INFECTION AND IMMUNITY, Sept. 1986, p. 606-6100019-9567/86/090606-05$02.00/0Copyright © 1986, American Society for Microbiology

Differences in Sensitivity of Schistosoma mansoni Schistosomula,Dirofilaria immitis Microfilariae, and Nematospiroides dubius Third-

Stage Larvae to Damage by the Polyamine Oxidase-PolyamineSystem

ANTONIO FERRANTE,l2* INGER LJUNGSTROM,2 CHRISTINE M. RZEPCZYK,3 AND DAVID M. L. MORGAN4

Department of Paediatrics, University of Adelaide, South Australia 5001, Australial; The National BacteriologicalLaboratory, Stockholm, Sweden2; Queensland Institute of Medical Research, Brisbane, Australia3; and Medical Research

Council Clinical Research Centre, Harrow, Middlesex, England4

Received 10 March 1986/Accepted 4 June 1986

The effect of the polyamine oxidase (PAO)-polyamine system on some helminths was examined in vitro. BothSchistosoma mansoni schistosomula and Dirofilaria immitis microfilariae were highly sensitive to this system,the latter more so than the former. In contrast, exsheathed third-stage larvae of Nematospiroides dubius wereresistant to the effects of the PAO-polyamine system. After incubation of microfilarie with either spermine orspermidine in the presence of serum containing PAO (bovine serum or human retroplacental serum) or

partially purified PAO, damage of worms occurred, compatible with our criteria for worm death. Similarresults were obtained with schistosomula by using spermine. The damage seemed to be mediated by PAOproducts other than hydrogen peroxide because catalase did not protect either parasite. Our data demonstratethat helminths may be damaged by products of the PAO-polyamine system.

Polyamine oxidases (PAOs) have been found in mostmammalian tissues (15, 24). Studies in the past have concen-trated mainly on PAOs from two sources, bovine serum andrat liver (13-15). PAO from bovine serum acts on theprimary amino groups of spermine to form an aminodialde-hyde, while rat liver PAO cleaves spermine at secondaryamino groups, with the formation of spermidine and 3-aminopropionaldehyde (13; Fig. 1). In both reactions, hydro-gen peroxide (H202) is formed. Preliminary studies have alsobeen conducted with macrophage and human pregnancyserum PAOs. These resemble the rat liver enzyme (14, 15,18; Fig. 1).Recently it was shown that trypanosomes are rapidly

killed when incubated with PAO or sera containing PAO andpolyamines (6, 7). Although this system was postulated to beof special relevance as a nonspecific trypanocidal mecha-nism operating in ruminants (6), it may be of more generalimportance since PAOs may be elevated during some infec-tions in nonruminants and during human pregnancy (13, 15,20). Also, macrophages, when activated, have been shownto contain higher quantities of PAOs and appear to secretethe enzyme(s) (20).

In view of these findings, it was of interest to determinethe susceptibility of helminths to the products generatedduring the oxidation of polyamines by both the bovine serum

and human pregnancy serum PAOs. We studied Schistoso-ma mansoni schistosomula, Dirofilaria immitis microfilariae,and exsheathed third-stage larvae of Nematospiroidesdubius.

MATERIALS AND METHODS

Parasites. Microfilariae of D. immitis were prepared fromfreshly collected, heparinized dog blood by filtration throughpolycarbonate filters (pore size, 5 ,um) and were used afterovernight incubation at 37°C (22). More than 95% of

* Corresponding author.

microfilariae showed normal motility after recovery by thisprocedure.

S. mansoni Puerto Rican strain was maintained by passagein laboratory-bred albino Biomphalaria glabrata snails andhamsters essentially as described previously (25). Theschistosomula used were prepared from cercariae by me-

chanical transformation (4, 10).Exsheathed third-stage larvae of N. dubius were kindly

provided by Charles R. Jenkin, Department of Microbiologyand Immunology, University of Adelaide.

Sera. Bovine serum was obtained from 9- to 14-month-oldcows. Human retroplacental serum (RPS), which was com-

posed mainly of intervillous blood and included somedecidual and placental interstitial fluid, was prepared as

previously described (19). All sera were heated to 56°C for 20min and stored in portions at -70°C.Enzymes. PAO (beef plasma, EC 1.4.3.6) was obtained

from Miles Laboratories, Goodwood, South Africa. This isdescribed as amine:oxygen oxidoreductase (deaminating)with a specific activity of 23.55 U/g of protein, where 1 U isthe amount of enzyme required to form 1 ixmol ofbenzylaldehyde per min from benzylamine at 25°C. Catalase(bovine liver, EC 1.11.1.6) was purchased from SigmaChemical Co., St. Louis, Mo.

