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. 1998 Feb;111(2):325–333. doi: 10.1046/j.1365-2249.1998.00499.x

Human platelet inhibition of Toxoplasma gondii growth

B F F Chumpitazi *, J Simon *, B Polack *, F Peyron †, S Picot †, J Ricard *, P Ambroise-Thomas *
PMCID: PMC1904904  PMID: 9486399

Abstract

The human platelet contribution against the intracellular growth of the parasite in vitro in human pulmonary fibroblasts was explored. It was observed that tachyzoites of Toxoplasma gondii induced activation of human platelets and additionally that platelets mediated inhibition of intracellular growth in a virulent T. gondii strain. A prominent role for platelet-derived growth factor (PDGF) was demonstrated in this phenomenon, by testing human recombinant PDGF-AA, -AB and -BB and antibodies to human PDGF-AB that partially reversed its effects. Moreover, the effect of PDGF was significantly higher if the host cells were treated 2 h before parasite infection. PDGF was not directly ‘toxic’ to free tachyzoites, but only affected parasites within host cells. PDGF-mediated inhibition may involve the cyclooxygenase cycle of the fibroblasts being partially reversed by the cyclooxygenase inhibitors, acetylsalicylic acid and indomethacin. However, a thromboxane synthetase pathway was not implicated. PDGF action against intracellular tachyzoites may also include increased IL-6 production in fibroblasts. Finally, transforming growth factor-beta 1 (TGF-β1), another component of α-granules released at the same time as PDGF, may not be antagonistic to the PDGF parasite inhibitory effect in confluent host cells.

Keywords: platelets, Toxoplasma gondii, IL-6, platelet-derived growth factor, transforming growth factor-beta 1

INTRODUCTION

Toxoplasma gondii is a ubiquitous, intracellular, coccidian parasite that infects birds and almost all mammals. Toxoplasmosis is widespread in humans and it is estimated that 30% (5–90%) of human adults are infected [1]. Fortunately, only a minority develop serious clinical disease, such as congenital or cerebral toxoplasmosis, associated with an immature and a deficient immunity, respectively. Thus, the parasite has increased in importance as the major cause of central nervous system infections in patients with AIDS [2, 3]. The important role played by T cells in immunoprotection has been shown earlier [4] and depends on interferon-gamma (IFN-γ) during both the acute and chronic phases of infection [5–8]. The protection is likely to be due to the participation of both CD4+ T cells and CD8+ T cells [7,9–11]. Neutrophils, monocytes and activated macrophages also participate in the control of infection [5, 12, 13]. In the Fischer rat model, a cytotoxic effect on T. gondii tachyzoites mediated by platelets and IgE antibodies has been reported [14]. Moreover, thromboxane was involved in a human platelet-mediated cytotoxic effect against free tachyzoites in the absence of antibodies [15]. The present study shows both a human platelet activation by free tachyzoites of T. gondii and human platelet-mediated cytoinhibition of T. gondii intracellular growth invitro, in the absence of antibodies. The results suggest a prominent role of platelet-derived growth factor (PDGF) in this phenomenon.

PDGF was originally isolated from the α-granules of platelets and has important growth-promoting activities and differentiation effects for several cell types which express PDGF α- and β-receptors [16–18]. PDGF is the result of two genes, PDGF A and B, which dimerize to form three possible isoforms, PDGF-AA, -AB and -BB [16–18]. In human platelets, only PDGF-AA and -AB are found.

MATERIALS AND METHODS

Platelets

Platelets were isolated from blood collected from healthy volunteers according to Polack et al. [19]. Briefly, ACD (85 mm citric acid, 66.6 mm sodium citrate, and 11 mm D(+) glucose) anticoagulated blood (1/6, v/v) was centrifuged for 15 min at 150 g to obtain platelet-rich plasma. Platelets were obtained by centrifugation at 1200 g for 10 min, and washed once in Tyrode buffer containing 3.5 g of human albumin/l, 1 μm prostangladin I2 (PGI2; Sigma, St Louis, MO), 10 U of heparin/ml (Choay, Paris, France) before being washed again in Tyrode buffer only. Platelets at a concentration of 640 × 106/ml were finally placed in modified Eagles' medium (MEM; BioMerieux, Lyons, France) containing 2 μl/ml of ATP diphosphohydrolase EC 5.6.1.5 (apyrase) (CRTS, Strasbourg, France). Contamination by lymphocytes was < 1/103 platelets, as shown by observation of platelet suspensions using interferential contrast microscopy.

