nitric oxide causes the macroschizonts of theileria annulata to disappear and host cells to become...

NITRIC OXIDE CAUSES THE MACROSCHIZONTS OFTHEILERIA ANNULATA TO DISAPPEAR AND HOST CELLSTO BECOME APOPTOTIC

J.O. RICHARDSON1, L.M.G. FORSYTH1, C.G.D. BROWN2 ANDP.M. PRESTON1*

1Institute of Cell, Animal and Population Biology, Ashworth Laboratories, Division ofBiological Sciences, University of Edinburgh, King's Buildings,West Mains Road,Edinburgh, EH9 3JT; 2Centre for Tropical Veterinary Medicine, Royal (Dick) Schoolof Veterinary Studies, University of Edinburgh, Roslin, Midlothian, EH25 9RG, UK*Correspondence

ABSTRACT

Richardson, J.O., Forsyth, L.M.G., Brown, C.G.D. and Preston, P.M., 1998. Nitric oxide causes themacroschizonts of Theileria annulata to disappear and host cells to become apoptotic.Veterinary ResearchCommunications, 22(1), 31^45

The proliferation of Theileria annulata macroschizont-infected cell lines in vitro was signi¢cantlyinhibited by nitric oxide (NO) generated by S-nitroso-N-acetyl-dl -penicillamine (SNAP). Incubationwith SNAP caused the macroschizonts to disappear and host cells to become apoptotic. SNAP-derivedNO also signi¢cantly inhibited the incorporation of tritiated thymidine by cultures of cells in which theschizonts had been induced to di¡erentiate into merozoites by maintenance at 418C instead of 378C, thetemperature used for culturing macroschizont-infected cells. These results point to NO as the mediatorof macrophage anti-T. annulata activity and provide new evidence that the protective immunemechanisms which allow cattle to recover from primary infection and resist challenge may be attributedprincipally to the products of activated macrophages. These ¢ndings indicate that e¡ective inactivatedvaccines against T. annulata should include antigens able to stimulate the type of CD4+ T cell responsewhich elicits macrophage activation and NO synthesis.

Keywords: apoptosis, macroschizont, merozoite, nitric oxide, SNAP, S-nitroso-N-acetyl-dl -penicill-amine, Theileria annulata

Abbreviations: ABC-HRP, avidin^biotin-conjugated horseradish peroxidase system; B cells, bonemarrow-derived cells; BL-20, bovine lymphosarcoma-20 line; CD, cluster of di¡erentiation; cpm,counts per minute; DAB, 3,3'-diaminobenzidine tetrahydrochloride; dpm, disintegrations per minute;dUTP-biotin, biotinylated deoxyuridine; IFN-a, interferon-alpha; IFN-g, interferon-gamma; IL-1,interleukin-1; IL-2, interleukin-2; NK cells, natural killer cells; PBM, peripheral blood mononuclearcells; SNAP, S-nitroso-N-acetyl-dl -penicillamine; Ta, T. annulata; TaH BL-20, BL-20 infected with T.annulata (Hisar); T cells, thymus-derived cells; TdT, terminal deoxynucleotidyl transferase; TH1 cell, T-helper 1 cell; TNF-a, tumour necrosis factor-alpha; TUNEL, TdT-mediated dUTP-biotin nick endlabelling

INTRODUCTION

Bovine tropical theileriosis (Dschunkowsky and Luhs, 1904) is caused by the tick-borne protozoan parasite Theileria annulata. Infection is initiated by the invasion ofcells by sporozoites injected by the tick vector and their transformation and prolifera-

Veterinary Research Communications, 22 (1998) 31^45Copyright#Kluwer Academic Publishers bv ^ Printed in the Netherlands

