Infection with Toxoplasma gondii does not Alter TNFα and IL-6 Secretion by A human Astrocytoma Cell Line

The secretion of tumour necrosis factor-α (TNFα), interleukin-1α (IL-α) and interleukin-6 (IL-6) by a human astrocytoma cell fine was studied 1 h, 3 h, 6 h and 24 h after infection with tachyzoites from three Toxoplasma gondii strains (virulent, RH; cystogentc, 76K and Prugniaud strains). The astrocytoma cell fine constitutively secreted TNFα and IL-6, but no IL-1α. A positive control was obtained by stimulation with phorbol esters inducing a significant increase (p < 0.05) in TNFα and IL- 6 secretion but not in IL-1α, while lipopolysaccharide (alone and after priming), interferon gamma, ionophore A 23187 and sera positive to T. gondii did not induce any increase in cytokine levels. None of the tachyzoites, whatever their virulence, induced a significant increase in cytokine production at any time in the study. Tachyzoites did not inhibit the secretion induced by phorbol esters.


Introduction
Toxoplasma gondii is a worldwide intracellular obligate parasite, which causes severe and frequent lesions (mainly cerebral) in AIDS patients. The pathophysiology of reactivation of toxoplasmosis in immunocompromised patients is still not clear, and the exact mechanisms of cyst rupture, including the role of cytokines remain unknown. ',3 While interferon-, (INFT) is involved in protective immunity, its precise role and mode of action is not known.
Tumour necrosis factor<z (TNF00, a cytokine which is involved in infectious and inflammatory diseases, has not been extensively studied in toxoplasmosis. Its role during toxoplasmic infection in mice is controversial, 5,e and TNF0t would seem to be implicated in human and animal toxoplasmosis. [7][8][9] Interleukin-6 (IL-6) and interleukin-lz (IL-Iz) play a role in parasitic diseases 1 and in mouse toxoplasmosis. 11 Their transcripts have been detected in brains of mice infected with T. gondii. " Thus, these three cytokines (TNFx, IL-6, IL-lcz) may be involved in the development of immunity that occurs in immunocompetent hosts after infection with T. gondii, as they are in the viral infections (particularly HIV). 3 Cerebral parasitic multiplication following cyst reactivation is the main event of toxoplasmic disease in patients with Acquired Immuno Deficiency Syndrome, but to our knowledge, few publications have studied the parasitic multiplication, the cyst forma-tion and rupture in astrocytes, which are cerebral host cells for T. gondii. Moreover, some authors have recently suggested that differences between T. gondii strains could be the cause of virulence differences in animals and perhaps in man, in association with the host immunological variations. [14][15][16][17] Thus, the aim of our work was to study the secretion of TNFz, IL-Iz and IL-6 by a human astrocytoma cell line infected by three T. gondii strains with different degrees of virulence.

Materials and Methods
Astrocytoma cell fine: The GHE (Glioblastome Humain E) cell line used for this study was derived from a surgical specimen of a primary brain tumour (grade II astrocytoma according to the classification of Kernohan and WHO). Briefly, the tumour specimen was minced and dissociated into single cells following incubation for 10 min in 0.5% trypsin in DMEM medium (Sigma, St Louis, MO). TM The cell line was then routinely grown in 25 crn" tissue culture flasks in DMEM medium supplemented with 10% foetal calf serum (DAP, Vogelgrun, France) and antibiotics (100 U penicillin and 50 t.tg streptomycin per ml). Culture was maintained at 37C in humidified air containing 5% CO2, and cells were subcultured when confluent.
After establishment, growth curves and cell doubling times were determined. The morphological features of the cell line were recorded with a phasecontrast microscope and camera. The glial origin of the cells was established by the presence of the glial fibrillary acidic protein (GFAP) and the S100 protein, markers for astrocytic differentiation, and with morphological histological criteria as described elsewhere. 18 The absence of Mycoplasma contamination was checked using the Mycoplasma detection kit from Boehringer (Meylan, France).

