Inhibition of Eimeria tenella replication after recombinant IFN-γ activation in chicken macrophages, fibroblasts and epithelial cells

Veterinary Parasitology 92 (2000) 37–49

Inhibition of Eimeria tenella replication after recombinant IFN-␥ activation in chicken macrophages, fibroblasts and epithelial cells C. Hériveau a , I. Dimier-Poisson b , J. Lowenthal c , M. Naciri a , P. Quéré a,∗ b

a INRA, Unité de Pathologie Aviaire et Parasitologie, 37380 Nouzilly, France CJF INSERM 93-O9 Immunologie des Maladies infectieuses-Equipe associée, INRA Immunologie Parasitaire, UFR des Sciences Pharmaceutiques, 31 Avenue Monge, 37200 Tours, France c CSIRO Division of Animal Health Research Laboratory, Geelong, Vic. 3213, Australia

Received 17 January 2000; received in revised form 9 May 2000; accepted 15 May 2000

Abstract We have previously shown that activation of primary cultures of chicken bone-marrow macrophages and embryo fibroblasts with supernatants of concanavaline A-stimulated or reticuloendotheliosis virus (REV)-transformed chicken spleen cells as source of IFN-␥ significantly decreases Eimeria tenella growth in vitro. In the present study, we used various chicken cell lines, HD11 macrophages and DU24 fibroblasts, both virally transformed, CHCC-OU2 fibroblasts and LMH hepatic epithelial cells, both chemically transformed, to replicate E. tenella in vitro. We confirmed the previous results by showing that HD11 macrophages pre-treated for 24 h with recombinant chicken IFN-␥ (either produced in E. coli or by transfected COS cells), at doses ranging from 1000 to 10 U/ml, drastically inhibited E. tenella replication as measured by [3 H] uracil uptake after a further 70 h of culture, as when treated with REV supernatant. Likewise the fibroblast and epithelial cell lines exhibited significant inhibitory activity on E. tenella replication after pre-treatment with recombinant chicken IFN-␥, but were less sensitive (1000–100 U/ml) than when treated with REV supernatant. Recombinant chicken IFN-␣ pre-treatment of all cell lines had no inhibitory effect on parasite development. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Eimeria tenella; Inhibition of in vitro replication; Recombinant chicken IFN-␥

∗ Corresponding author. Tel.: +33-247-42-79-12; fax: +33-247-42-77-74. E-mail address: [email protected] (P. Qu´er´e)

0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 2 7 5 - 2

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1. Introduction Avian coccidiosis is a major cause of economic loss to the poultry industry worldwide and is due to intracellular protozoan parasites from the genus Eimeria (E. acervulina, E. praecox, E. maxima, E. brunetti, E. mitis and E. necatrix). Following infection, parasite development is located in epithelial cells from different areas of the chicken intestinal mucosa according to the species. Host responses to coccidial parasites are complex, but cell-mediated immunity clearly appears to play an essential role in host protection (Rose and Hesketh, 1982; Lillehoj, 1987). T-lymphocytes from intestinal epithelium and lamina propria appear to be the major immune defense mechanisms against the parasite. Local intracellular multiplication of sporozoites during primary infection triggers the activation of CD4+ T cells, whereas CD8+ T cells pre-dominate in secondary infection (Rothwell et al., 1995; Trout and Lillehoj, 1995; Bessay et al., 1996). Eimeria infection also increases NK cell activity both in intestinal mucosa and in the spleen (Lillehoj, 1989). Interferon-gamma (IFN-␥) has been identified as one of the major cytokines involved in the limitation of parasite multiplication in mammals following primary infection with intracellular protozoans. In mammals, T cells and NK cells are the source of IFN-␥ which has been shown to play a major role limiting intracellular parasite replication. Indeed, the local production of IFN-␥ observed in intestinal secretions and mesenteric lymph nodes of mice following infection with Eimeria vermiformis, increases according to the genetic resistance status (Wakelin et al., 1993; Ovington et al., 1995). Moreover, in vivo depletion of IFN-␥ with specific antibodies induces increase in oocyst production and mortality after primary E. vermiformis infection (Rose et al., 1989). In the chicken, increase in IFN-␥ activity has been observed in the spleen (Byrnes et al., 1993) and blood leucocytes (Breed et al., 1997) of chickens infected with Eimeria, after stimulation of T lymphocytes with mitogens or parasite antigens in vitro. Various cells such as macrophages, fibroblasts and most particularly intestinal epithelial cells can be activated by IFN-␥, thereby inhibiting parasite development and limiting infection (Hughes et al., 1987; Rose et al., 1989; Woodman et al., 1991). The gene for ChIFN-␥ has recently been cloned and characterized (Digby and Lowenthal, 1995). Parental administration of recombinant ChIFN-␥ to chickens infected with E. acervulina has been shown to result in reduction of weight loss (Lowenthal et al., 1997; Lillehoj and Choi, 1998) and lowered oocyst production (Lillehoj and Choi, 1998). It, therefore, appears that ChIFN-␥ is likely to play a major role in limiting Eimeria infection in chickens. IFN-␥ does not act directly on the parasite, but through activation of various cells participating in the host defense immune network. Macrophages, fibroblasts, epithelial and endothelial cells become activated following treatment with IFN-␥, and are able to inhibit intracellular multiplication of protozoans such as Toxoplasma gondii and Eimeria in mammals (Hughes et al., 1987; Rose et al., 1989; Woodman et al., 1991). Similarly in chickens, primary kidney cells cultured with supernatants from chicken leucocytes stimulated with concanavaline A (Con A), inhibit E. tenella replication without blocking cellular invasion (Kogut and Lange, 1989). Chicken bone-marrow macrophages and embryonic fibroblasts were also shown to be able to limit E. tenella replication after culture in the presence of supernatants from Con A stimulated spleen cells and REV transformed T cells containing IFN-␥ activity (Dimier et al., 1998; Dimier-poisson et al., 1999). Furthermore, chicken fibroblasts transfected with the ChIFN-␥ gene display the same inhibitory activity