31

tion as macroschizont-infected cells. The parasites subsequently di¡erentiate to formmerozoites via the so-called macroschizont stage. Released from their host cells, themerozoites enter erythrocytes and the parasite is available to ticks. Macroschizont-infected cell lines may be grown in vitro at 378C (Brown, 1983), growth of the schizontand host cell being synchronous and interdependent (Hulliger et al., 1964). Di¡erentia-tion of the parasite from macroschizont to merozoite can be induced in vitro byincreasing the temperature at which the cultures are incubated to 418C (Hulliger etal., 1966), and involves a disruption of the synchrony of parasite growth and celldivision (Shiels et al., 1992).The work reported here was part of a study planned to identify the mechanisms

responsible for macrophage-mediated cytostasis of macroschizont-infected cell lines(Preston and Brown, 1988). When recombinant bovine tumour necrosis factor-alpha(TNF-a) was found to enhance rather than inhibit the growth of macroschizont-infected cells (Preston et al., 1992a), nitric oxide (NO) was assessed as a potentialmediator of macrophage anti-Theileria activity. The initial ¢ndings of that studyshowed that activated bovine macrophages can produce NO and that NO, derivedfrom S-nitroso-N-acetyl-dl -penicillamine, a well-documented producer of NO (Ignar-ro et al., 1981), prevents T. annulata trophozoite-infected cells transforming intomacroschizont-infected cell lines (Visser et al., 1995). Since established macroschi-zont-infected cell lines were una¡ected by the levels of NO (7,20 mmol/L) that inhibitedthe establishing trophozoite-infected cells, the experiments described below werecarried out to see whether macroschizont-infected cells were susceptible to increasedlevels of NO. This having been shown, the e¡ect of NO on cultures of cells containingschizonts di¡erentiating into merozoites was also examined.

MATERIALS ANDMETHODS

Cell lines (lines)

The uncloned Ankara stock of T. annulata, which had been established as amacroschizont-infected cell line in the peripheral blood mononuclear cells (PBM) ofcalf 2 (Ta Ankara 2), was used to assess the e¡ect of NO on macroschizont-infectedcells. A cloned line derived from Ta Ankara 2 (Ta Ankara 2/D7), which di¡erentiatesinto merozoites at a high level (Shiels et al., 1992), was used to test the e¡ect of NO oncells infected with di¡erentiating parasites. Macroschizont-infected lines were main-tained at 378C as described by Brown (1983). The cloned Ta Ankara D2/7 line wasinduced to di¡erentiate into merozoite-producing cells by increasing the temperatureof incubation to 418C, as described by Glascodine and colleagues (1990). A bovinelymphosarcoma cell line (BL-20) (Morzaria et al., 1984) and a line of BL-20 infectedwith the Hisar stock of T. annulata (Gill et al., 1976) were used to assess the e¡ect of theadditives on the integrity of bovine cells.

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Assays for the e¡ect of NO on cell lines

The e¡ect of SNAP-derived NO on cultures of macroschizont-infected cells wasassayed by monitoring the incorporation of tritiated thymidine by the host cells andby microscopic examination of smears of cultures, using methods based on thosedescribed by Visser and colleagues (1995). Test cultures of lines of infected cellscontaining di¡erentiating parasites were exposed to S-nitroso-N-acetyl-dl -penicilla-mine (SNAP) (Amersham Life Sciences) in complete medium (RPMI 1640 supple-mented with l -glutamine (2 mmol/L), penicillin (100 U/ml), streptomycin (100 mg/ml)and 20% fetal calf serum (all from Gibco BRL). Control cultures were incubated inmedium alone, in medium plus dl -penicillamine (Sigma), or in medium plus sodiumnitrate (NaNO2) (Sigma) to con¢rm that any e¡ects were due to NO production bySNAP and not to the penicillamine component of the SNAP or the nitrite ions (NO2