Parasites

Toxoplasma gondii tachyzoites, obtained from the peritoneal fluid of Swiss mice infected with the RH strain, were filtered through a 3 μm pore-size polycarbonate membrane (Cyclopore, Louvain-La-Neuve, Belgium), following by washing in 154 mm NaCl three times and centrifugation at 1200 g for 10 min. Viability was evaluated with acridine orange (Sigma), ethidium bromide (Sigma) and fluorescence microscopy as previously described [20].

Platelet activation

Activation of platelets was performed in 24-well cell culture plates (Nunc, Roskilde, Denmark). Ten millilitres of isolated platelets at a concentration of 640 × 106/ml were incubated with 10 μl of 3H-serotonin (1 μCi/ml; New England Nuclear, Boston, MA) in Tyrode buffer containing 3.5 g of human albumin/l, 1 μm PGI2, 10 U heparin/ml and washed by centrifugation at 1200 g for 10 min. In this experiment (N = 1), platelet/tachyzoite ratios of 10, 50 and 100, in the presence or absence of apyrase, were used with 1.6 × 108 platelets each time. Each assay was repeated six times (n = 6 replicates). Thrombin at 0.5 U/ml was used as a positive activator of platelets. Activation was measured by 3H-serotonin (1 μCi/ml; New England Nuclear) released from platelets after 5 min of cell contact. The cells from each well were harvested and radioactivity measured in a Minaxi liquid scintillation counter (Packard, Downers Grove, IL).

In vitro culture of T. gondii

The parasites were grown in human embryonic lung fibroblast cells (MRC5: Medical Research Council Number 5) (BioMérieux) [21] on sterile glass coverslips (Flobio, Courbevoie, France) deposited in 24-well cell culture plates (Nunc) at 37°C and in a humid atmosphere of 95% air–5% CO2. To Eagles' basal medium (BioMérieux) and MEM (BioMérieux) 20 ml of ultroser-G/l (GIBCO-BRL Life Technology Ltd, Paisley, UK), penicillin (100 mg/l), streptomycin (100 mg/l), kanamicin (50 mg/l), colistin (40 mg/l) and amphotericin B (5 mg/l) were added; the two media were used to grow the MRC5 cells to confluence (5 days), then for parasite multiplication, respectively. Platelets and other products were seeded at this time. After 24 h, the cultures were pulsed with 1 μCi/well of 3H-uracil (New England Nuclear) which is incorporated only by the parasite [22], or 1 μCi/well of 3H-thymidine which is incorporated only by the fibroblasts (New England Nuclear). After 18 h, the cells from each well were harvested and the radioactivity measured in a beta counter (Packard) in order to evaluate the parasite and fibroblast in vitro growth. Parasite, infected and uninfected cells without treatment were used as controls. In order to compare data from different experiments, ct/min values from controls were normalized to 100%.

Action of human platelets on uracil uptake by intracellular T. gondii

Human platelet preparations from four donors were assayed in order to inhibit T. gondii intracellular growth in human embryonic lung fibroblasts (MRC5 cells). Three platelet donors were negative for IgG antibodies to T. gondii. The fourth donor was positive and had 80 International Units (IU) of IgG antibodies to T. gondii. (One IU/ml corresponds to 1:4 titre in Dye test. IU are defined in relation to standard reference serum which is available on request from the World Health Organization.) MRC5 cells were used at confluence at the moment of the assay. Platelets and parasites were added at the same time. The parasite loads were 0.5 × 106 and 1 × 106 per well and the platelet loads were 1 × 106, 5 × 106 and 1 × 107. Normal-washed and frozen platelets were assayed using the same dilutions. Frozen platelets were investigated to ascertain if some free components of platelets were involved in the phenomenon. The control did not have parasite, nor did platelets. Each assay was repeated three times (n = 3 replicates). Parasite and MRC5 cell growth were evaluated by incorporation of 3H-uracil and 3H-thymidine, as indicated above.

Direct effect of human recombinant PDGF-AB (hrPDGF-AB) on extracellular tachyzoites of T. gondii

HrPDGF-AB (R&D Systems, Abingdon, UK) at 0, 30, 60 ng/ml was incubated with 106 tachyzoites for 0, 1, 2 and 4 h and the viability of parasites measured as indicated above. After washing in 154 mm NaCl three times and centrifugation at 1200 g for 10 min, treated and untreated parasites were subsequently seeded in invitro culture.