['4CJspermine oxidation assay. PAO activity was measuredby a radiochemical method (8, 20). To a 0.1 ml of a

1,000-Rg/ml mixture of purified beef plasma PAO was added0.1 ml of medium substrate solution (radioactive spermine,0.415 p.Ci/ml plus 5 mM cold spermine [1.17 ,ug/ml]), 0.05 mlof human serum (added as carrier protein with no effect onactivity), and 0.05 ml of medium. These were incubated at37°C for 90 min, and then 0.05 ml of 50% (wt/vol)trichloracetic acid was added to stop the reaction. Theprotein was sedimented by centrifugation at 400 x g for 10min, and the products in the supernatant were separatedfrom unreacted spermine by column chromatography on a

strong cation exchange resin (Dowex-50W-X2 resin).

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PAO-MEDIATED DAMAGE OF HELMINTHS 607

NH2(CH2)3NH(CH2)4NH(CH2)3NH2 + 202 + 2H20-Spermine

OCH(CH2)2NH(CH2)4NH(CH2)2CHO + 2NH3 + 2H202N,N1 -Bis(3-propionaldehyde)-1,4-diaminobutane

NH2(CH2)4NH(CH2)3NH2 + 02 + H20-

Spermidine

NH2(CH2)4NH(CH2)2CHO + NH3 + H202N-(4-Aminobutyl)-3-aminopropionaIdehyde

NH2(CH2)3NH(CH2)4NH(CH2)3NH2 + 02 + H20

Spermine

NH2(CH2)3NH(CH2)4NH2 + NH2(CH2)2CHO + H202

Spermidine 3-Aminopropanal

NH2(CH2)2NH(CH2)4 NH2 + 02 + H20Spermidine

NH2(CH2)2CHO + NH2(CH2)4NH2 + H202

3-Aminopropanal Putrescine

FIG. 1. Action of bovine plasma PAO (top) and rat liver or RPSPAO (bottom) on spermine and spermidine.

Spermine and reaction products were eluted with a stepwisegradient of hydrochloric acid, and the percent substrateconverted was calculated. The enzyme activity was ex-pressed in international units (micromoles of product formedper minute). The activity in RPS was similarly assayed byusing 0.1 ml of RPS in the assay. The PAO activity was

33,000 mU/g in the commercial PAO preparation materialand 5,000 mU/liter in the RPS.

Reagents. Spermine tetrahydrochloride and sperminetrihydrochloride were obtained from Sigma Chemical Co.;[14C]spermine (N'N'-bis[3-aminopropyl]-[1,4-14C]tetrameth-ylene-1,4-diamine trihydrochloride) was from AmershamCorp., Amersham, England.

Experimental assays. Experiments were set up in flat-bottomed microtiter plates (Linbro; Flow Laboratories,Inc., McLean, Va.). To 50 ,ul of the worm suspensioncontaining either 100 microfilariae, 100 N. dubius larvae, or50 schistosomula was added 50 ,zl of the PAO-containingserum or purified PAO, to the concentrations indicated inResults and Table 1, and 100 ulI of a polyamine concentra-tion. Controls were set up in which worms were incubatedwith one of the following: PAO preparation or serum,

polyamines in human serum albumin, or albumin only (theuse of bovine serum albumin was avoided since this may becontaminated with PAO). The plates were incubated at 37°Cfor 18 h (unless otherwise specified) in an atmosphere of 5%CO2 and high humidity. At the indicated times the organismswere examined with an inverted microscope under phase atx200 magnification. Death was scored as follows: forschistosomula, loss of motility, obvious tegument deforma-tions, and granular appearance were the main criteria; formicrofilariae and N. dubius larva, loss of motility was themain criterion indicating death (organisms which were im-mobile during the period of scoring were considered dead).

Indirect immunofluorescence. Antibody response to schis-tosomula injection was measured against cryostat sections offreshly recovered adult S. mansoni. Fluorescein-labeledsheep-antimouse immunoglobulin was used as a second

antibody to detect binding of mouse antibodies to schisto-somula antigens.

RESULTSEffects of bovine serum and polyamines. Examinations of

microfilariae and schistosomula after incubation with 10%bovine serum and 100 ,uM spermine for 18 h indicated thatthese conditions resulted in worm damage. Most schistosom-ula lost motility and showed morphological changes consist-ent with severe structural damage and schistosomula death.The surface of the tegument exhibited deformations andelaborations, and some of the worms showed extrusion oftheir content. All the microfilariae lost motility and straight-ened. Microfilariae and schistosomula incubated in eitherbovine serum or spermine alone did not display the effectsdescribed above and retained normal motility and appear-ances.

