Gamma Interferon Induces Fas-Dependent Apoptosis of Peyer s Patch T Cells in Mice following Peroral Infection with Toxoplasma gondii

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1 INFECTION AND IMMUNITY, Nov. 1997, p Vol. 65, No /97/$ Copyright 1997, American Society for Microbiology Gamma Interferon Induces Fas-Dependent Apoptosis of Peyer s Patch T Cells in Mice following Peroral Infection with Toxoplasma gondii OLIVER LIESENFELD, 1,2 JON C. KOSEK, 3,4 AND YASUHIRO SUZUKI 1,2 * Department of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto, California ; Division of Infectious Diseases and Geographic Medicine, Department of Medicine, 2 and the Department of Pathology, 3 Stanford University School of Medicine, Stanford, California 94305; and Department of Pathology, Veterans Administration Medical Center, Palo Alto, California Received 10 June 1997/Returned for modification 8 July 1997/Accepted 12 August 1997 Since we previously observed a remarkable decrease in the numbers of T cells in the Peyer s patches of the small intestines in C57BL/6 mice following peroral infection with Toxoplasma gondii, we performed studies to examine the mechanism(s) whereby this decrease in numbers of the T cells occurs. We found that apoptotic cell death of CD4 and CD8 T cells occurred in Peyer s patches following infection. Upregulation of Fas expression was observed in these T cells. C57BL/6-background mutant mice which lack functional Fas antigen did not develop apoptosis in their Peyer s patches following infection. Treatment of infected C57BL/6 mice with anti-gamma interferon (IFN- ) monoclonal antibodies prevented the upregulation of Fas on their Peyer s patch T cells and inhibited the occurrence of apoptosis of these T cells. These results indicate that IFN- induces Fas-dependent apoptosis in CD4 and CD8 T cells in Peyer s patches in C57BL/6 mice following peroral infection with T. gondii. During a recent study on the effect of Toxoplasma gondii on the small intestine following peroral infection, we observed that C57BL/6 mice developed severe necrosis of the villi and mucosal cells of their small intestines (9). We found that the presence of CD4 T cells and gamma interferon (IFN- ) was critical for the development of the necrosis and that treatment with anti-ifn- monoclonal antibodies (MAb) prevented development of the necrosis and prolonged the time to death for the infected mice (9). When necrosis was observed to have occurred in the small intestine, we noted that germinal centers were not discernible in the Peyer s patches (9). Quantification of the numbers of lymphocytes obtained from Peyer s patches and flow cytometric analysis revealed that a significant and remarkable decrease in the numbers of Peyer s patch T cells had occurred (9). In the present study, we attempted to analyze the mechanism(s) that underlies this remarkable decrease in numbers of Peyer s patch T cells following peroral infection with T. gondii and found that apoptotic cell death occurs in large numbers of CD4 and CD8 T cells in the Peyer s patches and that this apoptosis is Fas dependent and mediated by IFN-. MATERIALS AND METHODS Mice. Female C57BL/6 (Bantin and Kingman, Fremont, Calif.), Swiss-Webster (Simonsen, Gilroy, Calif.), and B6.MRL-FAS lpr (lpr) mutant mice and their control mice (Jackson Laboratories, Bar Harbor, Maine) were 6 to 8 weeks old when used. Infection with T. gondii. Cysts of the ME49 strain of T. gondii were obtained from the brains of chronically infected Swiss-Webster mice as previously described (21) and were used for peroral infection of mice (100 cysts per mouse given by gavage) (9). * Corresponding author. Mailing address: Department of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, 860 Bryant St., Palo Alto, CA Phone: (415) Fax: (415) ML.SUZ@forsythe.Stanford.edu. DNA fragmentation. Groups of three to five uninfected and infected mice were euthanized 7 days after infection. Peyer s patches were excised from the proximal and distal parts of the small intestines, and single-cell suspensions were prepared as previously described (9). Fragmented DNA in to Peyer s patch cells was examined by the method described by Moore and Matlashewski (12). Briefly, Peyer s patch cells were lysed in 10 mm Tris (Sigma, St. Louis, Mo.; ph 7.4) 5 mm EDTA (Sigma) 1% Triton X-100 (Sigma) for 20 min on ice. After centrifugation at 13,000 g for 20 min, supernatants containing DNA were collected and treated with 20 g of RNase A (Calbiochem, San Diego, Calif.) per ml for 1 h at 37 C, followed by digestion with proteinase K (Sigma) at a concentration of 