Action of hrPDGF on Toxoplasma-infected MRC5 cells

HrPDGF-AB (R&D Systems) at 0, 30, 60 ng/ml was incubated with MRC5 cells before (4 h, 2 h), during (0 h) and after (2 h, 4 h) infection by 106T. gondii tachyzoites per well, as indicated in the in vitro culture section. No significant variation in the invasion rate was observed. In subsequent experiments hrPDGF was incubated with MRC5 cells 2 h before infection by T. gondii tachyzoites. Parasite and MRC5 cell growth were evaluated by incorporation of 3H-uracil and 3H-thymidine as indicated above.

Effect of addition of anti-PDGF-AB antibodies

Affinity chromatography-purified antibodies against human PDGF-AB (R&D Systems) were used, at 1:400 dilution in MEM, to attempt the inhibition effect of hrPDGF. At this dilution, there was no direct effect on MRC5 cells or parasites. The antibodies, the hrPDGF-AB and the parasites were added to the invitro culture at the same time. Controls were: MRC5 cells plus antibodies, infected MRC5 cells plus antibodies, MRC5 cells and infected MRC5 cells alone. Parasite and MRC5 cell growth were evaluated by incorporation of 3H-uracil and 3H-thymidine, as indicated above.

IL-6 secretion

The level of IL-6 in the culture supernatants was measured by a sandwich immunoassay (Immunotech, Marseilles, France) at the end of each experiment.

Additional reagents used in experiments

To investigate possible mechanisms behind platelet- and PDGF-induced parasite growth inhibition, the following products were added to the in vitro culture of T. gondii: hrIL-6 (TEBU), Le Perray en Yvelines, France, hrPDGF-AA (Boehringer-Mannheim, Grenoble, France), hrPDGF-BB (TEBU), polyinosinic acid–polycytidylic acid (PI-PC) (Boehringer-Mannheim), human recombinant transforming growth factor-beta 1 (hrTGF-β1) (R&D Systems) and swine insulin (Organon, Saint-Denis, France). To elucidate PDGF mechanisms involved in the inhibition of the parasite, the following products were used for analysis of the arachidonic acid oxygenation and the cyclooxygenase cycle: acetylsalicylic acid (aspirin (Asp); Sigma), carboxyheptyl imidazole (CHI; TEBU), indomethacin (IND; Sigma), pinane thromboxane A2(PTA2; TEBU). Parasite and MRC5 cell growth under different treatments were evaluated by incorporation of 3H-uracil and 3H-thymidine, respectively.

Statistical analysis

For comparison of data, the paired Student's t-test was used. P ≤0.05 was considered significant.

RESULTS

Platelet activation

Platelet activation by T. gondii tachyzoites was observed both in presence and absence of added apyrase, though a better activation was noted in presence of apyrase (Fig. 1).

Fig. 1.

Fig. 1

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Release of 3H-serotonin from human platelets versus number of Toxoplasma gondii tachyzoites. Data are given as mean ct/min ± s.d. (six replicates). In the experiment, 1.6 × 108 platelets were used. The platelet/tachyzoite ratios were: infinite (0 tachyzoite), 10 (160 × 105 tachyzoites), 50 (32 × 105 tachyzoites) and 100 (16 × 105 tachyzoites). ▪, + apyrase; □, − apyrase.

Effect of addition of human platelets on uracil uptake by intracellular T. gondii

Human platelets inhibited T. gondii intracellular growth in human embryonic lung fibroblasts (MRC5 cells) in a non-dose-dependent manner, as shown by the 3H-uracil uptake (Fig. 2). The effect was the same whether 0.5 × 106 or 1 × 106 parasites/well were used (Fig. 2).

Fig. 2.

Fig. 2

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Inversely dose-dependent relationship between % growth of Toxoplasma gondii and number of human platelets. Data are given as mean % ± s.d. (triplicates). Human platelets inhibited parasite growth in MRC5 cells, in a non-dose-dependent manner. ▪, 106T. gondii; □, 0.5 × 106T. gondii.

