INFECTION AND IMMUNITY, Mar. 1987, p. 784-7910019-9567/87/030784-08$02.00/0Copyright C 1987, American Society for Microbiology

Activation of Murine Macrophages and a Bovine Monocyte CellLine by Bovine Lymphokines to Kill the Intracellular Pathogens

Eimeria bovis and Toxoplasma gondiitH. P. A. HUGHES,'* C. A. SPEER,' J. E. KYLE,' AND J. P. DUBEY2

Veterinary Research Laboratory, Montana State University, Bozeman, Montana 59717,1 and Protozoan DiseasesLaboratory, Animal Parasitology Institute, Agricultural Research Service, United States Department ofAgriculture,

BARC-E, Beltsville, Maryland 207052

Received 3 July 1986/Accepted 20 November 1986

Macrophage (M+)-activating lymphokines present in concanavalin A-stimulated bovine T-lymphocytecultures (ConAS) were studied by assessing their effects on Eimeria bovis and Toxoplasma gondii growth incultured bovine monocytes (BM) and mouse M+. The in vitro development of both parasites was assessed byincorporation of [3H]uracil and by microscopic examination of parallel cultures. Incorporation of [3lH]uracilinto infected cultures was an accurate indicator of growth of both E. bovis and T. gondii in BM and mouse M4+.Sporozoites of E. bovis underwent merogony in untreated BM but not in mouse M+, whereas T. gondiideveloped in both cell types. Inhibition of T. gondii growth was greatest in ConAS-treated BM, whereaspreincubation of mouse M+ with ConAS resulted in about 80% growth inhibition. There was no significantdifference between the inhibition of either T. gondii sporozoite- or tachyzoite-induced growth in ConAS-treatedcells, showing that activation pathways are equally effective against both stages. Treatment of ConAS withglycine-hydrochloride buffer (pH 2) resulted in a total loss of antiviral activity mediated by gamma interferon(IFN-y). When pH 2 dialyzed ConAS was used to activate BM, inhibition of T. gondii growth was only partiallyaffected. Because bovine IFN-,y does not activate mouse M4 and due to the partial effects of pH 2 onConAS-induced growth inhibition, the major component(s) of ConAS responsible for T. gondii growthinhibition is distinct from IFN--y. Furthermore, IFN--y may act synergistically rather than being part of a

priming sequence for M4 responsiveness to other lymphokines. Murine recombinant granulocyte-macrophagecolony-stimulating factor (rGM-CSF) was tested for any microbistatic activity against T. gondii sporozoites andtachyzoites. There was no significant difference in either colony formation or [3H]uracil incorporation betweenrGM-CSF-treated and control cultures, regardless of host cell type. Thus, rGM-CSF does not induce adequateM+ activation to kill T. gondii and is not a major microbistatic component of ConAS. rGM-CSF also had noeffect on T. gondii infection in vivo.

Infection of animals or humans with coccidian parasitesresults in immunity to subsequent lethal challenge with thesame species. Resistance to coccidian parasites, includingsome Eimie ia species and Toxoplasma gondii, is predomi-nantly cell mediated, and specifically committed T lympho-cytes or soluble mediators (lymphokines) appear to play afundamental role in the control of infection. Macrophage(MO~)activation is thought to play an essential role in controlof T. gondii and Eimeria bovis infection (20, 23, 26), andconsiderable effort has been spent in elucidating those fac-tors produced by sensitized T lymphocytes which are re-

sponsible for MX activation. Crude supernatants fromconcanavalin A (ConA)-stimulated (nonimmune) and anti-gen-stimulated (immune) lymphocytes can control the mul-tiplication of intracellular parasites in both human (1) andmodel animal (22) systems. Speculation as to the activecomponent(s) present in these supernatants abounds, butrecent evidence implicates gamma interferon (IFN-y) as amajor activating lymphokine. IFN-y can control the intra-cellular growth of Toxoplasma spp. and Leishmaniadonovani in vitro (9, 19, 27), and induces partial protectionagainst T. gondii in vivo (12). More recently, colony-

* Corresponding author.t Journal series no. 1872, Montana State University Agricultural

Experiment Station.

stimulating factors (CSFs), particularly granulocyte-macrophage (GM) CSFs, have been shown to have bothmicrobistatic (14) and tumoricidal (7) activity. Because su-pernatants from concanavalin A (ConA) stimulated lympho-cytes (ConAS) from the peripheral blood of a bovine was

found to control the development of E. bovis meronts invitro (26), the present study was undertaken to test theability of ConAS to control the growth of the relatedpathogen T. gondii and to determine whether the principlefactor(s) responsible for M+-mediated resistance is distinctfrom both IFN--y and GM-CSF.

