Inhibitory effect of heparin on red blood cell invasion by Theileria sergenti merozoites

bitory Effect of Heparin on Red CeZl Invasiun by Theileria sergerati Mer~zoi~~s KATSURO HAGIWARA,*t$ MASAYOSHI TSUJI,*

MIYUKI TAKAHASHI,* TSUTOMU ICHiKAWA,* KAZUYOSHI IKUTAt and CHIAKI ISHIHARA”

*School oJ’ Veterinary Medicine, Rakuno Gakuen University, Ebet.w, Hokkaido 069, Japun tSection of Serology, Institute of immunological Science, Hokkaido University. Kita-ku, Sapporo, Hokkaido 069. Japan (Received

29 November

1996: accepted

4 Februq

1997 I

Abstract-Hagiwara K., Takahaski M., Ickikawa T., Tsuji M., lkuta K. & lsbikara C. 1997. Inkibitory effect of keparin on red blood cell invasion by Z’heG’eria sergenti merozoltes. inter~thd Jwrd fi Pwws&&gy 27: 53!+539. Thderiu serge&i infection has been one of tke wost serious infectious diseases of cat& iti Japan. A major component in the patkogenesis of T. sergenti is anaemia. The erythcytk Rfe-eycle, wkkk is respoosibk for all of the clinkal manifestations of T. sergenti inkdon, is @h&d by inch of bovine red

factors Ltd.

for attachment

Keja words:

Theileria

to RBCs.

sergenti;

Q 1997 Australian

Society

for Parasitology.

bovine; invasion; hemagglutination;

Published

by Elsevier

Seknce

heparin

process of invasion by T. srrgenti hasbeendescribed

INTRODUCTION

at the light microscopelevel and by ultrastructural studieswith the electron microscope(Kawai et ~1.. 1993;Kawamotoet al., 1990a,b).However, the precise mechanismandmolecularbasisof T. sergeatiinvasion are still poorly characterized. Heparin, which is known as an inhibitor of the blood coagulation system(Jaques, 1979: McLean, 1959),hasbeenusedin the treatment of patientswith Plasmodiumfalciparum malaria (Mitchell. l974: Smitskamp& Wohhis, 1971).Heparintreatmentwas examinedin Rhesusmonkeysthat were infectedwith P. knowlesi(DennisSLConrad, 1968)and it hasalso beenshownto inhibit invasionand developmentof P. ,faleiparumin in vitro experiments(Butcher et al., L988; Kulane et al.. 1992; Sivaraman & Rai Chowdhuri. 1983).Low molecularweightheparin(LMW-hey&n) $ To whom correspondence should be addressed at: School has an antithrombotic effect without side-effectsNz of VeterinaryMedicine,RakunoGakuenUniversity,Bunuivo (Benderer al., 1988;@urna et al., 1990).II also kyoudai, Ebetsu 069, Japan. Tel: + 8 l-( 1 I)-386- 1111; Fax: has a slight inhibitory effect on invasion by f’ fi7i’+8141 I)-387-5890. Theileria sergenti is a protozoan parasite which is transmitted to cattle by ticks. The diseaseis widespreadin Japanand it isoneof the mosteconomically important diseases of cattle. The parasiteinfection by itself causesonly mild anaemia,but the majority of infected cattle becomelong-lasting carriers of this parasiteand occasionallythey developsevereandfatal anaemiaunder somestressconditions or following co-infection with other pathogenicmicro-organisms (Kawamoto et al., 1991; Minami et al., 1980; Takahashi, 1976).This diseaseresultsfrom repeated proliferation of the parasitesin the host’sred blood cells(RBCs). It is initiated by RBC invasion, and the

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K. Hagiwara

cipurwn merozoites (Kulane et (II., 1992). Recently, Shaw (1996) reported the inhibitory effect of proteoglycans such as heparin on sporozoite invasion of bovine lymphocytes by Theileria parvu. In this report, we focus on the inhibitory effect of heparin on the invasion process of RBCs by T. sergenti merozoites. We recently developed an in vitro experimental system for merozoite invasion (Hagiwara et al., 1996) and we have also developed a haemagglutination assay system to investigate the inhibitory effect of heparin and LMW-heparin on RBC attachment of T. sergenti merozoites. Using this system, we were able to show that heparin inhibited invasion of host RBCs by merozoites in a dose-dependent manner and, furthermore, both heparin and LMW-heparin blocked the haemagglutination by purified T. sergenti merozoites. Also, the haemagglutinating activity of merozoites was inhibited by treating them with heparinase. These results suggest that attachment of T. sergenti to host RBCs via a heparin-like molecule may play an important role in effective invasion.