There was no relationship between donor seropositivity to T. gondii and the parasite growth-inhibiting effect of their platelets in vitro. Taking into account all the experiments with one human platelet/tachyzoite ratio and 1 × 106 parasites or 0.5 × 106 per well, the parasite 3H-uracil uptake was inhibited with 75% of platelet preparations. The same results were obtained from repeatedly frozen and unfrozen platelets (data not shown).

PDGF-induced inhibition of uracil uptake by T. gondii

HrPDGF-AB induced a significant inhibition of the parasite (P < 0.05), as well as a significant proliferation of the infected or uninfected host cells. The results of one experiment (N = 1, n = 12 replicates) are shown in Table 1. The growth of T. gondii and uninfected MRC5 cells in the absence of hrPDGF-AB served as controls.

Table 1.

Effects of human recombinant platelet-derived growth factor (hrPDGF)-AB on Toxoplasma gondii and MRC5 growth

graphic file with name cei0111-0325-t1.jpg

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In order to compare data from different experiments the controls of each one were normalized to 100%. The overall mean percent inhibition of T. gondii growth (N = 31 experiments) due to hrPDGF-AB (40 ng/ml) was 28.3%, ranging from 8.2% to 69.4% (P < 0.05). The effect of different concentrations of hrPDGF-AB on T. gondii growth is shown in Fig 3a.

Fig 3.

Fig 3

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Human recombinant platelet-derived growth factor (HrPDGF)-AB inhibition of 3H-uracil uptake by Toxoplasma gondii. (a) Dose-dependent relationship between percent growth and concentration of hrPDGF-AB (mean % ± s.d. for triplicates experiments). (b) Action of hrPDGF-AA, hrPDGF-BB and both molecules together on the intracellular growth of T. gondii. Parasite intracellular growth and MRC5 cell growth were evaluated by incorporation of 3H-uracil and 3H-thymidine, respectively. Data are given as mean ct/min ± s.d. (12 replicates). □, Infected MRC5 cells; ▪, T. gondii.

In general, hrPDGF-AB (40 ng/ml) increased the growth of parasite-infected MRC5 cells (mean 115.1%, range 223.7–47.7% for N = 31 experiments) and MRC5 cells (mean 121.1%, range 202.5–71.5%). Both controls in the absence of hrPDGF were normalized to 100%. For most of the experiments, 3H-thymidine incorporation was higher in uninfected cells than the corresponding T. gondii-infected MRC5 cells.

Toxoplasma gondii growth in terms of different concentrations of hrPDGF-AB are shown in Fig. 3a.

HrPDGF-AA, hrPDGF-BB and hrPDGF-AA plus hrPDGF-BB also induced an inhibition of T. gondii growth. The respective incorporations of 3H-thymidine and 3H-uracil are shown in Fig 3b. HrPDGF-AA plus hrPDGF-BB yielded the best results. A significant difference (n = 12; P < 0.02) was observed between the inhibition of the parasite induced by hrPDGF-AA and that of hrPDGF-BB, (hrPDGF-BB>hrPDGF-AA) that is inversely proportional to 3H-uracil uptake (Fig. 3b). The significant differences between zero hrPDGF and hrPDGF-AA plus hrPDGF-BB, hrPDGF-AA, and hrPDGF-BB were P < 0.0001, P < 0.01 and P < 0.0001, respectively.

Polyclonal anti-PDGF-AB antibodies impaired the parasite growth-inhibitory effect of hrPDGF-AB in a significant way (n = 6; P < 0.0008) (Fig. 4). HrPDGF-AB induced a 37.3% (P < 0.001) growth inhibition of the parasite. Anti-PDGF (1/400) alone did not have any significant effect on T. gondii and MRC5 cell growth. Given together, hrPDGF-AB and anti-PDGF inhibited parasite growth by 21.3% (P < 0.008). The anti-PDGF thus impaired PDGF-mediated parasite inhibition by 37.3–21.3 = 16.0%, giving a net effect of anti-PDGF-AB as 16.0/37.3 = 42.9%.

Fig. 4.

Fig. 4

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Effect of rabbit anti-human platelet-derived growth factor (PDGF)-AB (anti) and hrPDGF-AB (PDGF) on intracellular Toxoplasma gondii growth. Parasite intracellular growth and MRC5 cell growth were evaluated by incorporation of 3H-uracil and 3H-thymidine, respectively (mean ct/min ± s.d., 12 replicates). □, Infected MRC5 cells; ▪, T. gondii. MEM, Modified Eagles' medium.

