SEARCH

SEARCH BY CITATION

Keywords:

  • Toxoplasma gondii;
  • progesterone;
  • sex steroid;
  • flow cytometry

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cell mediated immunity is very important for host defence against intracellular pathogens and many studies have shown the role of the production of nitric oxide (NO) by interferon (IFN)-γ/lipopolysaccharide (LPS)-activated macrophages. As the progesterone level increases during pregnancy in mammals, and as previous studies have shown that progesterone inhibits inducible nitric oxide synthase (iNOS) expression and NO production, we aimed to investigate whether progesterone might modulate intracellular replication of Toxoplasma gondii in macrophages. Our results showed that progesterone does not influence T. gondii replication in non-activated RAW 264·7 cells, and although progesterone inhibits NO production induced by IFN-γ/LPS, we observed that it fails to restore the growth of T. gondii blocked by IFN-γ/LPS. After discussing the reasons for this apparent discrepancy, we concluded that progesterone has no direct effect on the macrophage response. The real effect of the sex steroids in T. gondii infection and their implication in clinical toxoplasmosis therefore need to be investigated further to involve wider mechanisms of the immune system.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cell mediated immunity is crucial for host defence against intracellular pathogens, including Toxoplasma gondii (1). Studies have shown that, in the murine model, non-specific cellular activation plays a central role in the control of the early stages of infection and in the driving of an appropriate T-cell response (Th1). Macrophages play a key role in these cell interactions. One of the macrophage mechanisms to block T. gondii replication is the production of nitric oxide (NO) after priming with interferon (IFN)-γ. Although the precise role of NO in toxoplasmosis needs to be specified (2,3), in several in vitro studies, NO is involved in the killing of intracellular T. gondii (4,5). Potential positive or negative regulatory mechanisms of NO generation by inducible nitric oxide synthase (iNOS) are multiple: lipopolysacharide (LPS), cytokines and glucocorticoides (6–9). In murine macrophage cell line RAW 264·7, Miller et al. showed that progesterone inhibits iNOS expression and NO production (10).

Acute T. gondii infection of a mammal occurring during pregnancy may be followed by congenital toxoplasmosis in the child. As the progesterone level increases during pregnancy, we investigated whether progesterone might modulate intracellular replication of T. gondii in the macrophage.

Our results showed that progesterone does not influence T. gondii replication in non-activated RAW 264·7 cells. Although progesterone inhibits NO production induced by IFN-γ/LPS, we observed that it failed to restore the growth of T. gondii blocked by IFN-γ/LPS.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cells

RAW 264·7 cells (ATCC) were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Sigma, Saint Quentin Fallavier, France) supplemented with 1% penicilline-streptomycine (Sigma), 1% glutamine (Sigma) and 10% foetal calf serum (FCS) (Sigma) at 37°C in a 5% CO2 humidified atmosphere. For experiments with progesterone, we used DMEM without phenol red and the FCS concentration was decreased to 2%. In all experiments, cell viability was assessed by blue trypan exclusion.

Progesterone treatment

Progesterone was first dissolved in ethanol (1 mg in 1 ml); the concentrated stock solution at 5 × 104 m in DMEM (without phenol red) was stored in aliquots at −20°C until used. Progesterone was tested at 10−8 m to 4 × 10−5 m concentration range. Controls corresponding to equivalent concentrations of ethanol or medium alone were included in each experiment. For progesterone treatment, cells were first seeded in a 24-well plate (106 cells per well) in DMEM without phenol red and with 2% FCS. Cells were left to adhere for 90 min, after which the medium was replaced by a culture medium containing progesterone. Cells were incubated in the presence of the hormone for 6 h before infection. After the 1-h challenge with T. gondii (without progesterone), the culture medium containing progesterone was added as before.

Cytokine stimulation

Except where mentioned, cell stimulation was achieved with an association of IFN-γ (Sigma) and LPS (Sigma) at IFN-γ (10 U/ml)/LPS (10 ng/ml) concentration. Cytokines were added in the medium according to the exact same timing as progesterone treatment: 6 h before infection and during the replication period.

NGMMA treatment

NGMMA (Sigma) was tested in DMEM at 0, 25, 100, 400 µm concentrations and was added to the medium at the same time as cytokines.