suspensions were mixed with equal volumes of 2% bovine RBC suspension, and were incubated for I h at 37 C. The haemagglutination titre was determined as a reciprocal of the highest dilutions that showed haemagglutination as described previously (Beeley. 1985). For haemagglutination inhibition, 2511 of merozoite suspension containing a 7fold higher concentration than that required for complete agglutination, was incubated with an equal volume of 2-fold serial dilutions of sodium heparin or of low molecular weight sodium heparin (LMW-heparin, 50 units mg ‘: Sigma Chemical Co., St. Louis, MO) at 37’C for 1 h. The mixture was then added to 50~1 of a 2% bovine RBC suspension, incubated for another 1 h at 37,-C, and examined for haemagglutination. To investigate the inhibitory effect on haemagglutination of heparinase-treated merozoites, purified merozoites at a concentration of 2 x 10’organismsmll’ in PBS were incubated with 1 and 5 units of heparinase (heparinase 1: Sigma Chemical Co.. St. Louis, MO) for 1 h at 37°C and were then washed 3 times with PBS and resuspended at 2 x 10’mll’ in PBS. The merozoite suspension was then used for the haemagglutination assay. RBCs (2 x 10’ ml-‘) were also incubated with heparinase (5 U) and the treated RBC suspension (2% v/v) was used for the haemagglutination assay.

RESULTS MATERIALS

AND METHODS

Preparation of Theileria sergenti merozoites. Theileria sergenti merozoites (Chitose strain; Takahashi, 1976) were prepared as described previously (Shimizu et al., 1988; Sugimoto ef al., 1991). Briefly, RBCs were taken from a splenectomized T. serge&infected calf when its parasitaemia developed to about 15% and were haemolysed using high-pressure nitrogen gas. Free merozoites were obtained by density-gradient centrifugation, followed by 3 successive washings with phosphate buffered saline (PBS, pH 7.2). and resuspended at 2 x 109ml-’ in PBS. RBC invasion b.v Theileria sergenti merozoites and its inhibition by heparin. Blood was withdrawn from a naive Holstein heifer into 0.1 M citrate anticoagulant. RBCs were separated from platelet-rich plasma and white blood cells by differential centrifugation at 150g for 15min. The RBCs were passed through a cellulose column (CF-11; Whatman Biosystems Ltd.) to remove any remaining white blood cells. Then the RBCs were washed 3 times with PBS and resuspended in PBS at a final concentration of 2%. Merozoite invasion tests were done according to the method described by Hagiwara et al. (1996). In brief, 1 x lo* T. sergenfi merozoites were mixed with 2 x 10’ bovine RBCs in RPM1 1640 medium (GIBCO-BRL, Bethesda, MD) supplemented with 40% foetal bovine serum (FBS), followed by incubation for 2 h at 37°C. Then the mixtures were smeared on to a glass slide and stained with Giemsa’s stain. One, 10 or 50 units of heparin (140unitsmgg’: Sigma Chemical Co., St. Louis, MO) was added to the reaction mixture prior to the merozoite invasion test to examine the inhibitory effect of heparin on the merozoite invasion. The inhibitory effect of heparin on merozoite invasion was assessed by comparing the parasitaemia with the control RBCs. Haemagglutination and haemagglutination inhibition assay. For the haemagglutination assay. untreated or heparinasetreated merozoite suspensions containing 2 x lo9 organismsmll’ were diluted serially in 96-well round-bottomed microtitre plates. Fifty microlitres of the diluted merozoite

Inhibition of RBC invasion b-y Theileria sergenti merozoites by heparin To analyse the process of T. sergenti invasion, we first performed an in vitro invasion inhibition test by incubating 2 x 10’ bovine RBCs and 1 x 10’ T. sergenti parasites with various concentrations of heparin. Merozoite invasion of bovine RBCs was inhibited significantly by heparin; the invasion inhibition rate, in each of the 1, 10 or 50units of heparin-treated groups were 40.5%, 58.6% and 75.8%. respectively (Fig. 1).

