tnfcx il-6 astrocytoma · keywords: astrocytoma cells, cytokine, toxoplasmagondii infection with...

Research Paper

Mediators of Inflammation 3, 291-295 (1994)

TH secretion of tumour necrosis factor-ix (TNFtx),interleukin-ltx (IL-lx) and interleukin-6 (IL-6) by a hu-man astrocytoma cell fine was studied I h, 3 h, 6 h and24 h after infection with tachyzoites from threeToxoplasma gondtt strains (virulent, RH; cystogentc, 76Kand Prugniaud strains). The astrocytoma cell fine consti-tutively secreted TNFtx and IL-6, but no IL-Ix. A positivecontrol was obtained by stimulation with phorbol estersinducing a significant increase (p < 0.05) in TNFtz and IL-6 secretion but not in IL-ltz, while lipopolysaccharide(alone and after priming), interferon gamma, ionophoreA 23187 and sera positive to T. gondii did not induce anyincrease in cytokine levels. None ofthe tachyzoites, what-ever their virulence, induced a significant increase incytokine production at any time in the study. Tachyzoitesdid not inhibit the secretion induced by phorbol esters.

Key words: Astrocytoma cells, Cytokine, Toxoplasma gondii

Infection with Toxoplasma gondiidoes not alter TNFcx and IL-6secretion by a human astrocytomacell line

H. Pelloux,1,cA J. Ricard, M.-F. NissouflJ.-C. Renversez,3 J.-P. Vuillez,4 A. Meunierand P. Ambroise-Thomas

Ddpartement de Parasitologie-MycologieMdicale et Moldculaire, CNRS ERS15,Laboratoire de Neuro-Biophysique INSERMU 318, Laboratoire de Biochimie A andDdpartement de Biophysique CNRS URA 1287,Facultd de Mddecine, UniversitJoseph Fourier, Grenoble 1, 38043 GrenobleCedex, France

CA Corresponding Author

Introduction

Toxoplasma gondii is a worldwide intracellularobligate parasite, which causes severe and frequentlesions (mainly cerebral) in AIDS patients. Thepathophysiology of reactivation of toxoplasmosis inimmunocompromised patients is still not clear, andthe exact mechanisms of cyst rupture, including therole of cytokines remain unknown.’,3 While inter-feron-, (INFT) is involved in protective immunity, itsprecise role and mode of action is not known.Tumour necrosis factor

H. Pelloux et al.

features of the cell line were recorded with a phase-contrast microscope and camera. The glial origin ofthe cells was established by the presence of the glialfibrillary acidic protein (GFAP) and the S100 protein,markers for astrocytic differentiation, and with mor-phological histological criteria as described else-where.18

The absence of Mycoplasma contamination waschecked using the Mycoplasma detection kit fromBoehringer (Meylan, France).

Toxoplasma gondii strains: Three T. gondii strainswere used: the virulent RH, and the chronic 76K andPrugniaud strains. 15a9 The RH strain never forms cystsin mice, and infection kills mice after 3 or 4 days. Incontrast, mice infected with the 76K and thePrugniaud strains do not die and cysts are present inmouse brains. The RH strain was obtained by in vitroculture in human fibroblast MRC5 cells (bioM(rieux,Marcy l’Etoile, France). The RH tachyzoites werecollected from the culture supernatant (1200 xg for10 min) after 4 days and then counted in a Neubauercell. The Prugniaud and 76K tachyzoites were ob-tained after inoculation of Swiss mice (OF1 strain, IffaCredo, L’Arbresle, France) treated withcorticosteroids. Briefly, mice received 1 mg i.m. ofhydrocortisone acetate (Roussel, Paris, France) for5 days. Tachyzoites were injected intraperitoneally,and cortisone treatment was prolonged for 10 days.After tachyzoite multiplication, the peritoneal fluidwas harvested, the tachyzoites were washed(1200 xg for 10 min) and counted. Their viabilitywas assayed by the ethidium bromide-acridine or-ange assay (Becton Dickinson, Oxnard, CA), andonly parasite preparations with a viability > 95% wereused.

7Fot assay: The immunoradiometric assay kit fordetection of human TNFtz (Medgenix, Brussels, Bel-gium) was used. The medium containing astrocytesor parasites was free of TNFtx (< 6 pg/ml).

IL-Iz and IL-6 assay: The techniques used were thecompetitive sandwich immuno enzymatic methods(Immunotech, Marseille, France) for IL-6 and IL-ltxinterleukins. The medium containing astrocytes andparasites was free of these cytokines (< 20 pg/ml).