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(Lillehoj and Choi, 1998). Macrophages are crucial components of the immune response and are in contact with the parasite during passage through intestinal mucosa after infection (Lawn and Rose, 1982). Fibroblasts have also been cited as a nonspecific host defense barrier (Lavnivoka and Laskin, 1995). Intestinal epithelial cells, where the parasite multiplies, would seem to play the major role in limiting infection in the case of an Eimeria infection, however, no primary cultures or cell lines of intestinal epithelial origin are yet available in the chicken. The aim of the present study was to determine the ability of ChIFN-␥, either in native (from REV T cells) or recombinant (from COS cells or E. coli) form, to sensitize chicken cell lines from macrophage, fibroblast and epithelial origins to inhibit E. tenella replication.

2. Materials and methods 2.1. Avian cell lines The HD11 macrophage cell lines was obtained from chicken bone-marrow cell culture transformed with MC29 retrovirus (Beug et al., 1979). The DU24 fibroblast cell line was derived from a hepatic lesion in a chicken infected with MC29 retrovirus (Morais et al., 1988). The CHCC-OU2 fibroblast cell line was obtained from chicken embryo fibroblasts in vitro transformed by methylnitrosoguanidine (Ogura and Fujiwara, 1987) and the LMH hepatic epithelial cell line from a hepatic tumor in a chicken inoculated with diethylnitrosamine (Kawagushi et al., 1987). All cell lines were cultured in 25 cm2 flasks in Dubelcco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS). 2.2. Interferon-γ preparations Recombinant chicken IFN-␥ was produced in E. coli and was titrated to contain 105 U/ml according to the HD11 NO assay (reciprocal dilution of 50% maximal NO production) (Digby and Lowenthal, 1995). Recombinant IFN-␥ (Digby and Lowenthal, 1995) and recombinant chicken IFN-␣ (kind gift of Dr. Margaret Sekellick and Dr. Philip Marcus; Sekellick et al., 1994) were produced in COS cells after transfection with pcDNA-plasmid using lipofectamine and titrated at 5000 U/ml using the HD11 NO assay and 5×104 U/ml using the VSV inhibition assay, respectively. A control COS cell supernatant with only lipofectamine was made in parallel. The REV supernatant was taken after culture of the lymphoid cell line transformed by reticuloendotheliosis virus (REV) (Beug et al., 1981) seeded at 105 cells/ml and cultured for 72 h at 40◦ C (500 U/ml). 2.3. Parasites The wild-type strain of E. tenella (PAPt36,INRA, Pathologie Aviaire et Parasitologie, 37380 Nouzilly, France) was developed by isolating a single oocyst and then maintained by successive passages in vivo. Chickens, raised in specific-pathogen free conditions, were inoculated orally with 2×104 sporulated E. tenella. Ceca were removed on the seventh day