Effects of partially purified PAO. Microfilariae and schis-tosomula were incubated in the presence of 100 ,ug of PAOper ml and a range of concentrations of spermine or spermi-dine for microfilariae and spermine for schistosomula. ThePAO, in the presence of polyamines, mediated damage inschistosomula and microfilariae (Fig. 2, 3). The minimalconcentrations of spermine and spermidine producing dam-age in the majority of the microfilariae were approximately 5and 10 ,uM, respectively (Table 1). The minimal concentra-tion of spermine required to produce similar levels of dam-age in schistosomula was approximately 100 ,uM (Table 1).The PAO alone had no effect on the worms. The spermineand spermidine dose-related effects are presented formicrofilaria in Fig. 4, and the PAO dose-related effect ispresented in Fig. 5.Time to effect damage was dependent on the concentra-

tions of PAO and polyamines. In the presence of 50 ,ug ofPAO per ml and 25 ,uM spermine, 90% of microfilariae wereimmobilized after 90 min. In the presence of 100 ,ug of PAO

A

BFIG. 2. Photomicrograph showing the typical appearance of

schistosomula after treatment with PAO and spermine (A) and withspermine alone (B). The parasite lacked mobility, and showeddisruption of tegument, membrane elaborations, and granular ap-pearance after treatment with PAO and spermine. This contrastedwith the appearance of schistosomula in control cultures, e.g.,treated with spermine only, in which the parasites showed mobilityand were not damaged.

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608 FERRANTE ET AL.

A UL)

BFIG. 3. Photomicrograph showing the typical appearance of

microfilariae treated with PAO and spermine (A) and spermine only(B). The parasites lacked motility, straightened, and shrank aftertreatment with PAO and spermine. This contrasted with the appear-ance of microfilariae in control cultures, e.g., treated with spermineonly, in which the parasites retained normal motility similar to thatexhibited by worms freshly prepared from dog blood.

per ml and 200 ,uM spermine, >90% of schistosomula weredamaged after 120 min.

Preincubation of 100 ,ug of PAO per ml and 100 ,uMspermine for 1 h and subsequent heat inactivation of theenzyme (65°C for 1 h) did not decrease the capacity of thissystem to damage either microfilariae or schistosomula. Incontrast PAO heated (65°C for 1 h) before spermine additionhad no effect.Larvae of N. dubius were insensitive to the PAO-

polyamine system. Incubation in the presence of 1 mMspermine and 1 mg of PAO per ml for 3 to 4 days had noeffect on the motility of these worms.An attempt was made to assess the damaging effect of the

PAO-polyamine system in relation to the ability ofschistosomula to develop into adult worms in mice.Schistosomula were incubated in vitro with 100 ,ug of PAOper ml and 200 ,uM spermine for 18 h and then injectedintravenously into five mice. Other groups of mice were

TABLE 1. Effect of partially purified PAO and polyamines and ofRPS and polyamines on microfilariae and schistosomula

Minimal polyamine concn (,uM)Parasite required to damage':treatment

Microfilariae Schistosomula

Partially purified PAO(100 ,ug/ml)Spermine 5, 5, 2.5 200, 200, 100, 100, 100Spermidine 10, 10, 5 NDb

RPS (10%)Spermine 62.5, 62.5, 31.25 200, 100, 100Spermidine 125, 125, 62.5 NDa Damage criteria: microfilariae, >80% demonstrating immobility: schisto-

somula, >80% demonstrating immobility, opaque granular appearance, andmembrane deformations. Values represent determinations made in separateexperimental runs. The incubation time was 18 h.

b ND, Not done.

Polyamine WM)FIG. 4. Effect of different concentrations of spermine and sper-

midine on the antimicrofilarial action of the PAO-polyamine reac-tion. Symbols: 0 and *, spermine and spermidine, respectively, inthe presence of 100 ,ug of PAO per ml; 0, spermine alone; O,spermidine alone. Examination of cultures was made after 18 h ofincubation, and results are expressed as the means plus or minusstandard deviations of five experiments for spermine and of threeexperiments for spermidine.

injected with PAO- or spermine-treated schistosomula. After8 weeks mice were perfused for adult worm recovery. Serumwas also collected from these mice. No adult worms wererecovered from mice receiving schistosomula exposed toPAO plus spermine. Antibodies to tegument and somaticantigens but not to gut-associated antigens were present insera from these mice. Adult worms were recovered (12 to16% recovery) from mice receiving schistosomula treated

100

80-

=)60-/

40-

20-

0*0 5 10

PAO (jig/ml)FIG. 5. Effect of different concentrations of PAO on the antimi-

crofilarial action of the PAO-polyamine reaction. Cultures containedPAO and 50 ,uM spermine. Examination of cultures was made after18 h of incubation, and results are expressed as means plus or minusstandard deviations of three experiments.