MATERIALS AND METHODS

Parasites. E. bovis oocysts were separated from bovinefeces by sugar flotation, sporulated, and then stored inaqueous 2.5% K2Cr2O7 at 4°C for less than 5 months. E.bovis oocysts were suspended in phosphate-buffered saline,pH 7.2 (PBS) and broken by grinding in a motor-driven,Teflon-coated tissue grinder, and sporozoites were excystedfrom sporocysts aseptically as described previously (25, 26).

T. gondii RH tachyzoites were harvested from peritonealexudates of mice infected intraperitoneally 3 days previouslywith between 106 and 107 tachyzoites (10). T. gondii P1sporozoites were isolated as follows. T. gondii oocysts were

obtained from feline feces (4), sporulated by constant agita-tion in 2% (vol/vol) H2SO4 for 7 days at 22 to 26°C, and

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stored at 4°C for less than 6 months. Sporocysts were freedfrom oocysts by treatment for 30 min with sodiumhypochlorite at 4°C followed by extensive washing (fivetimes) in Hanks balanced salt solution and Vortex mixing(for -15 s) with 500-,um glass beads. Sporocysts werewashed once with Hanks balanced salt solution, and sporo-zoites were excysted in 5% bovine bile in 0.85% NaCl at370C. The optimum release of sporozoites was assessedmicroscopically and occurred after 60 to 90 min of incuba-tion. Sporozoites were washed free of bile by centrifugationat 550 x g and suspended in medium.

Sporozoites of T. gondii and E. bovis and tachyzoites of T.gondii were suspended in culture medium at desired concen-trations. All parasite preparations were carried out underaseptic conditions.Lymphocyte supernatants. Bovine peripheral blood mono-

nuclear cells were isolated and cultured as described previ-ously (26). Briefly, peripheral blood mononuclear cells wereisolated from venous blood by centrifugation on Ficoll-Hypaque, T cell enriched by passage over nylon wool, andincubated for 48 h at 37°C in a C02-enriched atmosphere inthe presence of adherent cells. T lymphocytes were stimu-lated with 4 ,ug of ConA per ml. Control cultures comprisedcells incubated with ConA and adherent or nonadherent cellscultured separately with ConA. ConAS was prepared from5-ml cultures of ConA-stimulated T cells (with adherentaccessory cells) at a density of 2 x 106 cells per ml and storedat -20°C before use (26). Control cultures comprised adher-ent or nonadherent lymphocytes alone stimulated withConAS and unstimulated T cells with accessory cells. In allcases, these supernatants did not induce microbistatic func-tion (full data not shown), and control results from M40treated with medium alone are shown unless otherwisestated.Host cells. Murine M4i were prepared from peritoneal cells

of 8- to 10-week-old BALB/c mice of either sex. Mice wereinjected 3 days before M4 were harvested with 0.5 to 1.0 mlof thioglycollate broth (Difco Laboratories, Detroit, Mich.).After cervical dislocation, 5 ml of ice-cold RPMI 1640medium containing penicillin (50 U/ml), streptomycin (50p,g/ml), and heparin (10 U/ml) was injected intraperitoneally,the abdomen was massaged, and the cell suspension waswithdrawn. After centrifugation (500 x g, 4°C), cells weresuspended in RPMI 1640 supplemented with penicillin,streptomycin, glutamine (20 mM), 2-mercaptoethanol (10-'M), and fetal bovine serum to a final concentration of 15%(RPMI-FBS). Cells were added to wells of 96-well clustertrays (Linbro Chemical Co., Flow Laboratories, Inc.,Inglewood, Calif.) or 8-chamber slides (Miles Scientific,Division of Miles Laboratories, Inc., Naperville, Ill.), atdensities of 107 cells per well (trays) or 2 x 106 cells per well(slides). After 3 h of incubation at 370C in 5% CO-95% air,nonadherent cells were washed away from MO monolayers,and 200 ,ul of RPMI-FBS or diluted lymphokine (ConAS orcontrol preparation) was added to each well.