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to collect the oocysts and kept afterwards in a solution of K2 Cr2 O7 at 2% for sporulation. The oocysts were then broken using glass beads to obtain the sporocysts. After incubation with 10% sodium hypochlorite for 20 min, the sporocysts were treated with phosphate buffer (0.4 g of trypsin and 0.5 g bile salts for 50 ml) for 1 h at 41◦ C to release the sporozoites used to infect cell cultures. 2.4. Flow cytometry analysis of MHC classes I and II cell surface antigen expression Flow cytometry analysis was performed on HD11 macrophages either untreated or treated with REV supernatant and recombinant chicken IFN-␥ for 24 h and fibroblast and epithelial cells treated in the same way but for 72 h. Cells were trypsinized, washed and suspended at 106 cells/ml in DMEM (10% FCS). Cells were incubated for 1 h at 4◦ C with an anti-major histocompatibility complex class I antigen (21.2: Crone and Simonson, 1987) and class II antigen (TAP1: Guillemot et al., 1986) monoclonal antibodies, kind gifts of Dr. O. Vainio (Turku University, Turku, Finland) and Dr N. Le Douarin (Collège de France and CNRS, Institut d’Embryologie, Nogent-sur-Marne, France), respectively. After two washes, the cells were then incubated with a phycoerythrin-stained polyclonal anti-mouse immunoglobulin G goat antibody (Jackson Lab.,West grove, PA, USA). Cells were analyzed by flow cytometry (FACStar® Plus, Becton Dickinson, San Jose, CA, USA) to determine the percentage of stained cells and the mean fluorescence intensity (MFI). 2.5. Assay for E. tenella intracellular replication and effect of REV supernatant and recombinant chicken IFN-γ Proliferation or inhibition of parasite development was assessed by [3 H] uracil uptake (Ouelette et al., 1974). Cells were seeded in 96-well culture plates (Falcon, Becton Dickinson labware, Franklin Lakes, NJ, USA) at a concentration of 2×104 /200 ␮l/well for 24 h at 40◦ C. Medium (DMEM, 10% FCS) was then replaced by various concentrations of REV supernatant or recombinant chicken IFN-␥ (E. coli or COS cells). The cultures were incubated for 24 h and then washed twice, and 4×104 sporozoites of E. tenella were added to the cells. After 2 h, 0.5 ␮Ci 5,6 [3 H] uracil (specific activity 50 Ci/nmol; Amersham SA, Les Ulis, France) was added to each well in 20 ␮l DMEM. The cultures were then incubated for a further 70 h and frozen before harvesting onto glass fiber filters with an automated cell harvester. The incorporated radioactivity was determined in a liquid scintillation counter (Kontron, Munich, Germany) and expressed as counts per minute (CPM)±S.E.M. (six replicates). 2.6. Statistical analysis The values (CPM) shown for E. tenella replication are the means of six replicates with S.E. bars. Statistical differences between the various groups were assessed by Student’s t-test. The level of significance was set at 0.05.