Study of cytokine secretions: The GHE cells weredistributed into 24-well plates at 1.5 x 104 cells perwell in 1 ml of culture medium and then left tomultiply for 1 week at 37C, 5% CO,.. Experimentswere performed when the cell monolayers wereconfluent (approximately 1.5 x 105 cells per welD.Fresh medium was added to the cells at TO.

T. gondii tachyzoites of the different strains(1.5 x 105 per well) were added to the wells at TO.The ratio cells:parasites was then of approximately1:1. In some experiments, 106 parasites were addedto the wells.

292 Mediators of Inflammation Vol 3. 1994

Depending on the experiments, lipopoly-saccharide (LPS) (10 or 1 l.tg/ml; Calbiochem,Meudon, France), positive sera, human recombinantinterferon-g (INF,,) (100 U/ml); Boehringer, Meylan,France), phorbol esters (phorbol-12-myristate-13-ac-etate (PMA) 10-8 M; Sigma, St Louis, MO), ionophoreA 23187 (10-6 M; Sigma), or polymyxin B (1 }.tg/ml;Pfizer, Orsay, France) in order to inhibit LPS action,were added to the wells at TO.

Supernatants were collected from correspondingwells at different times (1 h, T1; 3 h, T2; 6 h, T3;24 h, T4). For some experiments, parasites were al-lowed to grow in cells for 2 h before stimulation.

Immunofluorescence staining: The presence of para-sites in GHE cells was revealed by indirectimmunofluorescence staining. The cell monolayerwas fixed and then dried. A rabbit polyclonal anti-Toxoplasma antibody was added and incubated for30 min at 37C. After washing, a fluorescein-labelledanti-rabbit IgG antibody (Institut Pasteur, France)was used to reveal the presence of parasites with anUV light microscope.

Positive sera: Twelve sera were collected from twelvepatients with high levels of IgG antibodies to T.gondii (640 to 1280 IU/ml, indirect fluorescent anti-body technique).

Statistical analysis: A statistical analysis was per-formed using analysis of variance and Kolmogorov-Smirnov tests.

Results

Infection of GHE cells: The percentage of cells in-fected by T. gondii (assessed by immuno-fluorescence staining) was 10% after 1 h and 25%after 24 h when 105 parasites of the RH strain wereadded to the cells. When 106 parasites were added,those percentages were 20% and 45% respectively.Those infection rates were not significantly differentwith the parasites from the chronic strain, but theaverage numbers of parasites per infected cell werehigher for the virulent strain--approximately 5 para-sites/cell for the RH strain after 24 h, and less than2 parasites/cell for 76K. These infection rates werenot 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 withchronic strains.

Secretions without stimulation (negative control): Inour experimental conditions, the astrocytoma cellline 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 timeof sampling (the medium was changed at TO). Nodecrease in secretion was observed when polymyxin

T. gondii, astrocytoma cells and cytokines

1500

lOOO

5OO

Cells

J CelIs+LPS

[] Cells+lNF

I CelIs+PMAI"1 Cells+RH

Cells+76K

! CelIs+PRU

3 6 24

Time (h)

FIG. 1. TNF produced by astrocytoma cells. The values for TNF( representthe mean of nine experiments with astrocytoma cells alone (control), fiveexperiments with cells plus LPS (10 p.g/ml) or INF7 (100 U/ml), four experi-ments with cells plus PMA (10 M), four experiments with cells plus RH T.gondii strain, two experiments with cells plus 76K strain and three experi-ments with cells plus Prugniaud strain. The supematants were collected

h, 3 h, 6 h and 24 h after stimulation. The same results were obtained withLPS at Ig/ml. The results with astrocytoma cells alone were not modifiedby addition of polymyxin B (1 lg/ml). Error bars represent one standarddeviation.

B was added. After 48 h, the cytokine levels were notdifferent from those noted after 24 h (data notshown).

Positive controls: Cells from the GHE cell line wereable to secrete significantly (p < 0.05) higher amountsof TNF(z and IL-6 at T2, T3 and T4 when stimulatedby PMA than after each of the other stimulations (Figs1 and 2). In this case the viability of the cells re-mained high (> 95%). DMSO (dimethylsulfoxide;Sigma) alone, the diluent of the PMA, did not induceany secretion.Moreover, LPS alone (10 and 1 l.tg/ml), INF alone

(100 U/ml), ionophore alone (10-6 M), INF plus LPSat the same time, or cell priming by INF7 (8 h) andthen addition of LPS, did not induce a significantincrease in secretion of cytokine by these cells.Different parasite numbers (10 or 10 tachyzoites)did not modify the cytokine secretions. In our experi-ments, the cytokine (TNF(x and IL-6) levels weresignificantly lower (p < 0.05) at T1 than at T2, T3 orT4 after PMA stimulation.