Cultured bovine monocytes (BM) were originally obtainedfrom peripheral blood mononuclear cells of a 6-year-oldGuernsey cow and were grown in RPMI-FBS at 37°C in aC02-enriched atmosphere (26). BM are esterase positive andphagocytic, have a normal karyotype, and do not expressMHC class II antigens (G. A. Splitter, unpublished data).

Inhibition assays. Inhibition of parasite development orproliferation in BM or mouse MX was assessed by [3H]uraciluptake as well as visually by examining Giemsa-stainedmonolayers on eight-chamber slides. T. gondii growth inhi-bition was assessed as described previously (13), with the

following modification. BM or mouse MX monolayers inflat-bottomed 96-well cluster trays (above) were inoculatedwith 105 strain P1 sporozoites or strain RH tachyzoites in 100p.l ofRPMI-FBS. After 1 h of incubation (37°C, 5% C02-95%air), monolayers were washed with serum-free RPMI andincubated for 23 h. Then 0.5 p.Ci of [3H]uracil (New EnglandNuclear Corp., Boston, Mass.; specific activity, 41 Ci/mmol)was added to each well, and cultures were further incubatedfor 18 to 24 h. Cultures were then harvested onto glass-fiberfilter paper, and [3H]uracil uptake was assessed by -spectroscopy.

Inhibition of E. bovis development was assessed similarly.Monolayer cell cultures in 96-well cluster trays were inocu-lated with 5 x 103 E. bovis sporozoites per well. At 12 daysafter sporozoite inoculation cultures were pulsed for 48 hwith [3H]uracil (0.5 ,uCi per well) and harvested, and incor-poration of [3H]uracil was assessed. The 12-day incubationperiod and a 48-h pulse were found to be optimum conditionsfor assessing [3H]uracil uptake (data not shown).

In all experiments, the following control cultures wereused: (i) parasites cultured in the absence of host cells and(ii) host cells cultured alone. The effects of lymphokinesproduced by cultured T lymphocytes was assessed by addingdiluted supernatants (1:2) from ConA-stimulated andunstimulated cultures to cell monolayers before infectionwith parasites for 24 h. Immediately before parasite inocu-lation, monolayers were washed with serum-free RPMI (26).The ability of lymphokines to inhibit growth or developmentwas assessed by comparing [3H]uracil uptake by infected,untreated cultures and cells or parasites cultured alone (13).

Inhibition of E. bovis and T. gondii growth in eight-chamber slides was assessed as described previously (26).Cells (M or BM) were cultured as described above, andsupernatants were added to selected chambers before para-site inoculation. Monolayers were inoculated with either 105(T. gondii) or 5 x 103 (E. bovis) parasites. At 2 days (T.gondii) or 8 to 10 days (E. bovis) after sporozoite inocula-tion, monolayers on slides were fixed in Bouin solution andstained with Giemsa stain. Inhibition of parasite growth wasassessed by two different criteria. In T. gondii-infectedcultures, the numbers of parasites per infected cells and thetotal numbers of colonies in 20 microscopic fields (x200)were counted in at least five different chambers and com-pared with results from lymphokine-treated and untreatedcultures. Growth of E. bovis was assessed in chamber slidesas described previously (26). All data were statisticallyanalyzed with Student's t test. Experiments were carried outat least twice; typical or mean results are shown.pH stability studies. Samples (2.5 ml) of control and

ConA-treated lymphocyte supernatants were dialyzed for 24h against either 0.1 M glycine hydrochloride buffer (pH 2) or0.05 M PBS, pH 7 (17). After dialysis, the pH of the liquid inthe bag was measured directly to ensure equilibration.Samples were then dialyzed with multiple changes againstHBSS for 24 h and then against serum-free RPMI for 12 h,filtered through a 0.22-p.m filter, diluted as described above,and used to treat monolayers before parasite inoculation.rGM-CSF. Recombinant murine GM-CSF (rGM-CSF)

was produced in a yeast expression system and purified tohomogeneity by reversed-phase high-performance liquidchromatography (16). In all in vitro experiments, rGM-CSFwas used at 500, 100, 50, 25, 10, and 5 nglml; theseconcentrations have been shown to induce biological activityor activation in other systems (5, 14, 16). For in vivo studies,5 p.g of rGM-CSF in PBS was inoculated intraperitoneallyinto 20-g BALB/c mice before infection with 103 RH strain T.