^)produced by NO degradation. In brief, 200 ml of cells were plated out in medium intothe wells of a £at-bottomed 96-well plate and SNAP or the various control additivesdissolved in medium were added as 20 ml aliquots.Proliferation of macroschizont-infected cells was assessed by adding 18.5 kBq

tritiated thymidine (185 GBq/mmol [methyl-3H]thymidine, Amersham Life Sciences)in 20 ml medium to each well. After 18 h incubation, the cultures were processed using acell harvester and a conventional technique of scintillation counting (Preston andBrown, 1988). Incorporation of tritium by host cells was measured either as counts perminute (cpm) or as disintegrations per minute (dpm). A reduction in the incorporationof tritium by cells incubated with an additive as compared to the incorporation by cellsincubated in medium alone was taken to mean an inhibition of cell proliferation. Thee¡ect of NO on cultures incubated at 418C was also examined by assessing theincorporation of tritiated thymidine by such cultures. All experiments included sixreplicates of each combination of additive and cells. Di¡erences in incorporation oftritium by test and control cultures were analysed by the Mann^Whitney test fornonparametric data (Siegel, 1956). Only when p50.5 is the term signi¢cant used in thetext. Alterations in the proliferation of cultures in the presence of additives ascompared to proliferation in medium alone were expressed as the percentage inhibitionor enhancement of growth calculated from the formula

(mean cpm/dpm plus additive)100 ^ öööööööööööööö 6 100

(mean cpm/dpm in medium alone)

Smears were prepared for examination by light microscopy as follows. Duplicate`cytospin' smears of each culture were prepared with a cytocentrifuge (Cytospin,Shandon, UK), ¢xed in methanol and stained with Giemsa's stain diluted 1:10 inphosphate-bu¡ered saline (PBS), pH 7.2 (Giemsa's stain and Gurr's bu¡er tablets fromBDH Laboratory Supplies, Poole, Dorset, UK). In each smear, 250 cells wereexamined and the numbers of the following types of cells were recorded: cellscontaining macroschizonts; dead cells as indicated by degeneration of the nuclei and/or cytoplasm; and apoptotic cells as indicated by the characteristic formation ofcondensed bodies of nuclear material (Kerr et al., 1972). The results obtained fromduplicate smears were expressed as the mean percentage of the total cell populationwhich lacked schizonts, had undergone `cell death', or were apoptotic.

33


The occurrence of apoptosis, or programmed cell death, was con¢rmed using TdT-mediated dUTP-biotin nick end labelling (TUNEL) (Gavrieli et al., 1992) onmethanol-¢xed cytospin preparations. In brief, the nuclei of apoptotic cells were nickend labelled with biotinylated deoxyuridine (dUTP^biotin), which was introduced byterminal deoxynucleotidyl transferase (TdT) and visualized with the avidin^biotin-conjugated horseradish peroxidase (ABC-HRP) system using 3,3'-diaminobenzidinetetrahydrochloride (DAB) as the substrate. All reagents were from Dako, Glostrup,Denmark. The reaction is speci¢c, only the nuclei belonging to cells undergoingapoptosis being stained.

Detection of nitric oxide production by the Griess assay

The amount of NO2^ produced by di¡erent concentrations of SNAP was assessed in

96-well plates containing six replicates of each additive (SNAP, dl -penicillamine,NaNO2) in medium alone, i.e. 20 ml additive per 200 ml medium/well. These plates wereincubated for 18 h, in parallel with plates containing cells plus additives, and thenstored at ^208C prior to testing supernatants for NO2

^ with the Griess assay(Migliorini et al., 1991) as described by Visser and colleagues (1995).

RESULTS

Proliferation of macroschizont-infected cells altered by SNAP-derived NO

A dose-dependent inhibitory e¡ect of NO on the proliferation of macroschizont-infected cells was demonstrated in two sets of cultures of the parent Ta Ankara 2 lineafter 18 h incubation with 1000, 200 or 40 mmol/L SNAP (Figure 1A,B). The lowestdose of SNAP used was 40 mmol/L because this was double the dose which had failedto inhibit the growth of these cells in previous assays (Visser et al., 1995). In both sets ofcultures, one initiated with 104 cells, the other with 26104 cells, the proliferation oflines exposed to all three doses of SNAP was signi¢cantly less than the proliferation ofcells incubated in the control cultures containing medium or molar equivalent doses ofdl -penicillamine or NaNO2. Cells incubated in the presence of NaNO2 grew as well asin medium alone. Although the cells incubated with 200 mmol/L or 1000 mmol/L dl -penicillamine grew less well than cells in medium or equivalent molar concentrationsof NaNO2, they still grew considerably and signi¢cantly better than cells exposed toSNAP. Examination of cytocentrifuge preparations of selected cultures of macro-schizont-infected cells showed that SNAP had a direct e¡ect on the parasite. After 24 hincubation in 200 mmol/L SNAP, only 58% of the cells still contained macroschizontsas compared to 98% of cells in control cultures incubated with NaNO2 (Figure 2).Levels of NO2