No secretion depending on the strains: The multipli-cation of T. gondii tachyzoites did not induce the

140000

120000

100000

Cells

IJ CelIs+LPS

l! CelIs+INF

lJ CelIs+PMA

I--I CelIs+RH

Cells+76K

I1 CelIs+PRU- 8000060000

40000

_.20000

3 6 24Time (h)

FIG. 2. IL-6 produced by astrocytoma cells. Details are the same as givenin the legend to Fig. except that IL-6 was used instead of TNF.

secretion of the three cytokines by the astrocytomacells (Figs 1 and 2) whatever the strain used. Thetwelve IgG positive sera to T. gondii gave the sameresults.The penetration of parasites into the cells during

2 h before the experiment did not inhibit the secre-tions induced by PMA (Figs 3 and 4).

Discussion

The results show that the GHE cell line constitu-tively secretes TNF0t and large amounts of IL-6, butno detectable IL-I(z before 24 h. The constitutivesecretion of TNF0 is not constant in humanastrocytoma cell lines: Bethea et al. report thatTNF(x secretion was obtained only after stimulationin the CH235-MG cell line. The astrocytoma cells westudied do not respond to LPS and INF7 stimulation,even after priming by INFT, contrary to the datareported by Chung and Benveniste.2 These resultsare not surprising since cytokine expression or secre-tion by glial cells are very different depending on thecell origins and the stimulation. ’2-’s We did not ob-serve any secretion of IL-I induced by previousTNF secretion, as reported in a mouse model afterLPS stimulation.’6 Chang et al.n showed that, in mice,the anti-T, gondii action of INF7 depends partly onthe TNF production induced by INF7 itself.

Mediators of Inflammation. Vol 3. 1994 29:3

H. Pelloux et al.

1000 CelIs+PMA

Cells+RH strain+,PMA

u 500,,’z

03 6 24

Time (h)

FIG. 3. TNF produced by astrocytoma cells after infection and PMA. Dataobtained from one representative experiment obtained with the RH strain.Astrocytoma cells were infected with T. gondii and 2 h later PMA wasadded, simultaneously in the wells without parasites. Supernatants werecollected h, 3 h, 6 h and 24 h after PMA addition.

The virulent RH strain did not induce secretion ofany of the three cytokines, when compared with thesecretions observed with astrocytoma cells alone.Such data are consistent with our previous studies onhuman monocytes and macrophages concerningTNFo8 and with the study of Friedland27 on secretionof TNFot and IL-6 by a human monocytic cell line, butis different from the results obtained after infectionof murine mononuclear cells by Leishmaniainfantum which is another obligate intracellular pro-tozoan.28 However, the intracellular behaviour of T.gondii and L. infantum is not the same, especiallyconcerning the mechanisms used to escapeintracellular killing, which could lead to differencesin the induction of TNF secretion.The present model may mimic what happens after

cyst rupture: tachyzoites from cystogenic strains en-ter brain cells around the cyst where they multiply.Sera with high anti-Toxoplasma IgG antibody titresdid not induce this type of secretion, in contrast towhat was shown with blood monocytes andmonocyte-derived macrophages. The presence ofexogenous INFT, simultaneously with TNF andIL-6 secreted by GHE cells did not lead to a decreasein infection rates. Our results are similar to those

294 Mediators of Inflammation Vol 3. 1994

120000

100000

Cells+ P.MA

Cells+ RH strain+ PMA

80000

60000

40000

20000

3 6 24Time (h)

FIG. 4. IL-6 produced by astrocytoma cells after infection and PMA. Otherdetails are the same as given in the legend to Fig. 3.

from Chao et al.z9 and Peterson et al.3 comparingmicroglial cells and astrocytes in a murine model.Thus, the protective role of cerebral endogenous,astrocyte-secreted, TNFot is still unclear in man, eventhough it has been demonstrated to be an importantfactor of resistance to T. gondii infection in mice.6,31

The causes of the different degrees of virulencebetween T. gondii strains are not understood. Thegenotypic and phenotypic variations between the T.gondii strains have been demonstrated for enzymaticactivities, RFLP and pathogenicity in animals andhumans. 15-17 From our results, the different strains donot induce different cytokine profiles in response toa parasitic infection. Thus the assumed differencesbetween strains, which could explain the onset ofcerebral reactivation in AIDS patients, do not seemto be mediated by astrocyte cytokine production.However, cytokines produced by other cerebralcells, such as microglial cells, or recruited inflam-matory cells (lymphocytes, macrophages and/orpolymorphonuclear) might have an .influence onparasitic multiplication and cyst rupture. Moreover,human monocytes and macrophages may secreteTNF in the presence of specific anti-Toxoplasmaantibodies.8

In conclusion the T. gondii protozoan does notinduce (or prevent) cytokine secretion by humanastrocytoma derived cells.