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786 HUGHES ET AL.

gondii cells, with repeat inoculations every 24 h thereafter.Control mice received PBS alone.

RESULTS

[3lH]uracil uptake by E. bovis cultures. BM cultures in-fected with E. bovis showed a high incorporation of[3H]uracil after 12 days of growth and a 48-h pulse (Fig. 1).When BM cultures were pretreated with ConAS, infectedcultures consistently showed decreased [3H]uracil uptake,often significantly lower than host cell growth alone (Fig. 1).Microscopic inspection of infected BM monolayers beforeharvesting showed that ConAS-treated cultures showed littleor no development of first-generation meronts, comparedwith untreated cultures. These microscopic observationswere consistent with previously recorded results (26). E.bovis meronts did not develop in mouse peritoneal M4 (Fig.1).

[3H]uracil uptake by T. gondii-infected cultures. The uptakeof [3H]uracil by T. gondii in vitro was studied to ascertainwhether ConAS could affect the development of a parasiteunrelated to E. bovis. When T. gondii was cultured in theabsence of cells, [3H]uracil uptake was about one-half of thatof M4 cultured alone (Fig. 2A). T. gondii-infected culturesexhibited approximately a 10-fold increase of [3H]uraciluptake when compared with host cells cultured alone (Fig.2A), regardless of which stage (sporozoites or tachyzoites)or strain (P1 or RH) of the parasite was used to infectmonolayers. When mouse M4 were preincubated withmurine rGM-CSF, there was no significant difference in[3H]uracil uptake between rGM-CSF-treated and untreated,infected cultures (P > 0.1; Fig. 2A). However, when M(

10

ConAS - + +

FIG. 1. [3H]uracil uptake by E. bovis-infected cells. E. bovissporozoites were used to infect BM or (*) mouse Mcl monolayers,and [3H]uracil uptake of cells cultured alone (U) and of infectedcultures (0) was measured. The addition of ConAS (+) to culturesbefore sporozoite inoculation induced decreased [3H]uracil uptake.Results from two experiments are shown. Neither development ofmeronts nor elevated levels of [3H]uracil uptake was observed inmouse M4 infected with E. bovis (-*). Cells and parasites werecultured for 12 days with a 48-h pulse of 0.5 ,uCi of [3H]uracil. Errorbars represent standard errors of the means of triplicate cultures.[3H]uracil uptake of E. bovis cultured alone was 176 + 12 cpm.

T.gondli LKcpm, 103

10

FIG. 2. [3H]uracil uptake by T. gondii-infected cultures. (A) T.gondii tachyzoites (RH strain) or sporozoites (P1 strain) were eithercultured alone (-*) or used to inoculate mouse M4 monolayercultures as follows: -, host cells not treated before parasite inocu-lation; ConAS, host cells pretreated with ConAS; rGM-CSF, hostcells pretreated with murine rGM-CSF; (tested at 1:2 dilution).rGM-CSF was tested at 500, 100, 50, 25, 10, 5 ng/ml (results areshown for 10 ng/ml; similar results were obtained at all otherconcentrations tested). Infected cultures (O) showed an increaseduptake of [3H]uracil compared with host cells cultured alone (U).Pretreatment with rGM-CSF did not affect [3H]uracil uptake ofinfected monolayers compared with untreated cultures. ConASpretreatment significantly reduced [3H]uracil uptake (P < 0.05). (B)Conditions were identical to A, except the host cells were BM.ConAS pretreatment of BM inhibited utilization of [3H]uracil tolevels not significantly different from that of host cells only (P >0.01). rGM-CSF treatment did not affect [3H]uracil uptake whencompared with untreated BM. In both A and B, lymphokinetreatment did not affect [3H]uracil uptake of host cells cultured alone(data not shown). There was no significant difference (P > 0.1)between P1 and RH strains of T. gondii in both cell culture systems.

were incubated with ConAS, there was a significant decreasein [3H]uracil uptake (P < 0.01) compared with untreatedcultures (Fig. 2A).The same trends were apparent in BM cultures infected

with T. gondii (Fig. 2B). RH strain tachyzoites or P1 strainsporozoites both showed low [3H]uracil uptake when cul-tured alone (Fig. 2B). The uptake of label was highest inuntreated, infected BM cultures and was unaffected (P >0.1) by prior incubation with murine rGM-CSF (Fig. 2B).Incubation of BM with ConAS before parasite inoculationdrastically reduced incorporation of [3H]uracil to levelsinsignificantly different (P > 0.01) from those in BM cellscultured alone (Fig. 2B). As with mouse M+, the samedegree of growth inhibition occurred in BM infected withsporozoites (P1) or tachyzoites (RH) of T. gondii.