^ generated by 1000, 200 or 40 mmol/L SNAP were 340, 63 and 18mmol/L respectively. No NO2

^ was detected in cultures to which dl -penicillamine wasadded. Levels of NO2

^ recorded in cultures to which 1000, 200 or 40 mmol/L NaNO2

were added were 1000, 290 and 45 mmol/L, respectively.


34

35

12

10

% *

;6au

4

2

B

018 63 340

pM NO

1 I I I

018 63 340

fl NO

Figure 1. The cytostatic effect of SNAP on macroschizont-infected cells of the uncloned ZYanndata Ankara 2 line (experiment 1). The cultures were exposed to 40, 200 or 1000 u.rnol/LSNAP (equivalent to 18, 63 and 340 mmol/L of NO, respectively) (@), NaNOz (H), DL-

penicillamine (A) or medium alone ( x ). Proliferation of cells is expressed as the meanincorporation of tritium (cpm x 103) _+ SE for each set of replicate cultures (n = 6). Thecultures were initiated (A) with 2 x lo4 cells/well, (B) with lo4 cells/well

SNAP-derived NO causes macroschizon ts to disappear from cell lines

The second experiment was undertaken to confirm that incubation with SNAP causedmacroschizonts to disappear from the cells and, as high doses of DL-penicillamineinhibited cell proliferation, to assess the effect of all the additives on the host cells.Incubation with SNAP inhibited the proliferation of the Ta Ankara 2 and the TaH BL-20 infected lines, but enhanced proliferation of the uninfected BL-20 line (Table I). Thepatterns of proliferation of the three lines exposed to SNAP differed significantly fromthose found when the lines were incubated with the other two additives or in mediumalone. Neither NaN02 nor D L penicillamine significantly altered the growth of the TaAnkara 2 line. NaN02 caused a slight (12%), but significant, enhancement of thegrowth of the two BL-20 lines, while D L -penicillamine caused a slight (12%), but againsignificant, inhibition of their growth. The NO1 level generated by 200 pmol/L SNAPin this experiment was 172 umol/L; no NO2 was detected in wells to which DL -penicillamine was added.

Visual inspection of the cytocentrifuge preparations (Table I) confirmed that thecultures of Ta Ankara 2 and TaH BL-20 incubated with SNAP both contained morecells without macroschizonts than those incubated with the other two additives or in

36

Figure 2. Cytospin preparations of cultures of the Ta Ankara 2 macroschizont-infected cell linestained with Giemsa’s stain after incubation for 18 h with or without SNAP. (a) Cells incubatedin medium alone or control additives contained schizonts as indicated by the white arrows. (b)Cells incubated in 200 umol/L SNAP were schizont-free. x 1000

medium alone. While similar numbers of dead cells occurred in all the cultures of theTa Ankara 2 line, apoptotic cells also occurred in the cultures exposed to SNAP.Cultures of the TaH BL-20 cell line were very susceptible to SNAP. They contained farmore dead cells than the Ta Ankara 2 cell line or the uninfected BL-20 line, but very

TABLE ICytostatic and parasiticidal e¡ect of 200 mmol/L SNAP on macroschizont-infected cells of theparent, uncloned T. annulata Ankara 2 line or the T. annulata (Hisar) BL-20 cell lines(experiment 2)