Infection of GHE cells: The percentage of cells in-
fected by T. gondii (assessed by immunofluorescence staining) was 10% after 1 h and 25% after 24 h when 105 parasites of the RH strain were added to the cells. When 106 parasites were added, those percentages were 20% and 45% respectively.
Those infection rates were not significantly different with the parasites from the chronic strain, but the average numbers of parasites per infected cell were higher for the virulent strain--approximately 5 parasites/cell for the RH strain after 24 h, and less than 2 parasites/cell for 76K. These infection rates were not significantly modified by INF), (100 U/ml).
The microscopic examination showed areas of lysed cells in the case of infection by the RH strain, which were not present in the case of infection with chronic strains.
Secretions without stimulation (negative control): In our experimental conditions, the astrocytoma cell line constitutively secreted low amounts of TNF0t (Fig. 1), higher amounts of IL-6 ( Fig. 2) and no IL-I. The secretions of TNFcz and IL-6 increased with time of sampling (the medium was changed at TO). No decrease in secretion was observed when polymyxin B was added. After 48 h, the cytokine levels were not different from those noted after 24 h (data not shown).
Positive controls: Cells from the GHE cell line were able to secrete significantly (p < 0.05) higher amounts of TNF(z and IL-6 at T2, T3 and T4 when stimulated by PMA than after each of the other stimulations (Figs  1 and 2). In this case the viability of the cells remained high (> 95%). DMSO (dimethylsulfoxide; Sigma) alone, the diluent of the PMA, did not induce any secretion.
Moreover, LPS alone (10 and 1 l.tg/ml), INF alone (100 U/ml), ionophore alone (10 -6 M), INF plus LPS at the same time, or cell priming by INF7 (8 h) and then addition of LPS, did not induce a significant increase in secretion of cytokine by these cells. Different parasite numbers (10 or 10 tachyzoites) did not modify the cytokine secretions. In our experiments, the cytokine (TNF(x and IL-6) levels were significantly lower (p < 0.05) at T1 than at T2, T3 or T4 after PMA stimulation.
No secretion depending on the strains: The multiplication of T. gondii tachyzoites did not induce the secretion of the three cytokines by the astrocytoma cells (Figs 1 and 2) whatever the strain used. The twelve IgG positive sera to T. gondii gave the same results.
The penetration of parasites into the cells during 2 h before the experiment did not inhibit the secretions induced by PMA (Figs 3 and 4).

Discussion
The results show that the GHE cell line constitutively secretes TNF0t and large amounts of IL-6, but no detectable IL-I(z before 24 h. The constitutive secretion of TNF0 is not constant in human astrocytoma cell lines: Bethea et al. report that TNF(x secretion was obtained only after stimulation in the CH235-MG cell line. The astrocytoma cells we studied do not respond to LPS and INF7 stimulation, even after priming by INFT, contrary to the data reported by Chung and Benveniste. 2 These results are not surprising since cytokine expression or secretion by glial cells are very different depending on the cell origins and the stimulation. '2-'s We did not observe any secretion of IL-I induced by previous TNF secretion, as reported in a mouse model after LPS stimulation. ' The virulent RH strain did not induce secretion of any of the three cytokines, when compared with the secretions observed with astrocytoma cells alone. Such data are consistent with our previous studies on human monocytes and macrophages concerning TNFo 8 and with the study of Friedland 27 on secretion of TNFot and IL-6 by a human monocytic cell line, but is different from the results obtained after infection of murine mononuclear cells by Leishmania infantum which is another obligate intracellular protozoan. 28 However, the intracellular behaviour of T. gondii and L. infantum is not the same, especially concerning the mechanisms used to escape intracellular killing, which could lead to differences in the induction of TNF secretion. The present model may mimic what happens after cyst rupture: tachyzoites from cystogenic strains enter brain cells around the cyst where they multiply. Sera with high anti-Toxoplasma IgG antibody titres did not induce this type of secretion, in contrast to what was shown with blood monocytes and monocyte-derived macrophages. The 3 comparing microglial cells and astrocytes in a murine model. Thus, the protective role of cerebral endogenous, astrocyte-secreted, TNFot is still unclear in man, even though it has been demonstrated to be an important factor of resistance to T. gondii infection in mice. 6,31 The causes of the different degrees of virulence between T. gondii strains are not understood. The genotypic and phenotypic variations between the T. gondii strains have been demonstrated for enzymatic activities, RFLP and pathogenicity in animals and humans. [15][16][17] From our results, the different strains do not induce different cytokine profiles in response to a parasitic infection. Thus the assumed differences between strains, which could explain the onset of cerebral reactivation in AIDS patients, do not seem to be mediated by astrocyte cytokine production. However, cytokines produced by other cerebral cells, such as microglial cells, or recruited inflammatory cells (lymphocytes, macrophages and/or polymorphonuclear) might have an .influence on parasitic multiplication and cyst rupture. Moreover, human monocytes and macrophages may secrete TNF in the presence of specific anti-Toxoplasma antibodies. 8 In conclusion the T. gondii protozoan does not induce (or prevent) cytokine secretion by human astrocytoma derived cells.