Since there was no difference between the development or

RH -A

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TABLE 1. Effects of lymphokines on T. gondii (RH strain) incultures of BM and mouse M4

No. ofCells Lymphokinea infected cells PC

at 48 hb

BM - 134 ± 4.04BM rGM-CSF 127 ± 3.06 >0.1BM Con-AS 13 ± 3.51 <0.001M - 177 ± 5.13Mo rGM-CSF 142 ± 30.44 >0.1no ConAS 59 ± 18.15 <0.01a Cells were incubated with RPMI (-), ConAS, or rGM-CSF before

parasite inoculation.b Colonies were counted in 25 fields per chamber at 160 magnification.

Mean counts + standard errors of the means are shown for six chambers (150fields).

c Significance of difference between the ability of lymphokine-treated anduntreated cells to inhibit growth of T. gondii.

inhibition of growth between these two T. gondii strains, theRH straip was used for further studies.Growth of T. gondii RH in BM and mouse M4. Micro-

scopic examination of T. gondii-infected cells reflected theresults of the [3H]uracil assays (see above). There was nosignificant difference in numbers of parasite colonies inuntreated or rGM-CSF-treated BM or mouse MX cultures(Table 1). ConAS pretreatment significantly reduced parasitecolonies in both cell types, although more colonies wereobserved in ConAS-treated mouse MO than in ConAS-treated BM -(Table 1).

In untreated BM cultures, many colonies were observed,with the numbers of parasites per cell ranging from >60 toonly a few (Fig. 3C and D). In ConAS-treated cultures, fewparasites were generally seen in each infected cell (Fig. 3Aand B; Fig. 4). In untreated cultures (Fig. 4), the majority ofBM were infected with at least 60 parasites with fewer cells

1-10 11-20 21-GO 31-40 41-50 51-60 61-70

Nos. PARASITES per HOST CELL

FIG. 4. Effects of ConAS on the number of RH strain T. gondiitachyzoites in BM cultures. Monolayers ofBM were inoculated withRH strain T. gondii, incubated for 48 h, fixed, and stained, and thenumbers of organisms per cell was counted at 400x magnification;100 infected cells were counted from each experiment; results aremeans ± standard errors of the means of three different experi-ments. Significant differences between ConAS pretreated cultures(O) and parallel, untreated cultures (U) occurred at only the lowest(<10/cell; P < 0.001) and the highest (>61/cell; P < 0.01) parasitedensities. At all others, there was no significant difference (P > 0.1).

containing 10 or less parasites. Conversely, ConAS pretreat-ment resulted in few heavily infected BM (>61 organisms),with most cells infected with 10 parasites or less.

Closer microscopic examination of ConAS-pretreated cul-tures revealed that most parasites within BM were abnormal

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FIG. 3. Bright-field photomicrographs of RH strain T. gondii-infected BM cultures. (A) T. gondii growth 48 h after a 1-h pretreatment ofBM with ConAS. Few colonies were observed, generally with few parasites. Bar, 50 p.m. (C) T. gondii growth in a parallel untreated culture.Many cells are infected, and colony size varied from a few parasites (f) to many (m). Bar, 50 p.m. (B and D) Higher magnifications of A andC, respectively; colonies in ConAS-pretreated cells generally had fewer parasites (B) than those in untreated cells (D). Bars, 20 p.m.

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788 HUGHES ET AL. INFECT. IMMUN.