Parasites

The T. gondii RH strain was maintained by routine intraperitoneal passages of tachyzoites in female Swiss OF1 mice from IFFA Credo (Saint Germain sur l’Arbresle, France). To infect RAW 264·7 in vitro, tachyzoites were harvested from the peritoneal cavity 48 h after infection. After washing in Phosphate Buffered Saline (PBS) (Gibco, Cergy Pontoise, France), tachyzoites were resuspended in the culture medium at the appropriate concentration to obtain a ratio of one parasite per three cells. RAW 264·7 cells were incubated for 1 h at 37°C with 150 µl per well of T. gondii suspension, then washed once with PBS to remove non-adherent parasites before adding the medium in identical conditions as described before (progesterone alone, cytokines + progesterone or cytokines + NGMMA). Cell cultures were incubated for an additional 2 h for penetration studies (3 h in total) or 19 additional hours (20 h in total) for replication studies, and then harvested, fixed and stained.

Intracellular immunofluorescent staining

After incubation cells were harvested using EDTA-Trypsin (Sigma), fixed for 30 min at room temperature in 4% formaldehyde, washed and maintained in buffer (PBS-0·1% BSA) at 4°C until staining. Intracellular T. gondii labelling was performed as previously described (11). Briefly, after cell permeabilization using 0·3% saponin (Sigma) in PBS, intracellular parasites were first labelled with anti-P30 monoclonal antibody (IgG2a, P1E1E7, 19·4 µg/ml final concentration, provided by Bio Mérieux, Marcy l’Etoile, France) and then revealed by a fluorescein conjugated rabbit anti-mouse immunoglobulin F(ab′)2 specific fragment (1/50) from Dako (Trappes, France). After washing, cells were resuspended in 500 µl buffer without saponin and maintained at 4°C until flow cytometry or optical microscopy analysis.

Measurement of T. gondii penetration and replication with flow cytometry

In non-activated cells, penetration and replication were assessed using flow cytometry as previously described (11). Briefly, the percentage of positive cells in fl1 (green fluorescence), acquired on 3000 cells on the logarithmic scale, reflects the percentage of T. gondii infected cells. The mean of fluorescence intensity (MFI) acquired on 2000 infected cells on the linear scale is correlated with the intracellular replication (11). The negative controls used were samples of non-infected cells labelled as previously described.

Measurement of T. gondii penetration and replication with optical microscopy

In cytokine activated cells, replication was assessed with optical microscopy. After centrifugation of the samples, each pellet was resuspended in 30 µl buffer. Microscopic analysis was performed with propidium iodide (25 µg/ml). Parasites were revealed using a ultraviolet light microscope. Results were expressed as the number of tachyzoites per 100 infected cells.

Measurement of NO production

NO production was evaluated using the Griess reaction (12). 100 µl of the Griess reagent [1% sulfanilamide (Sigma)−0·1% naphtylethylenediamine (Sigma) in 2·5% phosphoric acid (Sigma)] were incubated in a 96-well flat bottom plate with 100 µl of supernatant and incubated for 10 min in darkness at room temperature. Samples were analysed in duplicate. The absorbance measurement was performed at 570 nm using a BEP III microplate reader (Dade Behring, Marburg, Germany). Sodium nitrite dilutions (Sigma) were used as standards.

Statistical analysis

Results are expressed as means ± SD of triplicate wells. The non-parametric Mann–Whitney test (because of the small size of our populations) was applied to test differences between progesterone and controls. P < 0·05 was considered statistically significant. NO production and T. gondii replication with NGMMA were studied with a simple regression test. Tests were performed using Statview software (Abacus, Cary, NC, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Role of progesterone on non-activated RAW 264·7 cells

Treatment of non-activated RAW 264·7 cells with progesterone had no direct effect (either inhibition or stimulation) on T. gondii infection and replication. Figure 1(a) shows percentages of infected cells assessed using flow cytometry, 3 h and 20 h after T. gondii challenge. Regardless of the progesterone concentration, we did not observe any significant difference between samples with or without progesterone. Figure 1(b) shows the MFI values, which are correlated with tachyzoite replication. We observed a substantial increase in MFI between 3 h and 20 h, reflecting T. gondii growth, but again there was no difference between progesterone and vehicle controls. In all experiments, cell viability was above 90%. Medium controls confirmed that replication was not affected by ethanol concentration (Figure 1b). Preincubation of the cell with progesterone for 24 h did not change the results (data not shown).

image

Figure 1. Effect of progesterone at various concentrations (with equivalent vehicle concentrations or medium as controls) on non-activated RAW 264·7 cells. Cells were harvested 3 h (Progesterone H3 and vehicle H3) and 20 h (Progesterone H20 and vehicle H20) after infection with tachyzoites. (a) The percentage of infected cells and (b) the mean fluorescence intensity (which correlates with replication) were obtained by flow cytometry analysis and are expressed as means ± SD of triplicate wells. *P < 0·05.