0.1 mg/ml for 1 h at 56 C. DNA extraction was performed with phenol-chloroform, followed by precipitation with ethanol. Precipitated DNA was dissolved in 30 l of Tris-EDTA buffer, ph 8.0, and 2 g of DNA was electrophoresed on a 1.2% agarose gel for 1 h at 85 V. Light and electron microscopy. Peyer s patches were excised from the small intestines of uninfected and infected mice at 7 days after infection. For light microscopy, Peyer s patches were fixed in a solution containing 10% formalin, 70% ethanol, and 5% acetic acid. Sections (5 m thick) of these organs were stained with toluidine blue, and the ratios of apoptotic to nonapoptotic lymphocytes in the lymphocyte population were evaluated by counting 500 lymphocytes in total for each sample by using photographs at a magnification of 400. Three or four samples were evaluated for each experimental group. For electron microscopy, Peyer s patches were fixed in 2% glutaraldehyde in cacodylate buffer (ph 7.2) (Sigma), postfixed with 1.5% osmium tetroxide, embedded in Epon, and observed with a Phillips 2O1 microscope. Flow cytometry. Each analysis was performed individually on three or four mice for each experimental group. Single-cell suspensions were prepared from Peyer s patches, axillary and inguinal lymph nodes, and spleens of uninfected or infected mice as previously described (9). The percentages of apoptotic cells in different T-cell subsets were determined by the terminal deoxynucleotidyl transferase-mediated dutp-biotin nick end labeling (TUNEL) technique (Boehringer Mannheim, Indianapolis, Ind.) following the manufacturer s protocol. Briefly, single-cell suspensions of the Peyer s patches or spleens (10 7 cells) from uninfected and infected C57BL/6 mice were fixed with 4% paraformaldehyde (Aldrich, Milwaukee, Wis.) in phosphate-buffered saline, ph 7.4, on ice for 30 min and were permeabilized with 0.1% Triton X-100 (Sigma) in 0.1% sodium citrate (Sigma) for 2 min. Thereafter, cells were incubated with 50 l ofthe fluorescein isothiocyanate (FITC)-labeled apoptotic cell-specific nucleotide for 1 h at 37 C. The cells were washed and then incubated with 10 l of optimal concentrations of phycoerythrin (PE)-labeled MAb against either T-cell receptor beta (TCR- ), TCR-, CD4, or CD8 (PharMingen, San Diego, Calif.) for 30 min on ice. Analysis of stained cells was performed with a FACScan (Becton Dickinson, Mountain View, Calif.) flow cytometer. For analysis of Fas expression, single-cell suspensions of Peyer s patch cells and inguinal or axillary lymph node cells were pretreated on ice for 10 min with 10 l of a predetermined optimal 4682

2 VOL. 65, 1997 IFN- -MEDIATED APOPTOSIS OF T CELLS 4683 FIG. 1. Detection of apoptosis by DNA fragmentation in Peyer s patch lymphocytes in C57BL/6 mice following peroral infection with T. gondii. Mice were each infected with 100 cysts of the ME49 strain of T. gondii, and 7 days later, their Peyer s patches were excised from the small intestines. DNA fragmentation analysis was performed with Peyer s patch cells isolated from uninfected and infected mice. DNA was extracted from Peyer s patch cells from the proximal (lanes 1 and 3) and distal (lanes 2 and 4) parts of the small intestines of mice. Lanes 1 and 2, uninfected mice; lanes 3 and 4, infected mice. concentration of anti-fc II/III receptor (PharMingen) to block non-antigenspecific binding of antibodies to the Fc II/III receptors. Thereafter, cells were incubated with 10 l of optimum concentrations of PE-labeled anti-cd4 MAb and FITC-labeled anti-cd8 MAb (PharMingen). CD4 and CD8 T cells were sorted by a FACStar (Becton Dickinson) flow cytometer. A total of 10 5 to sorted cells were incubated on ice for 30 min with 10 l of optimal concentrations of either PE- (for FITC-stained CD8 T cells) or FITC (for PE-stained CD4 T cells)-conjugated anti-fas MAb (PharMingen). Sorted CD4 and CD8 T cells were also stained with FITC- or PE-conjugated isotype control MAb (Caltag, South San Francisco, Calif.), respectively. Analysis of stained cells was performed with a FACScan (Becton Dickinson). Dead cells were gated out on the basis of propidium iodide staining. To compensate for any potential differences in cell size, the specific fluorescence index (SFI) of Fas expression was determined by subtracting the mean background fluorescence with isotype control antibodies from the mean fluorescence of Fas-stained cells. Results are expressed as the mean of the specific fluorescence index of mice in each experimental group. Treatment of mice with MAb against IFN-. C57BL/6 mice were injected intraperitoneally with 2 mg of