No significant difference was observed between hrPDGF-treated and untreated free tachyzoites. In addition, treatment of the parasite with hrPDGF-AB for 1 h, 2 h or 4 h and consecutive washings, before the host cell infection, did not have any effect upon subsequent in vitro parasite growth.

Incubation of fibroblasts with hrPDGF-AB, for 0 h, 2 h, and 4 h before infection, induced significant inhibitions of T. gondii3H-uracil uptake following infection. In this assay, the best intracellular parasite inhibition was obtained when hrPDGF-AB was added to the MRC5 cells 2 h before infection with T. gondii (32.3%, P < 0.003). The hrPDGF-AB, added 4 h after infection, elicited a 10.5% (P < 0.01) growth inhibition of the parasite. The hrPDGF-AB, added at the moment of the infection, elicited a 11.6% (P < 0.01) growth inhibition of the parasite.

Probable mechanisms involved in the inhibition of T. gondii growth

Swine insulin (10 IU/ml) alone induced an intracellular T. gondii growth inhibition of 36.8% (P < 0.03). Insulin and hrPDGF-AB (30 ng/ml) together generated an inhibition of 62.6% (P < 0.006). Other experiments with different quantities of insulin from 2 to 20 IU confirmed this added effect (data not shown).

Asp (1 mm) did not have any effect on the growth of uninfected MRC5 cells, but induced a 13.8% (P < 0.004) inhibition of the infected ones (Fig. 5a). In this experiment, Asp (1 mm) alone did not affect parasite growth. HrPDGF-AB (40 ng/ml) inhibited parasite growth by 57.3% (P < 0.002). Asp and hrPDGF-AB together induced a 45.3% (P < 0.002) inhibition of T. gondii growth. Asp, a cyclooxygenase inhibitor, impaired then the PDGF-mediated T. gondii growth inhibition by 12% (P < 0.01). The Asp net effect was therefore 12.0/57.3 = 20.9%.

Fig. 5.

Fig. 5

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Effect of aspirin (Asp), indomethacin (IND) and human recombinant platelet-derived growth factor (hrPDGF)-AB on intracellular Toxoplasma gondii growth. (a) Effect of Asp and hrPDGF-AB. (b) Effect of IND and hrPDGF-AB. Parasite intracellular growth and MRC5 cell growth were evaluated by incorporation of 3H-uracil and 3H-thymidine, respectively (mean ct/min ± s.d., 12 replicates). □, Infected MRC5 cells; ▪, T. gondii. MEM, Modified Eagles' medium.

In another experiment, IND (1 μm), a potent inhibitor of PGE2 synthesis and generally of the cyclooxygenase pathway in the arachidonic acid metabolism, generated a 53.4% (P < 0.0001) intracellular inhibition of T. gondii growth (Fig. 5b). An added effect was noted in association with hrPDGF-AB (40 ng/ml) (77.2%, P < 0.0001). In this case, hrPDGF-AB alone induced a 37.9% inhibition of T. gondii growth. IND impaired then PDGF-mediated T. gondii growth inhibition by 37.9 − (77.2–53.4) = 14.1%. The IND net effect was therefore 14.1/37.9 = 37.2%.

PTA2, a thromboxane synthetase inhibitor, did not have any effect on uninfected MRC5 cell growth, but hrPDGF-AB increased cell growth (25.8%, P < 0.005) (Fig. 6a). The association of PTA2 and hrPDGF-AB increased T. gondii-infected MRC5 cell growth (39.7%, P < 0.003). PTA2 (27 μm) inhibited parasite growth by 38.1% (P < 0.002), and hrPDGF-AB (40 ng/ml) induced an inhibition of 25.8% (P < 0.005) (Fig. 6a). An added effect was observed when associating PTA2 and hrPDGF-AB (62.3%, P < 0.001).Then, PTA2 impaired very weakly the PDGF-mediated T. gondii growth inhibition by only 25.8 − (62.3–38.1) = 1.6%. The PTA2 net effect was 1.6/25.8 = 6.2%.

Fig. 6.