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3. Results 3.1. Modulation of class I and class II MHC cell surface antigens with REV supernatant and recombinant chicken IFN-γ HD11 cells are virally transformed macrophages displaying an activated phenotype with high expression of class I and class II MHC antigen on the cell surface for the majority of cells (MFI of 99 and 72, respectively) (Table 1). Addition of REV supernatant up to dilution 1:100 for 24 h further increased the expression of class I and class II MHC, with 2.5 to 3×MFI. Recombinant chicken IFN-␥ at 25 U/ml had the same effect and at 3 U/ml 1.5 to 2-fold increases were still observed. Virally transformed DU24 fibroblasts did not express class I MHC cell surface antigens but the addition of REV supernatant (1:20) and of recombinant chicken IFN-␥ (50 U/ml) induced their expression on all cells with a MFI up to 120. Reducing the dose of recombinant chicken IFN-␥ to 25 U/ml decreased the MFI by half. On the other hand, chemically transformed CHCC-OU2 expressed high levels of class I MHC cell surface antigens (MFI 200) and a 3.5-fold increase in MFI was observed after the addition of REV supernatant (1:20) and of recombinant chicken IFN-␥ (50 U/ml). LMH hepatic epithelial cells weakly expressed class I MHC cell surface antigens and this expression was increased by six-fold with REV supernatant (1:50) and recombinant chicken IFN-␥ (50 U/ml). Reducing the dose of recombinant chicken IFN-␥ at 25 U/ml also decreased the MFI by half for LMH cells. None of the fibroblast or epithelial cells expressed class II MHC cell surface antigens and neither REV supernatant nor recombinant chicken IFN-␥ induced their expression, except very weakly for DU24 cells. 3.2. Inhibition of E. tenella replication in HD11 macrophages activated with REV supernatant and recombinant chicken IFN-γ [3 H] uracil uptake demonstrating E. tenella replication varied from 4000 to 40,000 CPM according to the experiment (Fig. 1). Pre-activation of HD11 cells for 24 h with REV supernatant (dilution 1:5 to 1:100, i.e. 100 to 5 U/ml IFN-␥) reduced [3 H] uracil uptake by 75 (Experiment 2) to 90% (Experiment 1), thus showing drastic inhibition of E. tenella replication (p<10−8 ). Recombinant chicken IFN-␥ produced in E. coli exhibited the same high inhibiting effect on [3 H] uracil uptake (up to 95%, from 1000 to 10 U/ml, p<10−8 –10−10 ). Recombinant chicken IFN-␥ produced in COS cells induced the same level of parasite inhibition (from 1000 to 10 U/ml). Nevertheless, control COS supernatant also displayed a significant inhibiting activity up to a dilution equivalent to 100 U/ml IFN-␥. Consequently, the inhibiting activity of COS IFN-␥ was significantly higher than that of COS supernatant at 100 U/ml (p<10−3 ) (Experiments 1 and 2). Recombinant chicken IFN-␣ (1000–10 U/ml) did not display any activity on E. tenella replication in HD11 cells in vitro (Experiment 2). 3.3. Inhibition of E. tenella replication in DU24 and CHCC-OU2 fibroblasts and LMH epithelial cells DU24 and CHCC-OU2 cells were able to support multiplication E. tenella in vitro, as demonstrated by the significant [3 H] uracil incorporation: 2900 (Experiment 1) to 22,600

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Table 1 Modulation of MHC classes I and II surface antigens on macrophage, fibroblast and epithelial chickens cell lines after activation with ChIFN-␥ IFN treatmenta

Nil REV: 1:20 COS (U/ml) 50 25 Nil REV: 1:20 COS (U/ml) 50 25 Nil REV: 1:20 1:100 COS (U/ml) 50 25 Nil REV: 1:20 1:100 COS (U/ml) 25 12.5 6 3

% positive (mean fluorescence intensity)b Class I

Class II

DU 24 fibroblasts 2.9 (1) 97.7 (119)

0 5.7 (8)

97.9 (122) 95.6 (52)

8.3 (15) 2.5 (10)

CHCC-OU2 fibroblasts 92.1 (208) 97.9 (769)

0 0

96.2 (642)

0

LMH epithelial cells 75.0 (29) 90.2 (128) 88.2 (83)

0 1.0 (0.1) 4.1 (2)

89.7 (118) 90.7 (65)

2.8 (1) 3.5 (3)

HD11 macrophages 95.8 (99) 87.9 (307) 90.0 (281)

93.0 (72) 87.1 (186) 88.8 (152)

90.3 (261) 90.0 (251) 90.6 (246) 92.4 (225)

86.9 (157) 86.1 (142) 85.9 (133) 90.0 (125)

a HD11 cells were stimulated for 24 h and LMH, DU 24, and CHCC-OU2 cells for 72 h with ChIFN-␥ (REV supernatant or COS-derived recombinant). No difference was observed between cells treated with medium alone and cells treated with control COS supernatant. b Cells were stained with monoclonal antibodies specific for MHC class I (21.2) (31) or class II (TAP1) (32), which were kind gifts from Dr. O. Vainio and Dr. N. Le Douarin, respectively. The cells were then incubated with phycoerythrin-labeled goat anti-mouse IgG serum (Jackson Lab., West grove, PA, USA). Cells were analyzed by flow cytometry (FACStar® Plus, Becton Dickinson, San Jose, CA, USA) to determine the percentage of positively stained cells and MFI.