anti-ifn- MAb (XMG1.2) (2, 9) 1 day before and 4 or 5 days after infection with T. gondii. Control mice were injected intraperitoneally with isotype control MAb (GL113; anti-escherichia coli -galactosidase) in the same manner as for anti-ifn- MAb. Statistical analysis. Levels of significance for differences in the relative percentages of apoptotic T cells and the expression of Fas in flow cytometry were determined by using the Student s t test and the alternate Welch t test. Levels of significance for differences in numbers of apoptotic cells in histological sections were determined by using Fischer s exact test. RESULTS Apoptosis occurs in Peyer s patch lymphocytes following infection. In light microscopy studies of the Peyer s patches, we noticed nuclear fragmentation of lymphocytes in C57BL/6 mice at 7 days after peroral infection with T. gondii (data not shown). To determine whether apoptosis of lymphocytes occurs in the Peyer s patches, we isolated lymphocytes from Peyer s patches excised from the proximal and distal parts of the small intestines at 7 days after infection and performed DNA fragmentation assays on these cells. Oligonucleosomal DNA fragmentation, which indicates the occurrence of apoptosis, was observed in Peyer s patches in the distal (where necrosis of the villi and mucosal cells occurred) but not the proximal parts of the small intestines of infected mice (Fig. 1). In contrast, DNA fragmentation was not demonstrable in Peyer s patches of either the distal or proximal parts of the small intestines of uninfected mice (Fig. 1). Electron microscopy confirmed the occurrence of apoptosis of lymphocytes in Peyer s patches in the distal parts of the small intestines of infected but not of uninfected mice (Fig. 2a and b). Apoptosis of lymphocytes was not observed histologically in inguinal or axillary lymph nodes (data not shown), indicating that apoptosis of lymphocytes is not a systemic phenomenon in lymphoid organs in the infected mice. Both CD4 and CD8 T cells of Peyer s patches undergo apoptosis following infection. To examine which lymphocyte population undergoes apoptosis in the Peyer s patches in C57BL/6 mice following infection, we performed flow cytometric analysis by the TUNEL technique at 7 days after infection. Apoptotic lymphocytes were observed only in the TCR population of Peyer s patch cells (Table 1). These results indicate that apoptosis occurred in T cells but not in T cells or B cells. Further analysis by flow cytometry demonstrated that both the CD4 and CD8 populations of T cells underwent apoptosis following infection (Table 1). The occurrence of apoptosis in both CD4 and CD8 T cells was confirmed by propidium iodide staining (14) of sorted CD4 and CD8 T cells (data not shown). Apoptosis of these T cells was not observed in spleens (Table 1) or peripheral lymph nodes of infected mice by the TUNEL technique (Table 1) or propidium iodide staining (data not shown). These results, along with our histological studies described above, indicate that the occurrence of apoptosis of T cells is a local phenomenon in the Peyer s patches following peroral infection. Fas is required for induction of apoptosis of Peyer s patch T cells following infection. Since the Fas-Fas ligand interaction has been shown to play the major role in induction of apoptosis of T cells in vitro (1, 4, 7, 13), we examined whether the expression of Fas antigen on the surfaces of T cells becomes upregulated in the Peyer s patches following infection. In infected mice, a significant increase in Fas expression over that in uninfected mice was observed in both CD4 and CD8 T cells on Peyer s patches (SFI values of Fas for infected versus uninfected mice were as follows: on CD4 T cells, versus [P ], respectively; and on CD8 T cells, versus [P ], respectively; Fig. 3). In contrast, expression levels of Fas antigen on T cells in the peripheral lymph nodes of infected and uninfected mice did TABLE 1. Frequencies of apoptotic cells in different T cell subsets in the Peyer s patches and spleens of control and infected C57BL/6 mice Cell source and mice a % Apoptotic cells in indicated T cell subset b TCR TCR CD4 CD8 Peyer s patch Control Infected Significance (P) N.S. c Spleen Control Infected Significance (P) N.S. N.S. N.S. N.S. a Mice were each infected perorally with 100 cysts of the ME49 strain of T. gondii. b At 7 days after infection, flow cytometry by the TUNEL technique in combination with MAb against TCR-, CD4, and CD8 was performed on Peyer s patch cells of at least three individual mice for each experimental group. c N.S., not significant.