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FIG. 5. Effect of ConAS on RH strain T. gondii development. (A) Bright-field photomicrograph of normal parasite development.Numerous distinct organisms are visible in the vacuole. (B) Bright-field photomicrograph of abnormal parasite development in ConAS-treatedBM. Elongated, multinucleate forms are visible within the vacuole, but no distinct organisms are visible. Bars, 20 ,um.

in appearance (Fig. 5B), whereas those within untreatedcells appeared normal (Fig. 5A). Occasionally, abnormal,multinucleate, and elongated organisms were present inConAS-pretreated BM (Fig. 5B).pH stability of ConAS. The ability of ConAS to induce

inhibition of T. gondii growth in BM was further studiedafter dialysis of supernatants against glycine hydrochloride(pH 2.0) or PBS (pH 7.4). BM were pretreated with ConASor control supernatants (from bovine lymphocytes culturedin the absence of ConA) before T. gondii inoculation (Fig. 6).Control supernatants did not interfere with T. gondii growth,as measured by [3H]uracil incorporation, regardless ofwhether they were left untreated or dialyzed against PBS orglycine hydrochloride (Fig. 6). The uptake of [3H]uracil byT. gondii-infected BM pretreated with ConAS was de-creased in all cases. However, although no treatment or PBSdialysis of ConAS reduced [3H]uracil uptake to levels similarto those in host cells grown alone, pH 2 dialysis partiallyabrogated the microbicidal capacity of BM (Fig. 6).Bovine IFN--y levels were assessed in these supernatants

by using a standard viral inhibition assay. Supernatantswhich had undergone no dialysis had 27.3 U of IFN--y per ml;pH 7.4 treatment decreased levels to 5.5 U/ml, and no IFN--yactivity could be detected after dialysis against glycinehydrochloride (pH 2.0) buffer. No control supernatant haddetectable IFN--y activity.These observations were substantiated by microscopic

examination of BM in chamber slides, treated, and subse-quently infected in a similar manner. The greatest number ofT. gondii colonized cells occurred in cultures pretreated withsupernatants from nonstimulated bovine lymphocytes.ConAS, either native or pH 7.4 treated, induced the greatestinhibition of colony formation, whereas pH 2 treatment ofConAS before it was used to pretreat BM induced somemicrobicidal capacity, which was still much less than that ofother ConAS treatments (Table 2).

DISCUSSION

In a previous study, supernatants from ConA-stimulatedbovine T-cell cultures induced an established cell line ofBMto inhibit development of E. bovis sporozoites to first-generation meronts (26). This activity has been confirmed inthe present study, and a [3H]uracil assay has been developedfor the assessment of E. bovis development in cell culture.The incorporation of uracil into developing E. bovis merontscorrelated with microscopic examination of microcultures(full data not shown). This assay system has distinct advan-tages over conventional (microscopic) methods of assessingparasite development; it requires less material (i.e., hostcells, parasites, and lymphokine) and offers an objectiveassessment of the ability of parasites to develop insidecultured cells. Pulsing of E. bovis-infected cultures with[3H]uracil before day 10 resulted in no significant uptake ofthe label, and a 48-h pulse at 10 to 12 days after sporozoiteinoculation was required to assess parasite growth. A rela-tively long incubation period was required for sufficientincorporation of [3H]uracil because sporozoites of E. bovisdevelop slowly in BM, with young meronts first appearing atabout 6 days after sporozoite inoculation and maturemeronts at 10 to 21 days after inoculation (Speer, unpub-lished data). In contrast, 18 to 24 h is an adequate timeinterval for pulsing with [3H]uracil to detect growth of T.gondii in vitro (13; present study).

Studies were then carried out to investigate whetherConAS could induce microbicidal effects against a relatedpathogen (T. gondii) with allogeneic (BM) and xenogeneic(mouse M4O) host cells. Previous studies of M4 killing of T.gondii have relied almost exclusively on the RH strain of T.gondii as the infectious agent (3, 13, 19, 22, 23). The RHstrain was originally isolated by Sabin (21) and no longerrepresents a wild type of T. gondii; it does not have theability to undergo a sexual cycle in the cat (6) and is highly

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TABLE 2

ConAS in murine M{> and BM are as effective against (P1)sporozoites as they are against (RH) tachyzoites, despitedifferences in antigenic structure (11), generation time, andpossible lymphocytic choriomeningitis virus infection. RHstrain tachyzoites can be produced in large numbers, with ahigh degree of purity from peritoneal exudates. Excystationof (P1) oocysts to produce sporozoites, however, requiresprocedures that are lengthy, and the resultant parasite sus-pension is often contaminated with unsporulated or unex-cysted oocysts, sporocysts, and other debris. For thesereasons, the RH strain was used in all further studies.The effect of ConAS on T. gondii growth was investigated

by using the [3H]uracil assay (13) and microscopic examina-tion of microcultures. When BM were used as host cells,[3H]uracil uptake was reduced to the level of BM culturedalone. This appeared to be due to killing of parasites, ratherthan parasite inhibition, since both the number of coloniesper culture and the number of parasites per cell werediminished. These observations indicate that killing occursintracellularly during early stages of parasite growth. Inaddition, those parasites that did develop in ConAS-

pretreated BM were abnormal in appearance when examinedmicroscopically. This implies that parasite inhibition may

