal enzymes from neutrophils is mediated by neutrophilic cyclic guanosine 3,5-monophosphate (cyclic GMP) and that ACh is capable of modulating enzyme release through the selective elevation of intracellular levels of this cyclic nucleotide. Lysosomal enzymes are mediators of acute inflammation and are discharged from polymorphonuclear leukocytes during phagocytosis or cell-surface contact with immunological reactants resulting in inflammatory lesions. As ACh appears to play an important role in this process it is possible that AChE secreted by nematodes may interfere with the process

Invited

Review

505

by hydrolysing the ACh before it reaches its target cells, thus reducing inflammation in the immediate vicinity of the nematodes. ACh also enhances phagocytosis by neutrophils through muscarinic receptors on the neutrophils (Ignarro & Cech, 1976) and enhances neutrophil chemotaxis (Hill et al, 1975). Polymorphonuclear leukocytes are able to mediate the immunologically specific cytolysis of antibody-coated target cells; as ACh elevates cyclic GMP and as this regulates lymphocyte-mediated antibody-dependent cellular cytotoxicity (ADCC) (Gale & Zighelboim, 1974) hydrolysis of the ACh by nematode AChE may interfere with ADCC thus giving some protection to the nematodes. Mast cells are a source of slow reacting substance of anaphylaxis (SRS-A) and of histamine, which are important mediators of immediate-type hypersensitivity; ACh enhances antigen-induced release of these substances from IgE-sensitized tissues, apparently through muscarinic receptors on the surface of the cells (Kaliner et al., 1972; Kaliner & Austin, 1975). ACh also enhances release of eosinophil chemotactic factor of anaphylaxis after exposure to specific antigen (Tauber, Kaliner, Stechschulte & Austen, 1973). Hydrolysis of the ACh by nematode AChE might inhibit release of these compounds thus enhancing the survival of the nematode. It has been claimed that enkephalins and substance P, which plays a role as a proinflammatory mediator through degranulation of mast cells and activation of short and long visceral reflexes and promotion of granulocyte infiltration into intestinal tissue, are inactivated by AChE (Chubb, Hodgson & White, 1980; Chubb, Ranieri, White & Hodgson, 1983; Small & Chubb, 1988). If this was so then it could be an important role for AChE secreted by nematodes. However, considerable doubt has now been thrown on this peptidase activity of AChE as Checker & Vincent (1989, 1995) have shown that it is probably attributable to contaminating peptidases. Platelet-activating factor is a mediator of inflammation and is synthesized by neutrophils, monocytes, macrophages, platelets, eosinophils, mast cells, vascular endothelial cells and fibroblasts (Blackburn & Selkirk, 1992b). It has been found at elevated levels in the gastrointestinal tract during expulsion of Nippostrongyfus in primary infections in rats. Secretions from adult Nippostrongylus inhibit platelet-activating factor and it is suggested that this may be due to direct cleavage of it by AChE secreted by the nematode (Blackburn & Selkirk, 1992b). These authors suggest that AChE may play a role in down-regulating immune effector mechanisms which promote expulsion of the nematode from the gastro-

intestinal tract. Riga et aZ. (1995) also suggested that the AChE secreted by S. trachea may act as an immunomodulatory agent. It is possible, therefore, that acetylcholinesterases secreted by nematodes in the alimentary tract, and in other locations in the body of the host, may have an important role in immunomodulation by inactivating the acetylcholine which plays such an important part in the release of substances which induce acute inflammation of the hosts tissues and are detrimental to the survival of the nematodes.

CONCLUSIONS

AChE is produced and secreted in varying amounts by different stages of the life cycle of many nematodes that inhabit the gastrointestinal system, or other locations, of their animal hosts. The theory that it acts as a biochemical holdfast appears to have been disproved, at least for some nematodes, as a vasoactive intestinal polypeptide-like protein appears to be responsible for the reduction in motility of the hosts intestine. Of the several other possible functions for secreted AChE the most likely is modulation of the hosts inflammatory and/or immune responses.

REFERENCES

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