Fig. 6

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Effect of pinane thromboxane A2 (PTA2), carboxyheptyl imidazole (CHI) and human recombinant platelet-derived growth factor (hrPDGF)-AB on Toxoplasma gondii growth. (a) Effect of PTA2 and hrPDGF-AB. (b) Effect of CHI and hrPDGF-AB. Parasite intracellular growth and MRC5 cell growth were evaluated by incorporation of 3H-uracil and 3H-thymidine, respectively. Data are given as mean ct/min ± s.d. (mean ct/min ± s.d., 12 replicates). □, Infected MRC5 cells; ▪, T. gondii. MEM, Modified Eagles' medium.

CHI (10 μm), a thromboxane synthetase inhibitor, had no effect on the growth of uninfected MRC5 cells, or on that of infected ones (Fig. 6b). The association of CHI and hrPDGF-AB (40 ng/ml) increased infected MRC5 cell growth (16.7%, P < 0.0001). CHI alone induced a T. gondii inhibition of 16.3% (P < 0.0004), while hrPDGF-AB (40 ng/ml) induced an inhibition of 8.2% (P < 0.03). A small added effect was observed with the association of CHI and hrPDGF-AB (9.7–8.2% = 1.5%, P < 0.01). Therefore, CHI did not impair clearly PDGF-mediated T. gondii inhibition.

Human rIL-6 (5 ng/ml, hrIL-6) affected neither infected nor uninfected MRC5 cell growth. The association of hrIL-6 (5 ng/ml) and hrPDGF-AB (40 ng/ml) increased infected MRC5 cell growth (34.8%, P < 0.0002). HrIL-6 (5 ng/ml) alone induced a T. gondii inhibition of 13.5% (P < 0.02). HrPDGF-AB (40 ng/ml) induced a parasite inhibition of 26.9% (P < 0.0002). HrIL-6 and hrPDGF-AB together inhibited parasite growth by 22.3% (P < 0.003). HrIL-6 (5 ng/ml) and hrPDGF-AB were antagonistic. IL-6 levels on cell culture supernatants of T. gondii-infected or uninfected MRC5 cells are shown in Table 2.

Table 2.

IL-6 secretion in MRC5 cells by human recombinant platelet-derived growth factor (hrPDGF)-AB and/or Toxoplasma gondii

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At higher concentrations (50 ng/ml) hrIL-6 had a synergistic effect with hrPDGF-AB. The two molecules together induced a 38.7% (P < 0.0001) inhibition of parasite growth against 29.7% (P < 0.0043) for hrPDGF-AB alone. HrIL-6 (50 ng/ml) alone did not generate any statistically significant data showing T. gondii growth inhibition. IL-6 levels on the spent culture fluids of the infected or uninfected MRC5 cells showed the same pattern as indicated above.

PI-PC (20 ng/ml), an inducer of IL-6, had only an effect on uninfected MRC5 cell growth (17.6%; P < 0.02). The combination of PI-PC and hrPDGF-AB increased infected cell growth (19.7%, P < 0.0009). PI-PC alone induced a non-significant T. gondii inhibition of 14.3%. HrPDGF-AB (40 ng/ml) generated a parasite inhibition of only 4.7% (P< 0.05). PI-PC and hrPDGF-AB together induced a parasite growth inhibition of 22.3% (P<0.003). PI-PC (20 ng/ml) and hrPDGF-AB exerted an antagonistic effect on parasite inhibition similar to that noted in the IL-6 experiment (5 ng/ml). IL-6 levels on cell culture supernatants of T. gondii-infected or uninfected MRC5 cells are shown in Table 3.

Table 3.

IL-6 secretion in MRC5 cells by Toxoplasma gondii, and polyinosinic acid–polycytidylic acid (PI–PC).

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TGF-β1 (5 ng/ml), another component of platelet α-granules, inhibited the growth of uninfected and infected MRC5 cells by 49.8% (P < 0.01) and 14.1% (P < 0.003), respectively. The association of hrTGF-β1 and hrPDGF-AB (40 ng/ml) increased the growth of both uninfected (14.1%, P > 0.05), and infected (4.8%, P < 0.04) MRC5 cells. HrPDGF-AB alone also increased the growth of uninfected (31.5%, P < 0.01), and infected (17.6%, P < 0.04) MRC5 cells. HrTGF-β1 alone induced a T. gondii inhibition of 35.5% (P < 0.005), whereas hrPDGF-AB alone generated a parasite inhibition of 20.1% (P < 0.02). With hrTGF-β1 and hrPDGF-AB together the level of inhibition was 34.5% (P < 0.01). TGF-β1 was not antagonistic to PDGF in inhibiting parasite growth in confluent host cells.