T.g. also occur at a second level, i.e., at a later stage of parasiteiAS + +-- + development, or that ConAS pretreatment induces abnormal

intracellular parasite development. In this light, abnormalpH - - 7.4 7.4 2.0 2.0 development of E. bovis meronts in ConAS-treated BM has

been reported previously (26).Effect of pH on ConAS activity, assessed by [3H]uracil Many havevindicateT. gondii-infected BM. BM were treated with superna-unstimulated (-) or ConAS-stimulated (+) bovine lym- sole factor which activates cells to kill T. gondii. MurineSupernatants either remained untreated (pH -) or were IFN--y has significant activity against T. gondii in vivo (10),gainst PBS (pH 7.4) or glycine hydrochloride (pH 2.0) and previous reports of murine Toxoplasma growth inhibi-abation with BM monolayers. After incubation, the BM tory factor bear a striking resemblance to IFN-y (24).ed and inoculated with T. gondii, and [3H]uracil incorpo- Treatment of human monocyte-derived MX (27) or fibro-,measured in BM cultured alone (U) and in T. gondii- blasts (19) with recombinant IFN--y also blocks T. gondiiM (O). T.g., Uptake by T. gondii cultured alone. Results development in vitro. It has been suggested that M+-

standard errors of the means of triplicate cultures. activating factor and IFN-y are identical, and that M+-activating factor should only be defined as a biological

when administered parenterally to mice. Recent activity (2). Further evidence that IFN-y is a M4)-activatingave shown that certain sublines are infected with lymphokine also arises from studies on intracellular killing ofrtic choriomeningitis virus (8). For these reasons, Leishmania donovani. Mitogen-stimulated lymphokines richdies were carried out with both tachyzoites of the in IFN--y from human T-cell cultures can induce intracellularand sporozoites of the P1 strain. The P1 strain was killing of L. donovani. Pretreatment with IFN--y neutralizingsolated from muscles of a naturally infected pig (5), antibody abolished both intracellular killing and oxidative-en passed in mice, does induce oocyst formation in metabolism (15). However, Hoover et al. (9) described ais therefore representative of a wild-type infection. lymphokine produced by ConA-stimulated human T cellsins grew equally well and were inhibited by ConAS which could be biochemically separated from IFN--y, havingne extent in both murine M4, and BM. Thus, it is a Mr of 25,000 to 30,000. The activity of this lymphokine wasI the killing or inhibitory mechanisms induced by not affected by IFN--y neutralizing antibody, providing fur-

ther evidence of its unique properties..Effects of pH treatmenton ConAS-induced T. gondii Other evidence indicates that IFN--y is not the only

growth inhnbition in cultured BMNo. infected

Factor Treatmenta cells at pc48 hb

_d None 224 ± 8.4ConAS None 39 ± 15.4 <0.001ConAS 7.4 56 + 14.6 <0.01ConAS 2.0 142 + 16 <0.01

a None, Untreated supernatants; 7.4, dialysis against PBS (pH 7.4); 2.0,dialysis against glycine hydrochloride (pH 2.0) before incubation.

b Colonies counted from 25 fields per chamber at 160x magnification.Values are means + standard errors of the means of three chambers.

c Significance of difference between T. gondii growth in ConAS-treated BMcompared with growth in control cultures of BM.d_, Supernatants from bovine lymphocytes cultured in the absence of

ConA.

TABLE 3. Effects of pH 2 treatment of ConAS or various hostcell type or utilization of [3H]uracil by T. gondii

Host Factor 3H incorporation % Inhibition"cell (mean Acpma)

BM None 21,879 0.00BM ConAS 32 99.86BM ConAS, pH 7.4 -20 100.00BM ConAS, pH 2.0 3,346 84.70MX None 5,430 0.00M4 ConAS 1,230 77.34

a Increase in counts per minute above cells cultured alone.I Percent inhibition: (1 - lymphokine-treated Acpm)/untreated Acpm x

100.