Haemagglutination of bovine RBCs by Theileria sergenti merozoites and haemagglutination inhibition bv heparin To analyse the mechanisms responsible for the inhibition of merozoite invasion, we attempted to establish a haemagglutination test by using purified T. sergenti merozoites. Merozoites exhibited a haemagglutination which was first detected at a concentration of 5 x 10’ organisms ml-’ and developed further with an increase in the concentration of merozoites (Fig. 2A). Furthermore, this haemagglutination was inhibited significantly by heparin as well as by LMWheparin (Fig. 2B). Their inhibitory activity, however, differed; heparin blocked the haemagglutination at 0.39 mg ml-’ (54 units) whereas LMW-heparin inhibited haemagglutination at 3.12 mg ml ’ (150 units) (Fig. 2B). To investigate whether the haemagglutination inhi-

Invasion inhibition of T. sergenti by heparin

1

10

50

Heparin concentrations (units) Fig. I Inhibition of T. sergenfi merozoite invasion of red blood cells (RBCs) by heparin. The inhibitory effect of heparin on the invasion of T. sergenti was estimated as below: invasion inhibition (9/o) of T. sergenti = (invasion rate of heparm-treated group/invasion rate of untreated group) x 100. The mean of invasion inhibition rates and their standard deviations are shown. These results are representative of 3 separate experiments, bition by heparin or LMW-heparin was the consequence of a competitive inhibition for the adhesion property of merozoite surface components, we performed the haemagglutination test using heparinasetreated T. serge& merozoites. As shown in Fig. 3, heparinase treatment of 7’. sevgenfi, 1 unit for 2 x IO9 merozoites. resulted in an inhibition of haemagglutination, and as little as 5 units of heparin were enough for complete inhibition. In contrast, the haemagglutinating activity of the merozoites was observed even after treatment of bovine RBCs with 5 units of heparinase. DISCUSSION We investigated T. sergenti invasion of bovine RBCs by examining whether this invasion could be inhibited by heparin, since sulfated glycoconjugates such as heparin and dextran sulfate have been shown to block RBCs reinvasion by P. jdciparum merozoites (Butcher ef al., 1988; Kulane er al., 1992; Sivaraman & Rai Chowdhuri, 1983). In the experiments reported here, we have demonstrated for the first time that heparin significantly blocked T. sergenti invasion in nirro (Fig. 1) and heparin and LMW-heparin inhibited haemagglutination (Fig. 2B). These effects of heparin or LMW-heparin were likely to be a consequence of inhibition of merozoite attachment to bovine RBCs. Moreover, treatment of T. sergenti merozoites with heparinase (Fig. 3) resulted in eradication of the haemagglutinating activity. A preliminary experiment using an indirect fluorescent antibody binding tech-

i ;7

nique also revealed that T. sergenti merozoites bound anti-heparin-sulfate monoclonal antibodies {data not shown). These results suggest that some molecules on the surface of T. sergenti merozoites, but not on bovine RBCs, have a characteristic of sulfated glycoconjugates such as heparin, and that these molecules might have a role in RBC attachment for invasion by T. sergenti merozoites. In previous studies on the invasion of lymphocytes by T. parzu sporozoites. invasion inhibition was shown by sulfated plycoconjugates, but treatment of lymphocytes with hsparinase did not inhibit the invasion and heparin -gold conjugate did not bind to either the sporozoite or the host cell surface (Shaw. 1996). Thus the mechanism by which sulfated glycoconjugates inhibit invasion by T. par/yn sporozoites might be partly different from that of T. sergmti merozoites. The different r(rle of sulfated glycoconjugates in invasion by Theikm IIMV be due to the stage or the life cycle. The inhibitory activities of heparin and LMW-heparin on haemagglutination differed significantl! and the degree of this difference was very similar to that shown by Kulane et al. (1992) for P.,falciparun~. ‘Phr~ inhibitory activity of heparin does not vary according to the strain of P. ~falciparum (Kulane et al.. 1992: Rowe et ul., 1994). One explanation of the difference in inhibitory activity between heparin and LMW-hrparin may be the degree of sulfation and the struclurc of heparin. Taken together, these and our result% supgest a common mechanism for merozoite attachment to host RBCs by heparin-like surface molecules &‘ the parasites. The relatively poor haemagglutinating potency of the merozoites, requiring at least 5 x 10” organisms ml ’ (Fig. 2A) for the reaction. may be due to contamination with immature merozoites which could not attach to RBCs in the merozoite suspcnsiou. It is impossible to eliminate immature mero~ones from the infected RBCs by our purification method. Although the detailed mechanism of inhibition by heparin is yet to be determined. it has been suggested that sulfated glycoconjugates are used by other developmental stages of malaria parasites as invasion and cytoadhesion receptors (Rogerson rt ul., 199s; Rowe et ul., 1994). Our results also suggest that heparin-like molecules have a role in attachment to RBC’s by 71 ser’genti. Thus, some sulfated glycoconjugates may serve as one of the cytoadherence molecules for infc:ction by the parasite and the molecular structure of sulfated glycoconjugates may be one of the important factors for effective attachment to RBC’s. Further studies will be necessary to clarify the mechanism of haemagglutination inhibition by sulfated gly~oconjugates and to identify r. scrgmti merozoite surfaces molecuie which play a role in cytoadheslon tc) host cells.