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PAO-MEDIATED DAMAGE OF HELMINTHS 609

40

20-

0

0 50 100 150 200 250Polyamine (juM)

FIG. 6. Effect of different concentrations of spermine and sper-

midine on the antimicrofilarial action of the RPS PAO-polyaminereaction. Symbols: * and *, spermine and spermidine, respec-

tively, in the presence of 10% RPS; 0 and O, spermine andspermidine, respectively in the presence of human male AB serum

(10%). Examination of cultures was made after 18 h of incubation,and results are expressed as means plus or minus standard devia-tions of three experiments.

with PAO only, and sera from these animals containedantibodies to tegument, somatic, and gut-associated anti-gens. Although no adult worms were recovered from micetreated with spermine alone, there was evidence of an

infection based on the serology results which showed thepresence of antibodies to gut-associated antigens.

Effects of catalase on PAO-mediated worm damage. PAO(40 jxg/ml) and 10 ,uM spermine were preincubated in thepresence or absence of 1,000 U of catalase per ml for 15 minbefore the addition of microfilariae. Worm damage was

scored after 18 h of incubation. Catalase was unable toabolish the PAO-mediated damage of microfilariae, indicat-ing that the effects were not due to hydrogen peroxide (datanot shown). Similar results were obtained with schistosom-ula by using 100 ,ug of PAO per ml and 200 jiM spermine.This result was obtained in four experiments.

Effect of acrolein. Microfilariae were incubated with a

range of concentrations of acrolein, and worm damage was

scored after 2 h of incubation. A typical experimental run

showed that 25 jiM acrolein or 5 FM spermine (the latter inthe presence of excess PAO) was required to achieve dam-age in at least 90% of worms.

Effect of RPS and polyamines. Microfilariae or schistosom-ula were incubated with 10% RPS and a range of concentra-tions of polyamines. Both were damaged when incubated inthe presence of RPS and polyamines. The appearance of theworms was similar to that after their incubation in bovinePAO and polyamines. The minimal concentrations ofspermine and spermidine producing damage of the majorityof the microfilariae were approximately 60 and 125 jiM,respectively (Table 1). The minimal concentration ofspermine to achieve marked damage to the majority of theschistosomula was 100 jiM (Table 1). A polyamine dose-related effect on microfilariae is presented in Fig. 6. Thiseffect was not observed with either RPS or polyamines alone(i.e., in human AB serum).

DISCUSSION

Previously, evidence was presented showing that proto-zoan parasites of the genera Plasmodium and Trypanosoma,in addition to tumor cells (1), are highly sensitive to productsof the PAO-polyamine reaction (6-8, 16, 17, 23). We nowpresent evidence that some helminth parasites were alsohighly sensitive to this system. Dirofilaria immitis micro-filariae were immobilized by relatively small concentrationsof spermine or spermidine in the presence of either PAO-containing bovine serum or a partially purified PAO prepa-ration. Similarly, schistosomula were markedly damagedwhen incubated with spermine and either bovine serum orPAO. However, exsheathed third-stage larvae of N. dubiuswere resistant to the damaging effects of the PAO-polyaminesystem. The killing of the parasites appeared to be a result ofextracellulary generated toxic products rather than an effectcaused by the uptake of PAO and spermine by the parasites.Evidence showed that preincubation of PAO and spermineand subsequent heat inactivation of PAO did not decreasethe ability of PAO to kill the worms.A number of products formed during the oxidation of

spermine were shown to be toxic to microorganisms andtumor cells. These included H202, aminodialdehyde or 3-aminopropionaldehyde, and acrolein. H202 is a productcommon to both bovine PAO- and RPS PAO-mediatedpolyamine oxidation. While microfilariae are highly sensitiveto H202 concentrations expected to be generated in thePAO-polyamine reaction (22), the inability of catalase toprevent damage suggests that other products besides H202are responsible for the effects of PAO-polyamine reaction.Acrolein, a breakdown product of the primary products,aminodialdehydes and aminoaldehydes, was found to betoxic for microfilariae, and under conditions similar to thosein the experiments, acrolein could be detected during bovinePAO-polyamine reaction (8). However, it is unlikely thatacrolein plays a significant part in the killing mediated byPAO-polyamines for two reasons. First, we found that 5times as much acrolein was required to get the same level ofdamage as that expected to be generated at the lowestconcentrations of polyamines causing >90% of worms to bedamaged. Second, the aminoaldehydes were highly reactive,and their reaction with microfilariae would greatly limit theconcentration of acrolein generated.H202 is unlikely to be the main product responsible for