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790 HUGHES ET AL.

lymphokine in ConAS with microbistatic function. First,ConAS had no effect on E. bovis development in Madin-Darby bovine kidney cells (26), which express receptors forIFN--y (P. E. Baker, personal communication). Second, wehave produced more conclusive evidence that IFN--y is notthe sole lymphokine in ConAS responsible for microbicidaleffects. For example, ConAS was able to induce 77% inhi-bition of [3H]uracil uptake in T. gondii-infected mouse M4(Table 3), which was also reflected in microscopic colonycounts of parallel infected cultures. Bovine IFN-y has pre-viously been shown to have no effect on murine M4 (J.Philip and P. E. Baker, personal communications), so theinhibitory effect of ConAS must be due to some otherlymphokine(s). In addition, anti-T. gondii activity in ConAS-treated BM was only partially abrogated after pH 2 treat-ment of lymphokine(s). If IFN-,y were solely responsible formicrobicidal function, then no growth inhibition would beexpected in BM pretreated with pH 2-treated ConAS. Inter-estingly, the effects of pH 2 treatment and subsequentincubation of allogeneic (BM) cells or incubation of xenoge-neic (murine) MX produced markedly similar states of acti-vation. As well as providing evidence that IFN--y is at bestonly a minor component of ConAS, these results indicatethat the major protective lymphokine(s) in ConAS is just aseffective in xenogeneic and allogeneic host cells. Theantiparasitic effects probably were not due to endotoxincontamination because medium or supernatants from controlcultures had no microbicidal effect.Recent evidence shows that CSF, particularly GM-CSF,

can activate MX as well as induce colony formation. CSFshave long been suspected of having tumoristatic activity(14), an effect which has recently been attributed to GM-CSF(7). GM-CSF can also induce antibody-dependent cellularcytotoxicity, neutrophil-mediated killing of schistosomula,and intracellular killing of Leishmania donovani (7). A seriesof studies was carried out to determine whether murinerGM-CSF could induce any T. gondii microbistatic effect inmurine MO) or BM. When rGM-CSF was added to cultures atconcentrations known to induce tumoristatic activity (7), ithad no effect on either [3H]uracil uptake or parasite multi-plication. Whereas a limited effect would be expected inrGM-CSF-treated BM (murine CSF shows optimal activityon murine progenitor cells [16]), a marked effect in rGM-CSF-treated murine M4 would have been expected if GM-CSF could induce T. gondii inhibition. In vivo studiesdemonstrated that rGM-CSF did not induce any protectionagainst a T. gondii challenge in BALB/c mice (data notshown).These studies indicate that rGM-CSF has no inhibitory

effect on T. gondii multiplication, although it can inducekilling of L. donovani (14, 16). Furthermore, they imply thatthe major active lymphokine(s) present in ConAS is notCSF. Work is in progress to produce large amounts ofConAS with functional activity against E. bovis and T.gondii, so that the active component(s) may be biochemi-cally isolated and purified to homogeneity. Preliminary acidlability studies have indicated that ConAS activity against E.bovis is, like that against T. gondii, not solely through theaction of IFN-y.These findings reinforce evidence that M4-activating fac-

tor activity cannot be attributed to a single lymphokine, butrather a complex mixture which appears to include at leastIFN--y (19, 27) and GM-CSF (16). Both IFN-y and GM-CSFhave been shown to differ in target cell surface receptors andactivation pathways (7). The results reported herein alsodemonstrate that the M+-activating capacity of ConAS in

activating cells to kill T. gondii is not due to the actions ofGM-CSF, nor solely that of IFN--y. IFN--y is also notrequired as a priming step for M( microbistatic function, asit is for tumoricidal activity (17). At present, it appears thatIFN--y may work in synergy with another, as yet undefined,lymphokine. Biochemical purification of other activelymphokine(s) and quantitation of their biological propertieswill elucidate the roles that different components of M+-activating factor have in the control of intracellular patho-gens.

ACKNOWLEDGMENTS

We thank Paul E. Baker (Immunex Corp., Seattle) for supplyingrGM-CSF and conducting bovine IFN--y asays, K. Knoblock andD. E. Burgess for technical assistance, and G. A. Splitter forsupplying the original cultures of bovine monocytes.

This work was supported in part by grants 85-CRSR-2-2688 and26000-976 from the U.S. Department of Agriculture to C.A.S.

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