Response to additives

Percentage of cells

%3H Dying Apoptotic Schizont-free

(A) Ta Ankara 2SNAP ^32a 16 11 23NaNO2 +4 17 1 10dl -Penicillamine ^3 13 1 8Medium 15 1 9

(B) TaH BL-20SNAP ^41a 78 3 28NaNO2 +12c,d 7 1 3dl -Penicillamine ^14b,d 20 1 5Medium 9 0 2

(C) BL-20SNAP +33a 2 7 UIe

NaNO2 +12c 10 1 UIdl -Penicillamine ^12b,d 12 0 UIMedium 6 0 UI

Cultures of (A) Ta Ankara 2, (B) TaH BL-20, (C) BL-20 were initiated with 26104 cells/well. The e¡ect onthe proliferation of cell lines (%3H) is expressed as the percentage inhibition (^) or enhancement (+) ofincorporation of tritium by cultures exposed to additive as compared to incorporation by cultures grown inmedium alone. The e¡ects on macroschizont presence, host cell integrity and apoptosis are expressed as thepercentage of cells lacking schizonts (schizont-free), or undergoing death (dying) or apoptosis (apoptotic),as shown by light microscopy. For details see Materials and Methodsa^dStatistically signi¢cant di¡erences (p50.05) observed between the incorporation of tritium in thesecultures and cultures incubated in (a) NaNO2, dl -penicillamine or medium alone; (b) NaNO2; (c) dl -penicillamine; (d) medium aloneeUI, uninfected line

37


38

a b

C

Figure 3. Preparations of cultures of the Ta Ankara 2 macroschizont-infected cell lineincubated for 18 h with or without SNAP and exposed to TdT-mediated dUTP-biotin nickend labelling (TUNEL) (Gavrieli et al., 1992). In brief, dUTP-biotin was combined with thecharacteristic nicks which occur in the DNA of apoptotic cells with TdT; the reaction wasvisualized with the ABC-HRP system and DAB, which gave a brown stain. The DNA ofnonapoptotic cells lacks such nicks, so dUTP-biotin did not link to the cells and incubationwith the ABC-HRP system and DAB failed to stain the cells. (a) Cells incubated in mediumalone or control additives are unstained by TUNEL ( x 60). (b) Cells incubated in the presenceof SNAP and in which apoptosis occurred are stained ( x 60). (c) Cells incubated in SNAP showthe crescentic, coalesced nuclei characteristic of apoptosis at high magnifications ( x600)

few apoptotic cells. In contrast, BL-20 cells exposed to SNAP included very few deadcells but more apoptotic cells than BL-20 cells exposed to NaN02, D L -penicihnine ormedium. The use of TUNEL confirmed that SNAP induced macroschizont-infectedcells to undergo apoptosis (Figure 3).

NO a¡ected cultures containing di¡erentiating parasites

Having found a positive e¡ect of NO on the parent, uncloned Ta Ankara 2 cell line,two duplicate experiments (experiments 3, 4) assessed the e¡ects of SNAP on thecloned Ta Ankara 2/D7 line, grown as macroschizont-infected cells at 378C and ascells containing parasites di¡erentiating to the merozoite stage at 418C. After 4 daysincubation at 418C, the cells contained large masses of small nuclei and theircytoplasm showed signs of coalescing; after 7 days, merozoites were visible asintracellular dots approximately 1 mm in diameter; after 11 days, most cells hadruptured, releasing their merozoites, and only a few cells remained. As the parasitesdi¡erentiated and host cells ceased to proliferate, the overall incorporation of tritiumby the control cultures exposed to 200 mmol/L penicillamine, 200 mmol/L NaNO2 ormedium dropped signi¢cantly (Figure 4a,b).In both experiments 3 and 4, 200 mmol/L doses of SNAP signi¢cantly inhibited the