CPM (Experiment 2) for DU24 (Fig. 2) and 26,500 (Experiment 1) to 36,700 CPM (Experiment 2) for CHCC-OU2 (Fig. 3). DU24 cells pre-treated for 24 h with REV supernatant inhibited significantly greater [3 H] uracil uptake (50–60%, p<10−3 ) until dilution 1:50 (equivalent to 10 U/ml IFN-␥). Recombinant chicken IFN-␥ produced in E. coli inhibited very efficiently (up to 70–80%) [3 H] uracil uptake at 1000 and 500 U/ml, but not at 100 U/ml,

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Fig. 1. HD11 macrophages inhibit E. tenella replication in vitro after activation with ChIFN-␥. HD11 cells were treated for 24 h with native ChIFN-␥ (REV supernatant), recombinant (E coli or COS-derived) ChIFN-␥ at doses ranging from 10 to 1000 U/ml, in Experiment 1, and also recombinant ChIFN-␣ in Experiment 2. Cells were also incubated in the presence of supernatant from mock-transfected COS cells. E. tenella sporozoites (two per cell) were added and their replication was measured by [3 H] uracil after a further 70 h of culture. Closed bars show the effect of equivalent concentrations of supernatant from mock-transfected COS cells. Results show mean CPM±S.E.M. of six replicates.

in a dose-dependant manner. Compared to recombinant chicken IFN-␥ produced in COS cells which inhibited about 35% [3 H] uracil uptake through 50 U/ml (p<0.05). Control COS supernatant was unable to modify [3 H] uracil uptake in these cells as was recombinant chicken IFN-␣ (1000–10 U/ml). CHCC-OU2 cells were more sensitive than DU24 cells but displayed the same pattern of sensitivity to the different sources of chicken IFN-␥ (Fig. 2). REV supernatant was able to induce an inhibitory activity of E. tenella replication at dilution 1:100: 45% (Experiment 1)

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Fig. 2. Virally transformed DU24 fibroblasts inhibit E. tenella replication in vitro after activation with ChIFN-␥. Cells were pre-incubated with ChIFN-␥ and cultured with E. tenella sporozoites as described in Fig. 1. Closed bars show the effect of equivalent concentrations of supernatant from mock-transfected COS cells. Results show mean CPM±S.E.M. of six replicates.

to 70% (Experiment 2) (p<0.05). Likewise the effect of recombinant chicken IFN-␥ produced in E. coli was clearly dose-dependent, with 85% inhibition at 1000 U/ml to 20% at 10 U/ml (p<0.05). This was not the case with recombinant chicken IFN-␥ produced in COS cells which inhibited 50–60% of [3 H] uracil uptake from 1000 to 10 U/ml (p<0.05) whereas control COS supernatant has no effect. Recombinant chicken IFN-␣ was also ineffective. LMH hepatic epithelial cells were able to replicate E. tenella well with 12,250–35,550 CPM for [3 H] uracil uptake (Fig. 4). Pre-incubation for 24 h with REV supernatant induced dose-dependent inhibition of E. tenella replication: 70–75% reduction of [3 H] uracil uptake at dilution 1:10 to 25 (Experiment 2) to 50 (Experiment 1) at 1:100 (p<10−5 ). Pre-incubation with recombinant chicken IFN-␥ produced in E. coli induced the same dose-dependent effect: 70% inhibition at 1000 U/ml (p<10−8 ) to 40% at 10 U/ml (p<10−4 ). After treatment, 35–15% inhibition (p<0.05) was noted with 1000–10 U/ml of recombinant chicken IFN-␥

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Fig. 3. Chemically transformed CHCC-OU2 fibroblasts inhibit E. tenella replication in vitro after activation with Sn REV and recombinant chicken IFN-␥.

produced in COS cells with no effect of control COS supernatant. Recombinant chicken IFN-␣ had no effect.

4. Discussion Our results showed that chicken cell lines of fibroblast origin, either chemically transformed (CHCC-OU2) or virally transformed (DU24), and of hepatic epithelial origin (LMH), chemically transformed, were able to respond to chicken IFN-␥ by modulating MHC antigens and by inhibiting intracellular parasite replication, as was the virally transformed macrophage cell line HD11. Chicken IFN-␥ was either native from supernatant of chicken

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Fig. 4. LMH hepatic epithelial cells inhibit E. tenella replication in vitro after activation with Sn REV and recombinant chicken IFN-␥.