DISCUSSION

Platelet activation with free T. gondii tachyzoites was observed in the presence or absence of added apyrase that destroys any extracellular ATP/ADP. Toxoplasma gondii produces an oligomeric protein with dithiol-activated nucleoside-triphosphate-hydrolase (NTPase) activity [23–28]. In fact, there are two isoenzymes termed NTPase-I and NTPase-II, the first one confined only to virulent strains and the second to all strains of T. gondii [29]. Thus, the use of apyrase with PGI2 in the isolation of platelets, the parasite NTPase activities, the absence of serum in the in vitro culture of the parasite, and the microscopic observations, all suggest that aggregation of platelets is not involved in the mechanism of T. gondii cytoinhibition, as found for Plasmodium falciparum [30, 31].

The observation that T. gondii causes platelet activation is consistent and complementary to the studies of Yong et al. [15]. Similar results on the activation of platelets by other protozoa such as P. falciparum have also been reported [19,31–33]. In toxoplasmosis, transient parasitaemia is only observed in the acute and the reactivated phases [34, 35]. Therefore, in this context, tachyzoites of T. gondii could activate human platelets during primary infection and reactivated toxoplasmosis. In our laboratory, following infection with tachyzoites of T. gondii, a thrombocytopenia was observed in experimentally infected rats (data not shown). Moreover, it is interesting to note that in congenital toxoplasmosis, six of the seven parasitologically proved cases examined had thrombocytopenia [36].

Experiments on the parasite intracellular inhibition with frozen and normal washed platelets suggest that the mechanism is complex, and at least one component of the platelets is involved in this phenomenon. It is well established that the activation of platelets initiates α-granule release. In the current study, PDGF, one component of α-granules, inhibited the intracellular growth of T. gondii. PDGF must act on the host cells, since PDGF does not have any direct cytotoxic effect on the free T. gondii tachyzoites, nor on host cell invasion, since hrPDGF-AB is able to induce parasite growth inhibition 4 h after infection. In addition, no significant difference was observed between 4 and 0 h after infection. It is interesting to note that invasion of cells by tachyzoites takes approximately 15 s. Other experiments showed that the maximal parasite cytoinhibition was obtained with hrPDGF-AB-treated host cells before infection. The specificity of the hrPDGF-AB action was illustrated when parasite cytoinhibition was partially impaired by polyclonal antibodies against PDGF. This suggests that PDGF is involved indirectly in parasite intracellular growth [22].

The medium used for the invitro culture of T. gondii lacks mammalian immunoglobulins, and three of the four platelet donors did not have antibodies to T. gondii. Moreover, in the present study the results obtained with frozen and normal washed platelets, hrPDGF-AB, -AA, -BB and hrTGF-β1 suggest that antibodies to T. gondii cannot be involved in this human platelet-mediated cytoinhibition against the parasite as found in other systems [37–40].

The mechanism of PDGF action involving host cell proliferation includes a requirement for insulin [41]. Our data on insulin and PDGF suggest that insulin is not necessary for platelet-mediated parasite inhibition, since the effects of both molecules were independent. Thus, it is possible that each molecule has a different action mechanism to inhibit parasite growth. In most of the experiments conducted for the present study, hrPDGF-induced host cell proliferation and parasite growth inhibition were measured by 3H-thymidine and 3H-uracil uptake, respectively. However, host cell proliferation and parasite growth inhibition are not necessarily related, since the TGF-β1 experiments showed that normal host cell proliferation is not essential for parasite inhibition. Thus, several mechanisms may act to inhibit parasite intracellular growth.

Human platelet granules comprise approximately 70% PDGF-AB. To know which chain is implicated in the cytoinhibition phenomenon, hrPDGF-AA and hrPDGF-BB were used separately. The results show that both chains are involved in the inhibition of T. gondii growth. PDGF-AA binds only to receptor dimers αα, PDGF-AB to αα or αβ, whereas PDGF-BB binds to αα, αβ, and ββ [16, 42]. Interestingly, α-β heterodimers can signal through unique transduction pathways, which could induce a stronger mitogenic signal in mammalian cells [16]. In addition, the β type is predominant among the PDGF receptors in human fibroblasts [43]. These facts may explain why, in the present study, hrPDGF-AA elicited the lowest T. gondii growth inhibitions, and hrPDGF-AB the highest ones.