3 FIG. 2. Detection of apoptosis by electron microscopy in Peyer s patch lymphocytes in C57BL/6 mice following peroral infection with T. gondii. Mice were each infected with 100 cysts of the ME49 strain of T. gondii, and 7 days later, their Peyer s patches were excised from the distal parts of the small intestines. (a) Electron microscopy of a representative Peyer s patch of an uninfected C57BL/6 mouse. Magnification, (b) Electron microscopy of a representative Peyer s patch of an infected C57BL/6 mouse. Magnification,

4 VOL. 65, 1997 IFN- -MEDIATED APOPTOSIS OF T CELLS 4685 FIG. 3. Upregulation of Fas on CD4 and CD8 T cells in the Peyer s patches of C57BL/6 mice following peroral infection with T. gondii. Sorted CD4 and CD8 Peyer s patch T cells were examined for their expression of Fas antigen by flow cytometry 5 days after infection. Fas expression on CD4 and CD8 T cells in the Peyer s patches of uninfected (light line) and infected (heavy line) C57BL/6 mice is shown. Fas SFI values for CD4 and CD8 T cells were significantly higher for infected than for uninfected control mice (P and , respectively). not differ (data not shown). The presence or absence of upregulation of Fas antigen in the Peyer s patches and peripheral lymph nodes correlated well with the presence or absence of apoptosis of T cells in these organs. These results strongly suggest that Fas is involved in the induction of apoptosis of Peyer s patch T cells following infection. To further study the role of Fas in the induction of apoptosis in Peyer s patch T cells, we examined whether lpr mutant mice (which are C57BL/6-background and lack functional Fas antigen [24, 26]) develop apoptosis in their Peyer s patches following infection. Peyer s patch T cells from the infected lpr mice did not show oligonucleosomal DNA fragmentation, whereas those from infected control C57BL/6 mice did (Fig. 4). These results clearly indicate that Fas is required for the induction of apoptosis of Peyer s patch T cells following infection. IFN- mediates upregulation of Fas expression on Peyer s patch T cells. Since we have previously shown that IFN- induces necrosis of villi and mucosal cells in the ilea (9) (in which apoptosis of Peyer s patch T cells was observed in infected C57BL/6 mice in the present study) and since IFN- has been reported to upregulate expression of Fas antigen on T cells in vitro (15), we examined whether IFN- mediates the upregulation of Fas expression on Peyer s patch T cells observed in the C57BL/6 mice following infection. For this purpose, we treated mice with anti-ifn- MAb to neutralize the activity of IFN- during infection and examined the expression of Fas antigen on their Peyer s patch T cells. Control mice infected and treated with control immunoglobulin G (IgG) showed significantly higher levels of expression of Fas on their Peyer s patch T cells (both CD4 and CD8 ) than did uninfected mice (SFI for CD4 T cells: versus , respectively [P ]; SFI for CD8 T cells: FIG. 4. Importance of Fas in the induction of apoptosis of Peyer s patch T cells in C57BL/6 mice following peroral infection with T. gondii. A DNA fragmentation analysis of Peyer s patch cells of C57BL/6 and lpr mutant mice is shown. DNA was extracted from Peyer s patch cells in the distal parts of the small intestines 7 days after peroral infection with 100 cysts of the ME49 strain of T. gondii. Lane 1, uninfected control C57BL/6 mice; lane 2, infected control C57BL/6 mice; lane 3, infected lpr mutant mice. versus , respectively [P ]; Fig. 5). Mice infected and treated with anti-ifn- MAb showed significantly lower levels of expression of Fas on their Peyer s patch T cells than infected control mice (SFI for CD4 T cells: versus , respectively [P ]; SFI for CD8 T FIG. 5. Effect of treatment with anti-ifn- MAb on the upregulation of Fas expression on Peyer s patch T cells in C57BL/6 mice following infection