incorporation of tritium by the cloned macroschizont-infected cell line maintained at378C (Figure 4a,b: A) and by ¢ve of the six cultures of cells with di¡erentiatingparasites maintained at 418C (Figure 4a: B, C, D; Figure 4b: B, C). In all theseinstances, the incorporation of tritium by cultures exposed to 200 mmol/L SNAP wassigni¢cantly less than that in control cultures incubated with dl -penicillamine,NaNO2 or medium. In the last set of cultures (Figure 4b: D), a signi¢cant di¡erencein incorporation of tritium was only observed between cultures incubated in SNAP andNaNO2. No signi¢cant di¡erence in growth occurred between the control culturesincubated in 200 mmol/L dl -penicillamine or medium or between those incubated in200 mmol/L NaNO2 or medium, except in experiment 4, where cells exposed toNaNO2 after 4 days incubation at 418C incorporated more tritium than culturesincubated in medium alone.While the levels of NO generated by 200 mmol/L SNAP a¡ected the cloned line when

parasites were di¡erentiating into merozoites and when infected with macroschizonts,the reduced concentrations of NO generated by 40 mmol/L and 20 mmol/L SNAP didnot a¡ect the line, either when infected with macroschizonts or when infected withdi¡erentiating parasites.The respective NO2

^ levels generated by 200, 40 and 20 mmol/L SNAP were 73, 13and 8 mmol/L in experiment 3, and were 88, 14 and 11 mmol/L in experiment 4. Levelsof NO2

^ in cultures to which dl -penicillamine had been added were 51 mmol/Lirrespective of dose in both experiments. The respective NO2

^ levels in control culturesto which 200, 40 or 20 mmol/L NaNO2 had been added were 198, 38 and 20 mmol/L inexperiment 3, and were 222, 41 and 18 mmol/L in experiment 4.

DISCUSSION

This is the ¢rst report of a soluble, physiological factor, with known a¤nity to theimmune system, which can kill intracellular macroschizonts and control the multi-plication of Theileria macroschizont-infected cells. In this study on T. annulata, SNAPinhibited the proliferation of macroschizont-infected cells and the incorporation of

39


40

2 a

1000

b

M N P S

M N P S

M N P S

B

M N P B

MN P S

C D

MN P S

MN P s MN p s

Figure 4. Cytostatic effect of SNAP on cells of the cloned ‘I: annulata Ankara 2/D7 line. In twoduplicate experiments ((a) experiment 3, (b) experiment 4), cells were grown (A) as macro-schizont-infected cells at 37°C; (B-D) as cultures including cells containing differentiatingparasites which had been incubated at 41°C for 4 days (B), 7 days (C) or 11 days (D) prior toincubation with 200 umol/L additives. Cultures were grown in medium alone (M) or exposedto 200 wol/L NaN02 (N), DL-penicillamine (P) or SNAP (S). The viability of cultures wasmonitored by incorporation of tritium, expressed as the mean disintegrations per minute(dpm) x lo2 + SE for each set of replicate cultures (n = 6). Cultures were initiated with 2 x lo4cells/well

tritiated thymidine by cultures which included cells with parasites di¡erentiatingtowards merozoites. This e¡ect was attributed to NO since the SNAP had asuppressive e¡ect which could not be attributed either to the penicillamine componentof the molecule or to the production of NO2

^ by the degradation of NO. SNAP at 200mmol/L inhibited the proliferation of all sets of cultures of macroschizont-infected cellsand the incorporation of tritiated thymidine by ¢ve of the six sets in which cellscontained di¡erentiating parasites. The ¢nding that 40 mmol/L SNAP only inhibitedthe parent line by 27^35% and failed to inhibit any other cultures indicated that theamount of NO produced by 40 mmol/L SNAP did not consistently a¡ect T. annulata-parasitized cells. The level of NO produced by 20 mmol/L SNAP appeared completelyinsu¤cient. The positive results were attributed to the use of higher concentrations ofSNAP and, consequently, the generation of higher levels of NO than in previous work(Visser et al., 1995).Exposure to SNAP caused macroschizonts to disappear and host cells to become