The toxic, antibody-independent effect of human platelets on certain tumour cell lines is blocked by inhibitors of the arachidonic acid metabolism [44]. In the present study, the action of PDGF on parasite intracellular growth was only partially blocked by cyclooxygenase inhibitors (IND and Asp). Moreover, IND also inhibited the intracellular growth of T. gondii. Consequently, the cyclooxygenase pathways could be very important for parasite survival and growth [45].

However, thromboxane A2 cannot be involved in the PDGF intracellular growth inhibition of the parasite, as shown by the inaction of the two inhibitors used in the current study. On the contrary, T. gondii might use this metabolite or its effects on the host cell for its intracellular survival. This does not contradict the results of Yong et al. [15], who used free tachyzoites that were without the protection of the parasitophorous vacuole membrane.

Prostaglandin E2 (PGE2) is produced by T. gondii [15] and it is well known that prostaglandins (PG) may inhibit or promote tumour cell replication, depending on the cell system under study. In the present study, the inhibitors of the cyclooxygenase cycle usually increased MRC5 cell thymidine uptake but decreased the T. gondii one. It was suggested that PG endoperoxidase synthase activity could be almost completely inhibited by PDGF [46]. This is in agreement with the current study and in particular with the experiment using indomethacin, a potent inhibitor of PGE2 synthesis. For these reasons, PG inhibition may be involved in T. gondii inhibition. However, the PDGF-induced inhibition could not be mediated by only the cyclooxygenase cycle.

In a recent report, it was suggested that leukotriene B4 induced T. gondii cytotoxicity [47]. However, T. gondii-induced inhibition of leukotriene B4 released in stimulated monocyte-derived macrophages is reversed by IFN-γ [47]. PDGF might act in a similar way to IFN-γ, since PDGF can amplify lipooxygenase products [48]. Thus, PDGF-mediated T. gondii inhibition may use multiple mechanisms.

In the present study, T. gondii, PI-PC and PDGF all elicited IL-6 secretion by MRC5 cells. Similar results were reported concerning PI-PC and PDGF as inducers of IL-6 in fibroblasts [49]. IL-6 may be involved in intracellular parasite growth, as confirmed by our data on T. gondii, hrIL-6, PI-PC and hrPDGF. It is however very difficult to show that PDGF-induced IL-6 in the infected host cell is implicated in parasite inhibition, since T. gondii can also elicit an IL-6 response in the host cell. This IL-6 secretion may depend on the type of cells used [50]. Our data suggest that high concentrations of hrIL-6 are necessary for a synergistic effect between IL-6 and PDGF to inhibit the parasite. Thus, PDGF-induced parasite inhibition may also involve IL-6.

In the present study, the fact that platelet inhibition was not dose-dependent suggests that more than one component was involved. One of the most important cytokines released from platelets at the same time as PDGF is TGF-β, which is characterized by its extreme multifunctionality [51]. In the current study, TGF-β1 inhibited thymidine uptake of the confluent uninfected MRC5 cell cultures, and the mitogenic action of PDGF, as previously reported [52]. In the T. gondii-infected cells this was also true, but to a lesser extent. TGF-β increased the mRNA level in the PDGF-A chain and induced the up-regulation of PDGF receptor-β mRNA in human embryonic lung fibroblasts [53]. This fact may partly explain why PDGF-AB-induced parasite inhibition was greater in association with TGF-β.

In conclusion, human platelets induce intracellular T. gondii growth inhibition mediated by PDGF in human embryonic pulmonary fibroblasts. The PDGF mechanism involved is complex. The cyclooxygenase cycle and the high concentration of IL-6 may explain in part the effect of PDGF on the parasite inhibition. Moreover, TGF-β1 may also be involved in human platelet inhibition of the parasite. In addition, other cytokines and other biochemical pathways, such as the 5-lipooxygenase products [47, 48], ATP production [54], intracellular free calcium concentrations [55], and reactive oxygen species [56] might participate in parasite growth inhibition. This might also involve transformation from tachyzoites to bradyzoites [57].

Acknowledgments

The authors would like to thank Dr F. Santoro, Mrs G. Tavernier, D. Lamotte and Mr T. Josan for their assistance.

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