splenocytes transformed by REV (Lowenthal et al., 1994), or recombinant, produced in E. coli and in COS cells (Digby and Lowenthal, 1995). HD11 macrophages were very responsive to IFN-␥, dramatically increasing cell surface expression of classes I and II antigens at doses as low as 5–10 U/ml, as also shown by Lowenthal et al. (Lowenthal et al., 1997; Song et al., 1997). The dramatic increase in class II antigen cell surface expression has also been observed with primary cells from the macrophage lineage such as monocytes (Kaspers et al., 1994) and bone-marrow macrophages (Dimier et al., 1998) elicited with Con A- and REV-induced supernatants. By contrast, chicken cell lines of fibroblast and epithelial origin appeared very weakly to modulate their class II cell surface expression upon stimulation with REV supernatant and recombinant IFN-␥, despite using experimental conditions suitable for embryo fibroblasts (Dimier et al., 1998). The level of class I antigen expression was different according to the cell line, but was particularly sensitive to stimulation with IFN-␥, either from the REV supernatant or recombinant source, as has been observed in mammals (Seliger et al., 1997), though much less than for HD11 cells (i.e. up to 25–50 U/ml). In fact, E. tenella was able to effectively replicate in macrophages, fibroblasts and epithelial cells. Parasite replication was measured by means of specific incorporation of [3 H] uracil into the parasite (Ouelette et al., 1974). HD11 macrophages pre-incubated with REV supernatant (up to dilution 1:100) and recombinant IFN-␥ produced in E. coli (1000–10 U/ml),

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for 24 h before infection with E. tenella inhibited almost all parasite replication. The inhibiting effect of stimulation with REV supernatant and recombinant IFN-␥ produced in E. coli was also observed, although to a lesser extent, with fibroblast and epithelial host cells. The activity of REV supernatant containing native IFN-␥ appeared more effective than recombinant IFN-␥ when relative to NO production by HD11 cells. REV supernatant inhibited 60–70% of the parasite multiplication at dilution 1:5 and could be still effective at dilution 1:100 (5 U/ml). Recombinant IFN-␥ produced in E. coli appeared effective in all cases up to 100 U/ml (60–85% inhibition at 1000 U/ml to 0–40% at 50 U/ml). So chicken IFN-␥, as mammalian IFN-␥, has the capacity to limit the replication of Eimeria in different in vitro culture systems and this also appeared to be the case in vivo as well (Lillehoj and Choi, 1998). Recombinant IFN-␥ produced in COS cells also inhibited E. tenella either in macrophage, fibroblast, or epithelial cell lines. Nevertheless, a linear dose relationship, according to the titration of IFN-␥ using HD11 as NO producers, was not clearly obtained. Moreover, the control COS supernatant produced with lipofectamine, used for the cell transfection was thought necessary because of the weak production of the recombinant protein in this system, appeared to inhibit parasite multiplication in HD11 at the first dilutions used, but not in other types of cells. This action may be linked to the capacity of the control COS supernatant to induce weak NO production in HD11, but not in the fibroblast and epithelial cell line. NO is produced by macrophages but not fibroblasts to limit in vitro E. tenella replication in chicken (Dimier-poisson et al., 1999). No effect of the COS control supernatant was noted on classes I and II cell surface antigen modulation. This observation emphasizes the importance of the source of recombinant IFN-␥ in the type of in vitro activity tested. The first studies upon avian interferons led to controversy concerning the very existence of two different types of interferon in chickens. In fact chicken leukocytes stimulated with Con A (Putzai et al., 1986) and even chicken virally infected fibroblasts (Heller et al., 1997) are able to produce the two types of interferon, in contrast to mammals. At present, the IFN-␥ gene (Digby and Lowenthal, 1995) and genes belonging to the IFN-␣/␤ family (Sekellick et al., 1994; Schultz et al., 1995) have been identified ascertaining the existence of the two types of interferon in chickens. Our results proved that the induction of E. tenella replication inhibiting activity in chicken macrophages and fibroblast cells was restricted to chicken recombinant IFN-␥, and not to chicken recombinant IFN-␣, as in mammals. In conclusion, macrophage, fibroblast and epithelial chicken cell lines were sensitive to chicken recombinant IFN-␥ and to REV supernatant with identified IFN-␥ activity, both to modulate classes I and/or II cell surface antigen expression and to inhibit the replication of the intracellular parasite, E. tenella, thus corresponding to what is known in mammals for such cell types and thus emphasizing their importance in chicken defenses against Eimeria. The response of chicken primary intestinal epithelial cultures and an intestinal cell line is currently under investigation. References Bessay, M., Le Vern, Y., Kerboeuf, D., Yvore, P., Quéré, P., 1996. Changes in intestinal intra-epithelial and systemic T-cell subpopulations after an Eimeria infection in chickens: comparative study between E. acervulina and E. tenella. Vet. Res. 27, 503–514.

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