Download figure to PowerPoint

Role of progesterone on IFN-γ/LPS-activated RAW 264·7 cells

To investigate the role of progesterone on IFN-γ/LPS-activated RAW 264·7, preliminary experiments showed that both signals (IFN-γ and LPS) were necessary in our model for inhibiting T. gondii growth with a dose-dependent action. Incubation in the presence of IFN-γ (10 U/ml) and LPS (10 ng/ml) for 6 h before T. gondii challenge, and during the replication period, leads to an approximate 50% growth inhibition (data not shown). These concentrations were retained for the following experiments. We confirmed that progesterone inhibits IFN-γ/LPS-induced NO production (P < 0·05) (Figure 2a) but, in these conditions (low amounts of NO generation), progesterone did not restore T. gondii replication which was inhibited by the IFN-γ/LPS association (P > 0·05) (Figure 2b).

image

Figure 2. Effect of progesterone at various concentrations (with equivalent vehicle concentrations or medium as controls) in activated RAW 264·7 cells on (a) NO production and (b) tachyzoite replication at 20 h. (b) Shows a control of replication without cytokines. The results are expressed as means ± SD of triplicate wells. *P < 0·05.

Download figure to PowerPoint

Role of NGMMA on NO production and T. gondii replication

In order to verify that IFN-γ/LPS action can be blocked, we used NGMMA, a competitive inhibitor of l-arginine known to block NO production (4). As expected, increasing doses of NGMMA suppressed NO synthesis (NO production is inversely correlated to NGMMA concentration, r = 0·888) and restored the intracellular growth of T. gondii in a dose-dependent way (r = 0·832) (Figure 3). NGMMA had no effect on T. gondii replication in non-activated cells. Moreover, we aimed to investigate whether progesterone could counter the effects of NGMMA and then looked at the combined action of progesterone and NGMMA on IFN-γ/LPS-activated cells. Even though progesterone slightly adds to NO inhibition by NGMMA, it does not modify the parasite replication obtained with NGMMA alone on IFN-γ/LPS-activated cells (data not shown).

image

Figure 3. Effect of various concentrations of NGMMA on NO production and T. gondii replication, on IFN-γ/LPS-activated RAW 264·7 cells. Results are expressed as means ± SD of triplicate wells.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The present study showed that progesterone does not modulate T. gondii (RH strain) replication in the murine macrophage cell line (RAW 264·7), either in non-activated or IFN-γ/LPS-activated cells, despite its effect on NO production.

Concerning non-activated cells, our results indicated that progesterone has no direct effect on intracellular parasite replication. These results are consistent with many reports showing that the modulation of the immune system by sex steroids only happens on activated cells (13–18). Thus, we investigated whether the action of progesterone could be revealed on activated cells. First, we explored the effect of IFN-γ/LPS activation on T. gondii intracellular replication and NO production and observed an inhibition of the parasite replication and high amounts of NO production. Additionally, this effect can be modulated in a dose-dependent fashion by NGMMA, a specific competitive inhibitor of l-arginine for the NO synthase (NOS). These results appear to be consistent with previous studies suggesting that NO generation has a role in macrophage defence against intracellular pathogens, including T. gondii (2,4,19–21). After we determined activation conditions, we explored the action of progesterone. We used optical microscopy in this analysis because, in our model, IFN-γ treatment induced an intense autofluorescence of RAW 264·7, which prevented flow cytometry analysis. We did not observe any modulation of T. gondii replication with progesterone, although it had an effect on NO production in response to IFN-γ/LPS activation. In exactly the same conditions, and unlike progesterone, NGMMA succeeded in restoring parasite growth. The reason for this discrepancy is unclear. It may be because steroids and NGMMA do not reach the same target: progesterone inhibits iNOS induction at a pretranscriptional level (10) while NGMMA acts as a competitive inhibitor of arginine transport (7). More probably, our results suggest that the microbicidal action of IFN-γ is not mediated exclusively by NO. Studies have shown that IFN-γ (even more so when combined with LPS) induces iNOS but blocks arginase activity (9). While there is evidence that progesterone blocks IFN-γ/LPS-induced stimulation of iNOS (10), its ability to increase arginase activity needs to be investigated further. The fact that NGMMA can restore parasite growth in the presence of IFN-γ/LPS (according to our results) while potentiating TGF-β induced arginase activity (22) suggests that increased arginase activity could be beneficial to the replication of the parasite. The arginase pathway induces the production of polyamines, and many studies have shown that difluoromethylornithine (DFMO), a competitive inhibitor of ornithine decarboxylase, and thus inhibitor of polyamine synthesis, could inhibit parasite growth (23–27). This raises the question of the role of the arginase pathway in the defence against T. gondii. This hypothesis has already been considered but not confirmed. It was shown that DFMO affects neither in vivo toxoplasmic infection in mice nor replication of the RH strain in murine peritoneal macrophages (28). Similar results were obtained in another study (29) and appear to dismiss any role of polyamines in T. gondii growth. However, it is difficult to generalize this conclusion, as many results obtained with DFMO appear to be closely linked to the model used and, furthermore, in vitro conditions may hide some metabolic requirements of the intracellular parasite (30).

