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Fas Ligand-Defective Mice
CELLULAR IMMUNOLOGY ARTICLE NO.
168, 220–228 (1996)
0069
Inhibition of Apoptosis and Augmentation of Lymphoproliferation in bcl-2 Transgenic Fas/Fas Ligand-Defective Mice AKIHO TAMURA,* MAKOTO KATSUMATA, MARK I. GREENE,
AND
KATSUYUKI YUI†
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; *Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, 390 Japan; and †Department of Medical Zoology, Nagasaki University School of Medicine, Nagasaki, 852 Japan Received September 7, 1995; accepted November 5, 1995
Mice defective in Fas (CD95 or APO-1) or its ligand (lpr or gld mice) develop age-dependent lymphadenopathy and systemic autoimmune disease. T cells accumulating in the lymph nodes of these mice express reduced levels of Bcl-2 protein and are susceptible to spontaneous and glucocorticoid-induced apoptosis. We backcrossed bcl-2 transgenic mice to lpr and gld mice to homozygosity to determine the effects of Bcl2 overexpression. T cells in these mice were resistant to spontaneous and glucocorticoid-induced apoptosis in vitro. Moreover, the accumulation of CD40CD80 T cells in the lymph nodes and the spleens was augmented, suggesting that a Bcl-2-dependent mechanism regulating the number of T cells residing in the peripheral lymphoid organs in addition to the Fas-mediated pathway exists. q 1996 Academic Press, Inc.
INTRODUCTION Mice homozygous for the lpr or gld gene develop an age-related massive lymphadenopathy characterized by the accumulation of CD40CD80 (DN)1 T cells and systemic autoimmune disease and have been used as models for human systemic lupus erythematosus (1– 3). These lpr and gld loci encode genes for the Fas (CD95 or APO-1) (4) and Fas ligand (5), respectively. Both gene products are expressed on activated T cells, and the occupancy of the Fas receptor by the ligand can induce apoptosis in activated T or tumor cells. This receptor–ligand interaction can occur in an autocrine or paracrine fashion and may limit the expansion of T cells. In fact, activation-induced death of T cells appears to be mediated by this receptor–ligand system (6–8). Therefore, the defects in the Fas-mediated 1 Abbreviations used: Dex, dexamethasone; DN, CD40CD80; PI, propidium iodide; SEM, standard error of the mean; Tg-/// mice, bcl-2 transgenic mice backcrossed to C3H mice; Tg-lpr/lpr mice, bcl2 transgenic mice homozygous for the lpr locus; Tg-gld/gld mice, bcl2 transgenic mice homozygous for the gld locus.
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MATERIALS AND METHODS Animals The production and characterization of the bcl-2 transgenic mice have been described previously (15, 16). The transgene construct contained portions of
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0008-8749/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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apoptosis in activated T cells may lead to uncontrolled expansion of activated T cells resulting in their accumulation in the lymphoid organs as seen in lpr and gld mice. Apoptosis appears to play a critical role in T cell development and the maintenance of self-tolerance (9– 11), but its molecular basis is not fully understood. Despite the defects in the Fas-mediated apoptosis pathway, T cells accumulating in the lymph nodes of lpr and gld mice are susceptible to spontaneous apoptosis in vitro and to glucocorticoid-induced death (12, 13). We have recently shown that the expression of the Bcl2 protein, a repressor of apoptosis (11, 14), is reduced in DN and CD4/CD80 T cells accumulating in the lymph nodes of lpr and gld mice (13). We hypothesized that this reduction in Bcl-2 expression is involved in the accelerated apoptosis of accumulating T cells and may participate in the removal of these T cells in vivo. To test this possibility, we backcrossed bcl-2 transgenic mice to lpr and gld mice to homozygosity. As expected, T cells of bcl-2 transgenic lpr and gld mice were resistant to spontaneous and dexamethasone (Dex)-induced apoptosis. Furthermore, the number of accumulating T cells in the lymphoid organs of these mice was significantly increased mostly due to the increased DN T cells. However, the bcl-2 transgene had little effect on autoantibody production in these mice, while serum creatinine levels in the transgenic lpr and gld mice were higher than in their nontransgenic littermates. These results suggest the presence of a Bcl-2-dependent apoptosis pathway, in addition to the Fas system, that is important in the maintenance of lymphocyte homeostasis.