538

K. Hagiwara

A t3N

0 tnerozo~tcs 2XlOVml Dilution

sertcs

1.6 x 1O’hn I

B 1 No merozoites

Dilution series

2 No merozoites

Dilution series

NO LMW-Hepnrin Fig. 2. (A) Haemagglutination of bovine RBCs by T. serge& merozoites. Purified T. sergenti merozoites (serially diluted) and bovine RBC suspension were incubated for 1 h at 37°C and then the mixture was assessed for haemagglutmation. (B) Haemagglutination inhibition by heparin. Two-fold serial dilutions of heparin or LMW-heparin (50-0.39mgml-‘) were added to the haemagglutination assay. Lane 1: heparin; lane 2: LMW-heparin.

Invasion

Heparinase

5U

Heparinase

1U

Heparinase

Xl

inhibition

of T. sergerlri

(vtytlirocyios)

Untreated Fig. 3. Effect of merozoites on their haemagglutination RBCs were treated agglutination test

Control

the heparinase treatment of T. sergenti haemagglutinating activity. Prior to the assay, purified T. sergenri merozoites and with heparinase (1 U or 5 U). The haemwas carried out as described in the text.

Acknowledgements---The authors wish to thank Dr T. Kurosawa, Mr K. Takahashi and MS Y. Kuribayashi (Rakuno Gakuen University) for their helpful advice and encouragement. This work was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

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by heparin

;:9

Kawamoto S., Takahashi K., Onuma M., Kurosawa 1”. 8 Sonoda M. 1990. Invasion of bovine erythrocytes by TheiIeria sergenti piroplasms in &ro. Japanese Journal o! F’cierinary Science 52: lZ!61-1263. Kawamoto S., Takahashi K., Onuma M., Kubota S., NeJo H., Kurosawa T. & Sonoda M. 1991. Rebound phenomenon of parasitemia in splenectomized calves primarily infected with Theileria sergenti. Japanese Journal of I’,;!erinarj, Science 53: 1:!7-128. Kulane A.. Ekre H.-P.. Perlman P., Rombo L., Wahlgren M. & Wahlin B. 19921. Effect of different fractions of heparin on Plasmodium~‘hlciparum merozoite invasion of red blood cells in f)itro. A,nerican Journal of Tropic& M~,dic~mr and Hygiene 46: 589-594. ('irculafiwl 19: McLean J. 1959. The discovery of hepdrin. 75-78. Minami T.. Fujinaga T.. Furuya K. & Ishihara T. l%(l. Clinico-hematologic and seroIogic comparisons Of Japanese and Russian strains of Theikeria ,wrgrnr~. National Institute of’ Animul Health Quarterl~~ , Jupa>l) 20: 44-52. Mitchell A. D. 1974. Recent experience with severe and cerebral malaria. South African Medical Journal 4& ! 353 1354. Oguma Y.. Sakuragawa N.. Maki M.. Nakagawa M. 6i Hasegdwa H. 1990. Clinical effect of low molecular weight heparin (fragmin) on DIC: a multicenter cooperative stud! in Japan. Thrombosis Research 59: 37-49. Rogerson S. J., Chaiyaroj S. C.. Ng K., Reeder J. C-. & Brown G. V. 1995. Chondroitin sulfate A is a cell surface receptor for Plasmodrum,f&iparum-infected erythrocyte,. Journal qf Experimmtal Medicine 182: 15 -20, Rowe A.. Berendt A. I?.., Marsh K. & Newbold C I. IY94. Plasmodium f&iparum: a family of suif:dted glkcoconjugates disrupti erythrocyte rosettes. E.rprrimentn/ Parasitology 79: 506 5 16. Shaw M K. 1996. Characterization of the parasite-ghost cell interactions involved in Theileria parou sporozolte invasion of bovine lymphocytes. Para.siroiog\. 113: X7 277. Shimizu S.. Suzuki K.. Nakdmura K., Kadota K.. Fujisaki K., Ito S. & Minami 1‘. 1988. Isolation of Theileriu .