with T. gondii. Mice were infected perorally with 100 cysts of the ME49 strain. Mice were injected intraperitoneally with 2 mg of anti-ifn- MAb 1 day before and 4 days after infection. Control mice were injected with 2 mg of control IgG. At 5 days after infection, Peyer s patch lymphocytes were sorted for CD4 and CD8 cells and were examined for the expression of Fas by flow cytometry. Fas SFI values for CD4 and CD8 T cells of control mice infected and treated with control IgG ( ) were significantly higher than those of uninfected mice ( ) (P and , respectively); Fas SFI values for CD4 and CD8 cells of mice infected and treated with anti-ifn- MAb ( ) were significantly lower than those of infected control mice (P and , respectively).

5 4686 LIESENFELD ET AL. INFECT. IMMUN. cells: versus , respectively [P ]; Fig. 5). These results demonstrate that IFN- plays a critical role in the upregulation of the Fas antigen on Peyer s patch T cells in the infected mice. IFN- induces apoptosis of Peyer s patch T cells. Since IFN- was found to mediate upregulation of Fas expression on Peyer s patch T cells in the infected mice and since Fas was found to be necessary to induce apoptosis of these T cells in

6 VOL. 65, 1997 IFN- -MEDIATED APOPTOSIS OF T CELLS 4687 Downloaded from FIG. 6. Effect of treatment with anti-ifn- MAb on the development of apoptosis in Peyer s patch T cells in C57BL/6 mice following peroral infection with T. gondii. Mice were injected intraperitoneally with 2 mg of anti-ifn- MAb 1 day before and 5 days after infection with 100 cysts of the ME49 strain. Control mice were injected with 2 mg of control IgG in the same manner. Light and electron microscopy were performed on Peyer s patches excised from the distal parts of the small intestines 7 days after infection. (a) Light microscopy of a representative Peyer s patch in an infected C57BL/6 mouse treated with control IgG. Magnification, 288. (b) Light microscopy of a representative Peyer s patch in an infected C57BL/6 mouse treated with anti-ifn- MAb. Magnification, 288. (c) Electron microscopy of a representative Peyer s patch of an infected C57BL/6 mouse treated with control IgG. Magnification, (d) Electron microscopy of a representative Peyer s patch of an infected C57BL/6 mouse treated with anti-ifn- MAb. Magnification, these mice, we examined whether IFN- mediates induction of this apoptosis. To neutralize the activity of endogenous IFN-, infected mice were treated with anti-ifn- MAb and were thereafter examined by light and electron microscopy for the occurrence of apoptosis of Peyer s patch T cells. The numbers of apoptotic lymphocytes were significantly less in the Peyer s patches of the anti-ifn- MAb-treated mice than in those of control mice treated with control IgG (ratios of apoptotic lymphocytes in the total lymphocyte population counted were % [n 4] versus % [n 3], respectively [P ]; Fig. 6). These data reveal that IFN- mediates Fas-dependent apoptosis of Peyer s patch T cells following infection. DISCUSSION The results of the present study provide the first evidence that IFN- induces apoptosis of peripheral T cells in vivo. Peyer s patch T cells were revealed to undergo apoptosis in C57BL/6 mice following peroral infection with T. gondii, and their apoptosis was prevented by treatment with anti-ifn- MAb. Although the effect of IFN- on the induction of apoptosis of peripheral T cells in vitro has been reported by other investigators, the effect has been controversial (3, 6, 10, 15, 16). By using cross-linking of TCRs, IFN- has been demonstrated to play a critical role in the induction of apoptosis in murine cloned T cells (10) and human activated T cells (6, 15). In contrast, using the same in vitro T-cell activation system, other investigators reported that IFN- either does not affect (16) or inhibits (3) the induction of apoptosis of T cells. The in vivo data obtained in the present study clearly demonstrate that IFN- induces apoptosis in CD4 and CD8 T cells in the Peyer s patches of mice following peroral infection with T. gondii. Apoptosis of T cells was observed in the Peyer s patches but on March 18, 2019 by guest