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the human bcl-2 gene and a t(14;18) breakpoint region with the human immunoglobulin heavy-chain enhancer. The founder mouse generated in an (SWR/J 1 SJL/J)F1 background was backcrossed to SWR/J mice for several generations. One of the lines of the transgenic mice which expresses the transgenic human Bcl-2 protein predominantly in the T lineage (line 2) (15, 16) was used in this study. This line of transgenic mice was backcrossed to C3H/HeJ (C3H)-lpr, -gld, or C3H/HeJ strains for two to six generations. The presence of the transgene in the offspring was screened by PCR as described (15). The homozygosity of the lpr locus was tested by Southern blot analysis of the BamHI-digested tail DNA using the HincII fragment of the mouse Fas cDNA probe as described (17). The homozygosity for the gld locus was determined by the presence of lymphadenopathy in all of their parental generation as described (18). In all experiments, nontransgenic and transgenic littermates were compared to evaluate the effect of the bcl-2 transgene expression. C3H, C3H-lpr, and -gld mice were originally purchased from The Jackson Laboratories (Bar Harbor, ME) and maintained in the animal facility of the University of Pennsylvania. Cell Preparation and Flow Cytometry Thymus, spleen, and lymph nodes (axillary, cervical, and inguinal) removed from each mouse were weighed. Single cell suspensions were prepared from each organ and the total viable cell numbers were counted using trypan blue. Lymph node T cells enriched using nylon wool columns contained ú96% TCR positive cells as measured by flow cytometry. Thymocytes (5 1 105) were stained with PE-conjugated rat anti-CD4 (Becton–Dickinson, Mountain View, CA) and FITC-conjugated rat anti-CD8 (Becton– Dickinson) mAb in PBS containing 0.5% BSA and 0.01% sodium azide for 30 min on ice, washed three times, and analyzed using a FACScan (Becton–Dickinson). Lymph node and spleen cells (5 1 105) were incubated with biotinylated anti-TCR ab mAb (Pharmingen, San Diego, CA) or biotinylated anti-B220 mAb (clone RA3-6B2, Gibco BRL, Gaithersburg, MD), washed, and stained with RED670-conjugated streptavidin (Gibco BRL) plus PE–anti-CD4 and FITC–antiCD8 mAb. These cells were also stained with FITCconjugated anti-mouse Ig Ab (Jackson Immunoresearch Lab., West Baltimore Pike, PA). Flow cytometric analysis of Bcl-2 expression was performed as described (13). Briefly, 1 1 106 cells were fixed with 1% paraformaldehyde in PBS (pH 7.2), permeabilized with 0.1% Triton X-100 in PBS, and incubated with hamster anti-human Bcl-2 (6C8; Pharmingen) or hamster antimouse Bcl-2 mAb (3F11; Pharmingen) followed by biotinylated anti-hamster Ig Ab (Jackson Immunoresearch Lab.). After washing these cells, unoccupied
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sites of the secondary antibody were blocked with 50 mg/ml rat IgG (Sigma, St. Louis, MO) for 10 min. Cells were stained with RED670 –streptavidin plus PE–antiCD4 and FITC–anti-CD8 mAb. All flow cytometric analyses were carried out using the LYSIS II software package (Becton–Dickinson). T Cell Survival and Apoptosis Assay Nylon wool nonadherent lymph node cells (2 1 106 cells/ml) were cultured in 24-well plates in RPMI 1640 medium supplemented with 10% FCS, 5 1 1005 M 2ME, 100 U/ml penicillin G sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere of 5% CO2 at 377C for 4 days. The number of viable cells was counted daily using trypan blue. At the end of culture, cells were stained with PE–anti-CD4 and FITC–antiCD8 mAb in addition to propidium iodide (PI, Sigma; 10 mg/ml) and analyzed using a FACScan. The survival of the lymph node T cells was also tested after culture in the presence of Dex. The apoptosis of T cells was determined as previously described (13). Briefly, T cells were fixed in 75% ethanol overnight at 0207C, washed, incubated in PBS containing PI (20 mg/ml) and DNasefree RNase (250 mg/ml; Sigma) for 30 min at room temperature, and analyzed using a FACScan. Serum Ig and Creatinine Levels Serum samples were obtained from the retroorbital plexus of each mouse and stored frozen until use. The total serum Ig levels were determined by ELISA (19). Flat-bottom 96-well microtiter plates (Immulon 4, Dynatech Laboratories, Chantilly, VA) were coated with goat anti-mouse IgG (g-chain specific) or goat antimouse IgM (m-chain specific) Ab (2 mg/ml; Sigma) overnight at room temperature and blocked with boratebuffered saline containing 0.05% Tween 20, 1 mM EDTA, 0.25% BSA, and 0.05% NaN3 for 1 hr. These plates were incubated with sera diluted in blocking buffer for 2 hr at room temperature and with alkaline phosphatase-conjugated anti-mouse Ig Ab (Sigma) overnight at 47C. Finally, p-nitrophenyl phosphate substrate (Sigma) was added and absorbance at 405 nm was measured using a plate reader. The detection of anti-dsDNA antibodies was performed as described (20). Briefly, Immulon microtiter plates were coated with poly-L-lysine (50 mg/ml, Sigma), S1 nucleasetreated calf thymus dsDNA (2.5 mg/ml), and poly-L-glutamine (50 mg/ml, Sigma). After blocking with PBS containing 3% BSA and 0.05% Tween 20, plates were incubated with a serial dilution of serum samples and the plate-bound serum Ig levels were determined. Serum creatinine levels were measured by a modified Jaffe reaction (21) using a calorimetric determination kit (Sigma) according to the manufacturer’s specifications.