schistosomula damage for two reasons. First, we wereunable to prevent damage of schistosomula in the PAO-polyamine system by the addition of catalase. Second, it hasbeen reported that schistosomula are not sensitive to H202(11). Superoxide does not appear to be detectable during thereaction of polyamines with PAO (15). Thus, we have notconsidered this oxygen metabolite in the mechanisms ofPAO-mediated worm damage.

Ferrante et al. (8) demonstrated that human RPS PAO,when reacted with polyamines (spermine or spermidine),generates trypanocidal products. Both microfilariae andschistosomula were sensitive to the RPS PAO-polyaminesystem. Studies with Babesia and Plasmodium species haveshown that, although these parasites are susceptible to theaction of bovine serum PAO, the enzyme present in RPSappears not to be able to mediate killing of intracellularhematoprotozoa (16, 17) including P. falciparum (C. M.Rzepczyk and A. Ferrante, unpublished data). The presentresults with RPS thus suggest a more general relevance ofthe PAO-polyamine system.

Since it is believed that the human serum (RPS) PAO

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610 FERRANTE ET AL.

resembles the macrophage PAO (14, 15, 18), macrophagesmay be involved in the damage and killing of some microor-ganisms by utilizing the PAO-polyamine system. Morganand Christensen (16) have shown that spleens of miceinfected with B. microti contained a threefold increase inPAO specific activity. In addition, the spleen size was asmuch as fourfold that of normal mice, showing a markedincrease in total enzyme activity per spleen. Macrophagesexposed in vitro to lipopolysaccharide appear to secretePAO (18). These preliminary studies suggest that, uponactivation, the macrophage may have increased quantities, ifnot increased activity, of PAO. However, it remains to beexplored whether these changes significantly contribute tomacrophage-mediated killing mechanisms.Macrophages in cooperation with immunoglobulin E im-

munocomplexes mediate schistosomula cytotoxicity (3). Inaddition, other studies have shown that macrophages, acti-vated by injecting mice with Mycobacterium bovis BCG orCorynebacterium parvum, killed schistosomula (12). Simi-larly, lymphokine-activated macrophages developed anti-schistosomula activity (2). The mechanisms by which acti-vated macrophages mediate killing of schistosomula are stillunder investigation. Our results suggest that the PAO-polyamine system could contribute to schistosomula killingby activated macrophages. However, the significance of ourfinding in relation to PAO-polyamine killing of D. immitismicrofilariae is less clear since the principal effector cellinvolved in the killing of this parasite (5, 22) is the neutrophiland we have no information on whether PAO is present inthis cell type.There was clear evidence from our study that, under in

vitro conditions, microfilariae of D. immitis and schistosom-ula of S. mansoni are damaged by the PAO-polyaminesystem. Although there was some difficulty in interpretingthe effects of this system on the ability of schistosomula todevelop into adult worms, serological evidence did suggestthat PAO-spermine-treated parasites, but not those treatedwith spermine or PAO alone, developed into adults. It isplausible to envisage an in vivo relevance for this system.First, tissue fluids of some animals (ruminants) containnaturally occurring high levels of PAO activity. In otheranimals the levels may rise during an infection, such as theincreases that occur in spleens of Babesia-infected mice or insera of some patients with hepatitis (13, 16). Second,polyamines are present in all living tissues, and their con-centrations in tissue fluids rise dramatically as a conse-quence of tissue damage and regeneration (9). Parasites canthemselves be a source of polyamines. Indeed, it has beenrecently reported in relation to African trypanosome infec-tions that resistant cattle have significantly higher levels ofPAO than susceptible breeds (21). However, any in vivo rolefor this system should be cautiously extrapolated since thelevels of enzyme and substrate as well as the existence ofoptimal conditions for the oxidation reaction at sites ofparasite microenvironments are not known.

ACKNOWLEDGMENTS

We are grateful to R. B. Atwell, Department of VeterinaryMedicine, University of Queensland, and Gunnel Huldt, NationalBacteriological Laboratory, Stockholm, for their advice.

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