apoptotic, as demonstrated in cytocentrifuge preparations stained conventionally withGiemsa's stain and by preliminary work using TdT-mediated dUTP-biotin nick endlabelling (Gavrieli et al., 1992). In the absence of autoradiographic studies, it is notknown whether the inhibition of incorporation of tritiated thymidine seen in culturesmaintained at 418C and exposed to SNAP re£ected suppression of proliferation of hostcells and/or of parasite di¡erentation. Although the exact e¡ect of SNAP on thesedi¡erentiating cultures is not yet known, NO clearly a¡ected the macroschizont, amajor pathogenic stage in the Theileria life cycle, and its host cell. The occurrence ofsuch an event in vivo would clearly decrease the parasite's ability to complete its lifecycle and produce the merozoites associated with haemolytic anaemia.The inhibitory e¡ects obtained with the high doses of dl -penicillamine illustrate the

di¤culties associated with using SNAP to assess the e¡ect of NO on the growth of cellsor parasites. dl -Penicillamine, in particular 1000 mmol/L, was clearly toxic to cells,especially at low cell concentrations, suggesting that this component of SNAP mayhave made a signi¢cant contribution to the inhibition of proliferation of cells exposedto 1000 mmol/L SNAP. However, 200 mmol/L SNAP caused both signi¢cantly andconsistently greater levels of inhibition of cell proliferation than 200 mmol/L dl -penicillamine. Since there were no indications that nitrite ions were toxic to the cells,the signi¢cantly greater suppression of cell proliferation found with SNAP than withdl -penicillamine is taken to mean that NO alone can have a detrimental e¡ect on theintracellular schizonts of T. annulata and their host cells. The relative insusceptibility ofthe uninfected BL-20 cell line was interesting. It suggested that the death of theparasites had caused the macroschizont-infected host cells to stop proliferating, aswas found when macroschizonts of Theileria parva were eliminated from cell lines bydrug treatment (Dobbaelaere et al., 1988).The ¢nding that a macrophage-derived product like NO can inhibit the proliferation

of macroschizont-infected cells, induce apoptosis and cause schizonts to disappear, aswell as suppress the transformation of cells containing trophozoites and sporozoiteinvasion of host cells (Visser et al., 1995), provides new evidence that macrophage-dependent innate immune mechanisms play a prominent role in controlling infectionswith T. annulata. This idea is supported by the recent proposal that macrophages use a

41


number of pro-apoptotic mediators, including reactive oxygen and nitrogen speciesand TNF-a, to kill target cells by apoptosis (Aliprantis et al., 1996). The earlier ¢ndingthat sporozoites are also susceptible to NO (Visser et al., 1995) suggests that theextracellular merozoites will be vulnerable to such mechanisms. As the levels of NO2

^

(63^176 mmol/L) produced by 200 mmol/L SNAP were only double or triple thosefound in culture supernatants of peripheral blood mononuclear cells (PBM) harvestedfrom infected cattle (Visser et al., 1995), the levels of NO which inhibited theproliferation of parasitized cells described here are likely to be within those levelsgenerated by activated macrophages in vivo.Evidence that both macrophage- and natural killer (NK) cell-dependent innate

immune mechanisms may directly control the proliferation of the di¡erent stages ofT. annulata in cattle has now been provided by the following observations. (1)Macrophages harvested from infected and immune cattle (i) exhibited a sustained andconsistent cytostatic e¡ect on macroschizont-infected cell lines (Preston and Brown,1988); (ii) spontaneously produced NO in vitro; (iii) produced increased amounts ofNO when exposed to interferon-gamma (IFN-g) (Visser et al., 1995). (2) The patternsof occurrence of cytostatic macrophages in infected cattle (Preston and Brown, 1988)and the spontaneous production of NO by cells from infected calves (Visser et al.,1995) were similar. (3) Macrophage-derived cytokines (TNF-a, IFN-a, interleukin-1(IL-1)) suppressed the transformation of trophozoite-infected cells (Preston et al.,1992a). (4) Scid mice, which lack B and T cells but possess normal macrophage andNK cell functions, controlled the growth of T. annulata macroschizont-infected cells(Fell and Preston, 1993). In this case, the macrophages and not the NK cells appearedto be responsible for curtailing infections in scid mice. (5) NK cells were implicated inthe healing stages of primary infections of T. annulata in cattle (Preston et al., 1983).The rapidity with which innate immune mechanisms can be evoked in vivo ^ within