Others have considered the possibility that reactive nitrogen intermediates do not affect T. gondii directly, but via activation of other mechanisms that inhibit the intracellular proliferation of T. gondii (31). More recently, Taylor et al. suggested that IGTP, a family of IFN-γ regulated genes, might have a potential role. Indeed IGTP-deficient mice demonstrated a complete inability to restrict acute T. gondii infection despite the retention of a significant increase in iNOS level. This suggests that the IFN-γ regulated pathway is involved in microbial defence but not restricted to a direct effect of NO (32), which is consistent with our results.

Our study shows that progesterone does not influence in vitro T. gondii infection in murine macrophages. There is neither a direct effect on non-activated cells nor a modulation in IFN-γ/LPS-induced inhibition. Thus, we conclude that progesterone does not act directly on the macrophage response. Consequently, although many effects of sex steroids on the immune system have been described (33–35), their real implication in the defence against pathogens needs to be explored, and this is especially important for clinical toxoplasmosis. Indeed, one of the most important problems of T. gondii infection in humans is congenital toxoplasmosis. Mechanisms which control materno–foetal transmission are poorly understood and pregnancy hormonal impregnation may play a role. Future studies should consider the role of sex steroids on the overall immune system, especially taking into account the macrophage–lymphocyte interactions in the defence against intracellular pathogens.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by grants from the Ministère de la Recherche (EA 3087) and by Hospices Civils de Lyon. We would like to thank Dr Dechaud for his help concerning hormones and Thierry Bonhomme for reviewing the English.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Denkers EY, Gazzinelli RT. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin Microbiol Rev 1998; 11: 569588.
  • 2
    Hayashi S, Chan CC, Gazzinelli R, Roberge FG. Contribution of nitric oxide to the host parasite equilibrium in toxoplasmosis. J Immunol 1996; 156: 14761481.
  • 3
    Khan IA, Schwartzman JD, Matsuura T, Kasper LH. A dichotomous role for nitric oxide during acute Toxoplasma gondii infection in mice. Proc Natl Acad Sci USA 1997; 94: 1395513960.
  • 4
    Adams LB, Hibbs JB Jr, Taintor RR, Krahenbuhl JL. Microbiostatic effect of murine-activated macrophages for Toxoplasma gondii: role for synthesis of inorganic nitrogen oxides from 1-arginine. J Immunol 1990; 144: 27252729.
  • 5
    Gazzinelli RT, Camargo MM, Almeida IC et al. Identification and characterization of protozoan products that trigger the synthesis of IL-12 by inflammatory macrophages. Chem Immunol 1997; 68: 136152.
  • 6
    Boucher JL, Moali C, Tenu JP. Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for 1-arginine utilization. Cell Mol Life Sci 1999; 55: 10151028.
  • 7
    Morris SM Jr, Billiar TR. New insights into the regulation of inducible nitric oxide synthesis. Am J Physiol 1994; 266: E829E839.
  • 8
    Morris SM Jr, Kepka-Lenhart D, Chen LC. Differential regulation of arginases and inducible nitric oxide synthase in murine macrophage cells. Am J Physiol 1998; 275: E740E747.
  • 9
    Shearer JD, Richards JR, Mills CD, Caldwell MD. Differential regulation of macrophage arginine metabolism: a proposed role in wound healing. Am J Physiol 1997; 272: E181E190.
  • 10
    Miller L, Alley EW, Murphy WJ, Russell SW, Hunt JS. Progesterone inhibits inducible nitric oxide synthase gene expression and nitric oxide production in murine macrophages. J Leukoc Biol 1996; 59: 442450.
  • 11
    Gay-Andrieu F, Cozon GJ, Ferrandiz J, Kahi S, Peyron F. Flow cytometric quantification of Toxoplasma gondii cellular infection and replication. J Parasitol 1999; 85: 545549.
  • 12
    Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol 1988; 141: 24072412.
  • 13
    Chao TC, Van Alten PJ, Walter RJ. Steroid sex hormones and macrophage function. modulation of reactive oxygen intermediates and nitrite release. Am J Reprod Immunol 1994; 32: 4352.
  • 14
    Chao TC, Van Alten PJ, Greager JA, Walter RJ. Steroid sex hormones regulate the release of tumor necrosis factor by macrophages. Cell Immunol 1995; 160: 4349.
  • 15
    Chao TC, Phuangsab A, Van Alten PJ, Walter RJ. Steroid sex hormones and macrophage function. regulation of chemiluminescence and phagocytosis. Am J Reprod Immunol 1996; 35: 106113.
  • 16
    Miller L, Hunt JS. Sex steroid hormones and macrophage function. Life Sci 1996; 59: 114.
  • 17
    Piccinni MP, Giudizi MG, Biagiotti R et al. Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established Th1 cell clones. J Immunol 1995; 155: 128133.
  • 18
    Savita Rai U. Sex steroid hormones modulate the activation of murine peritoneal macrophages: receptor mediated modulation. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 1998; 119: 199204.
  • 19
    Sibley LD, Adams LB, Krahenbuhl JL. Macrophage interactions in toxoplasmosis. Res Immunol 1993; 144: 3840.
  • 20
    Scharton-Kersten TM, Yap G, Magram J, Sher A. Inducible nitric oxide is essential for host control of persistent but not acute infection with the intracellular pathogen Toxoplasma gondii. J Exp Med 1997; 185: 12611273.
  • 21
    Khan IA, Matsuura T, Kasper LH. Inducible nitric oxide synthase is not required for long-term vaccine-based immunity against Toxoplasma gondii. J Immunol 1998; 161: 29943000.
  • 22
    Boutard V, Havouis R, Fouqueray B, Philippe C, Moulinoux JP, Baud L. Transforming growth factor-beta stimulates arginase activity in macrophages. Implications for the regulation of macrophage cytotoxicity. J Immunol 1995; 155: 20772084.
  • 23
    Bacchi CJ, Nathan HC, Hutner SH, McCann PP, Sjoerdsma A. Polyamine metabolism: a potential therapeutic target in trypanosomes. Science 1980; 210: 332334.
  • 24
    McCann PP, Bacchi CJ, Clarkson AB Jr et al. Further studies on difluoromethylornithine in African trypanosomes. Med Biol 1981; 59: 434440.
  • 25
    Gillin FD, Reiner DS, McCann PP. Inhibition of growth of Giardia lamblia by difluoromethylornithine, a specific inhibitor of polyamine biosynthesis. J Protozool 1984; 31: 161163.
  • 26
    Hollingdale MR, McCann PP, Sjoerdsma A. Plasmodium berghei: inhibitors of ornithine decarboxylase block exoerythrocytic schizogony. Exp Parasitol 1985; 60: 111117.
  • 27
    Van Voorhis WC. Therapy and prophylaxis of systemic protozoan infections. Drugs 1990; 40: 176202.
  • 28
    Hofflin JM, Guptill DR, Araujo FG, Remington JS. Difluoromethylornithine and formycin B in toxoplasmosis. J Infect Dis 1985; 152: 1101.
  • 29
    Harris C, Salgo MP, Tanowitz HB, Wittner M. In vitro assessment of antimicrobial agents against Toxoplasma gondii. J Infect Dis 1988; 157: 1422.
  • 30
    Gonzalez NS, Sanchez CP, Sferco L, Algranati ID. Control of Leishmania mexicana proliferation by modulation of polyamine intracellular levels. Biochem Biophys Res Commun 1991; 180: 797804.
  • 31
    Langermans JA, Van der Hulst ME, Nibbering PH, Hiemstra PS, Fransen L, Van Furth R. IFN-gamma-induced 1-arginine-dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous tumor necrosis factor-alpha. J Immunol 1992; 148: 568574.
  • 32
    Taylor GA, Collazo CM, Yap GS et al. Pathogen-specific loss of host resistance in mice lacking the IFN-gamma-inducible gene IGTP. Proc Natl Acad Sci USA 2000; 97: 751755.
  • 33
    Grossman CJ. Interactions between the gonadal steroids and the immune system. Science 1985; 227: 257261.
  • 34
    Roberts CW, Satoskar A, Alexander J. Sex steroids, pregnancy-associated hormones and immunity to parasitic infection. Parasitol Today 1996; 12: 382388.
  • 35
    Olsen NJ, Kovacs WJ. Gonadal steroids and immunity. Endocr Rev 1996; 17: 369384.