7 4688 LIESENFELD ET AL. INFECT. IMMUN. not in the spleens or peripheral lymph nodes. These results suggest that IFN- locally produced in Peyer s patches contributes to the induction of apoptosis of Peyer s patch T cells. In histological studies, large numbers of parasites were detected only in the small intestines, including the Peyer s patches, of mice at the time when apoptosis of Peyer s patch T cells occurred (9). Since T. gondii is known to be a potent stimulator of IFN- production (5, 11, 19, 20), the presence of the large numbers of parasites may have induced a local and intense production of IFN- in the Peyer s patches. Khan et al. (8) recently reported that splenic CD4 T cells obtained from mice with acute T. gondii infection underwent apoptosis after in vitro culture for 48 h. Since spleen cells from T. gondii-infected mice produce IFN- in vitro without antigen stimulation (5, 22), IFN- may have been involved in the induction of the apoptosis of splenic T cells in vitro observed by Khan et al. (8). In regard to the mechanism(s) of induction of apoptosis in peripheral T cells, Fas has been shown to play the major role in activation-induced cell death of T cells in vitro (1, 4, 7, 13). A critical role for Fas in the activation-induced death of T cells in vivo has recently been shown in studies using murine models (17, 18). Scott et al. (17) reported that following the administration of superantigen (staphylococcal enterotoxin B [SEB]), lpr mice which lack functional Fas antigen failed to delete the V 8 T-cell subset which reacts to SEB. Singer and Abbas (18) demonstrated that lpr mice bearing transgenic TCRs specific for a peptide of pigeon cytochrome c did not show the deletion of peripheral T cells following injection of the peptide. The results of these studies indicate that Fas is required for activation-induced apoptosis of T cells in vivo and thereby contributes to the deletion of peripheral T cells (17, 18). In the present study, we demonstrated that oligonucleosomal DNA fragmentation, which is a direct indication of apoptosis, did not occur in Peyer s patch T cells of lpr mice following peroral infection with T. gondii. These results clearly indicate that Fas is required for the induction of apoptosis in Peyer s patch T cells following infection. In addition to the Fas-dependent pathway for apoptosis of T cells, the existence of a Fas-independent pathway(s) in lpr mice has recently been reported. Tucek-Szabo et al. (25) observed that apoptosis of peripheral T cells occurred in lpr mice following activation of these cells by injection of anti-cd3 MAb, and Sytwu et al. (23) reported that tumor necrosis factormediated apoptosis of hemagglutinin (HA)-reactive peripheral T cells occurred following in vivo stimulation with HA in lpr mice that have a transgenic TCR specific for HA. The latter investigators suggested the possibility that the Fas-mediated pathway is predominant over or more primary than the tumor necrosis factor pathway (23). In the present study, we demonstrated that treatment of infected mice with anti-ifn- MAb prevented the upregulation of Fas expression on their Peyer s patch T cells. These results indicate that IFN- mediates the upregulation of Fas expression on Peyer s patch T cells following infection. In relation to this, Oyaizu et al. (15) reported that IFN- upregulates the expression of Fas antigen on human peripheral blood T cells in vitro. As mentioned above, the present study also demonstrated that Fas and IFN- are required for the induction of apoptosis in Peyer s patch T cells following infection. 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