less than 24 h (Bancroft, 1993) ^ suggests that the macrophage and NK cell activitydescribed above could reduce the parasite load during primary infections with T.annulata and contribute to the resistance to challenge stimulated by nonlethalinfections (Preston et al., 1992b), in particular during the period between infection orchallenge and the appearance of antigen-speci¢c T cell-mediated responses. Sincemacroschizont-infected cells produce IFN-a (Entrican et al., 1991; Preston et al.,1993), a recognized stimulator of NK cell activity (Finkelman et al., 1991; Romagnani,1992), and can stimulate macrophages to produce TNF-a (Preston et al., 1993),parasite proliferation per se could induce NK cells and macrophages to produce IFN-g and TNF-a, respectively, and so promote NO synthesis during the early stages ofprimary and challenge infections.Innate and adaptive immune systems are now understood to cooperate during

infections, with the innate response helping to drive the adaptive T cell responsetowards a T-helper 1 (TH1) cell reponse and the promotion of macrophage-dependentimmune responses (Romagnani, 1992; Bancroft, 1993; Gazzinelli et al., 1993; Scott,1993; Fearon and Locksley, 1996). The macrophage and NK cell responses evoked byTheileria infections may therefore help to resolve primary infections and resistchallenge infections by driving the adaptive response towards TH1 cell responses. Suchresponses would include the production of (i) lymphocyte-derived cytokines, which can


42

both activate macrophages to produce reactive nitric intermediates, IFN-g or TNF-a(Stout, 1993) and inhibit trophozoite-infected cells (Preston et al., 1992a); and (ii) Tcells cytotoxic for macroschizont-infected cells (Preston et al., 1983). Evidence for Tcell responses helping to promote macrophage anti-T. annulata activity, in turn, is asfollows. Bovine lymphocytes exposed to interleukin-2 (IL-2) or macroschizont-infectedcells produced IFN-g; IFN-g stimulated bovine macrophages to synthesize TNF-a(Preston et al., 1993) and NO (Visser et al., 1995). When exposed to macroschizont-infected cells, PBM from sensitized cattle produced NO but PBM from a naive animaldid not (Visser et al., 1995).Recognition that innate and adaptive immune responses, rather than cytotoxic T

cells, may act synergistically to resolve primary infections and resist challengeinfections with T. annulata, with activated macrophages and their products playingsigni¢cant roles in both responses, as with other intracellular pathogens (Kaufman,1988), has several implications with respect to recombinant vaccine development. Themost e¡ective immunity should be induced by vaccines able to stimulate CD4+ T cell-mediated macrophage activation. Inclusion of antigens stimulating such speci¢cmacrophage activation should promote an environment in which the adaptive responseis driven towards TH1 cells and the generation of T-cytotoxic cells and cytokines withparasiticidal properties. The ability of cell line vaccines to induce resolving infectionsfollowed by immunity to challenge (Pipano, 1995) may be attributed to their generatingconsistent macrophage cytostatic activity against macroschizont-infected cells, undercircumstances in which only very transient cytotoxic T cell responses are detectable(Preston and Brown, 1988).

ACKNOWLEDGEMENTS

We are grateful to the European Commission (P.M.P., L.M.G.F.) and the Departmentfor International Development (DFID) of the United Kingdom (C.G.D.B.) for their¢nancial support of this work and to Dr B. Shiels, University of Glasgow for the T.annulata D7 merozoite-producing cloned cell line.

REFERENCES

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the establishment of macroschizont-infected cell lines and is produced by macrophages of calvesundergoing bovine tropical theileriosis and East Coast fever. Parasite Immunology, 17, 91^102

(Accepted: 1 April 1997)

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nitric oxide causes the macroschizonts of theileria annulata to disappear and host cells to become...

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