T. gondii, astrocytoma cells and cytokines

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211-222.2. Beaman MH, Wong SY, Remington JS. Cytokines, Toxoplasma and intracellular

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14. Cristina N, Liaud MF, Santoro F, Oury B, Ambroise-Thomas P. A family of repeatedDNA sequences in Toxoplasma gondff: cloning sequence analysis, and in straincharacterisation. Exp Parasitol 1991; 73." 73-81.

15. Cristina N, Oury B, Ambroise-Thomas P, Santoro F. Restriction-fragment-lengthpolymorphisms among Toxoplasma gondtt strains. Parasitol Res 1991; 77." 266-268.

16. Darde ML, Bouteille B, Pestre-Alexandre M. Isoenzyme analysis of 35 Toxoplasmagondii isolates and the biological and epidemiological implications.JParasito11992;75t 786-794.

17. Sibley LD, Boothroyd JC. Virulent strains of Toxoplasma gondtt comprise singleclonal lineage. Nature 1992; 359: 82-85.

18. Nissou MF. Etablissement et caractrisation de lignes cellulaires partir detumeurs cr(brales humaines. PhD thesis. Universit Joseph Fourier GrenobleGrenoble. France. 1989.

19. Zenner L, Darcy F, Cesbron-Delauw MF, Capron A. Rat model of congenitaltoxoplasmosis: rate of transmission of three Toxoplasma gondii stains to fetuses andprotective effect of chronic infection. Infect Immun 1993; 61t 360-363.

20. Bethea JR, Yancey-Gillespie G, Benveniste EN. Interleukin-l[3 induction of TNFtxgene expression: involvement of protein kinase C. J Cell Physiol 1992; 152."264-273.

21. Chung IY, Benveniste EN. Tumour necrosis factor-a production by astrocytes.Induction by lipopolysaccharide, IFN-7, and IL-l[3. J Immunol 1990; 144:2999-3007.

22. Hetier E, Ayala J, Bousseau A, Den6fle P, Prochiantz A. Amoeboid microglial cellsand not astrocytes synthesize TNF-tx in Swiss brain cell cultures. Eur JNeurosci 1990; 2: 762-768.

23. Lee JC, Simon PL, Young PR. Constitutive and PMA-induced interleukin-1 produc-tion by the human astrocytoma cell line T24. Cell Immunol 1989; 118: 298-311.

24. Lieberman AP, Pitha PM, Shin HS, Shin ML. Production of tumor necrosis factor andother cytokines by astrocytes stimulated with lipopolysaccharide neurotropicvirus (interleukin 1/type interferons/interleukin 6). Proc Natl Acad Sci USA 1989;$6: 6348-6352.

25. Wesselingh SL, Gough NM, Finlay-Jones JJ, McDonald PJ. Detection of cytokinemRNA in astrocyte cultures using the polymerase chain reaction. Lymphokine Res1990; 9." 177-185.

26. Zuckerman SH, Evans GF, Butler LD Endotoxin tolerance: independent regulationof interleukin-1 and tumour necrosis factor expression. Infect Immun 1991; 59t2774-2780.

27. Friedland JS, Shattock RJ, Johnson JD, Remick DG, Holliman RE. Differentialcytokine gene expression and secretion after phagocytosis by human monocyticcell line of Toxoplasmagondii compared with Mycobacterium tuberculosis. Clin Explmmuno11993; 91: 282-286.

28. Chiofalo MS, Delfino D, Mancuso G, La Tassa E, Mastroeni P, Ianello D. Inductionof tumor necrosis factor-ix by Letshmanta infantum in murine macrophages fromdifferent inbred mice strains. Microbiol Pathogen 1992; 12.. 9-17.

29. Chao CC, Hu S, Gekker G, Novick wJ, Remington JS, Peterson PK. Effects ofcytokines multiplication of Toxoplasma gondii in microglial cells. J Immunol1993; 150t 3404-3410.

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31. Johnson LL. A protective role for endogenous tumor necrosis factor in Toxoplasmagondii infection. Infect Immun 1992; 60". 1979-1983.

ACKNOWLEDGEMENTS. This work supported by A.N.R.S. (Agence Nationale deRecherche le SIDA). We indebted to M. L. Darde for kindly providing thePrugniaud strain, to C. Boudin for statistical analysis and to B. F. F. Chumpitazi forcritical reading of the manuscript.

Received 21 March 1994;accepted 12 April 1994

Mediators of Inflammation. Vol 3. 1994 295

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tnfcx il-6 astrocytoma · keywords: astrocytoma cells, cytokine, toxoplasmagondii infection with...

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