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FIG. 1. The expression of human and mouse Bcl-2 protein in lymph node T cells of bcl-2 transgenic mice. Nylon wool nonadherent lymph node cells from ///, Tg-///, lpr/lpr, and Tg-lpr/lpr mice (all 6 months of age) were stained without the first antibody (0) or with anti-human Bcl-2 (human Bcl-2) or anti-mouse Bcl-2 (mouse Bcl-2) mAb and analyzed using a FACScan. Transgenic Bcl-2/ cells in bcl-2 transgenic mice were defined as cells expressing human Bcl-2 protein at levels higher than the control nontransgenic mice as shown in the middle panels.
RESULTS Expression of the Transgenic Human Bcl-2 Protein in T Cell Subsets The transgenic mice used in this study express transgenic human Bcl-2 protein predominantly in cells of the T lineage (15, 16). The expression of transgenic Bcl-2 in T cell subsets was analyzed using flow cytometry to determine whether the transgene is expressed in all T cells (Fig. 1, Table 1). Permeabilization of the cells in staining Bcl-2 protein had little effect on the background staining and cell size and enabled us to evaluate the levels of the Bcl-2 expression in a heterogeneous population of lymphocytes (Fig. 1 and Ref. 13). The bcl-2 transgenic mice backcrossed to C3H (Tg-// /) expressed transgenic Bcl-2 protein in 10 to Ç30% of both CD4/ and CD8/ T cells and this ratio did not change significantly between 2 and 6 months of age. The bcl-2 transgenic mice backcrossed to C3H-gld (Tggld/gld) or C3H-lpr (Tg-lpr/lpr) mice also expressed the transgenic Bcl-2 protein in a similar proportion of T cells at a young age, but the proportion increased with age reaching to 49–62% at 6 months of age in DN
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T cells (Fig. 1, Table 1). In the thymus, 24–30% of thymocytes expressed human Bcl-2 in both Tg-/// and Tg-gld/gld mice (data not shown). The expression of endogenous mouse Bcl-2 protein appears not to be affected by the presence of transgenic human Bcl-2 protein (Fig. 1). The expression of mouse Bcl-2 in T cells of the transgenic mice was superimposable on that of nontransgenic mice and was reduced with age in transgenic lpr and gld mice, as we previously reported in C3H-lpr and -gld mice (13) (Fig. 1 and data not shown). Apoptosis of T Cells in Tg-lpr/pr and -gld/gld Mice T cells in lpr and gld mice show accelerated spontaneous apoptosis ex vivo (12). To determine whether transgenic Bcl-2 protein can prevent this accelerated apoptosis, spontaneous and glucocorticoid-induced apoptosis of T cells in Tg-lpr/lpr and -gld/gld mice was evaluated. First, T cells from bcl-2 transgenic and nontransgenic mice (at 6 months) were cultured for 3 hr, and the proportion of apoptotic cells was determined using flow cytometry (Fig. 2, top). As expected, the proportion of T cells undergoing apoptosis was signifi-
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TABLE 1 Expression of the Transgenic Human Bcl-2 Protein in T Cell Subsets Age (months) Mice Tg-/// Tg-gld/gld
Tg-lpr/lpr
T subset CD4/ CD8/ CD4/ CD8/ DN CD4/ CD8/ DN
2 18.1 28.5 15.8 22.6 24.5
{ 10.2a { 17.5 { 2.1 { 2.9 { 2.6 NDc ND ND
4
6
12.6b 20.2 20.2 { 5.3 27.1 { 3.5 33.6 { 5.6 15.5 { 0.4 29.5 { 2.6 30.5 { 2.5
10.8 { 1.8 23.4 { 0.2 29.8b 29.5 49.3 29.4 { 10.1 47.5 { 13.6 62.4 { 14.0
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tion of T cells with the DN, CD4/B220/, and Thy-10 phenotype, which is characteristic for the accumulating T cells in these mice, also increased (Tables 2–4). This increase in T cell number became clearer at 4 months of age (Table 2, Fig. 4). The increased T cell number and organ weight were also observed in the spleens of these mice (Table 3). At 6 months of age, the number
a The ratio (%) of human Bcl-2high cells in each lymph node T cell subset is shown. The values represent means { standard error of the mean (SEM) of the data collected from three to seven mice. b From one mouse. c ND, not determined.
cantly reduced in T cells of Tg-lpr/lpr mice (mean 6.9%), when compared with nontransgenic lpr mice (mean 12.4%). The ratio of T cells undergoing apoptosis did not reach the levels of control mice, probably because the transgenic Bcl-2 was expressed only in a subpopulation of T cells. In addition, the survival of T cells from Tg-lpr/lpr mice in a longer culture period (up to 4 days) was prolonged when compared with nontransgenic lpr T cells (Fig. 2, bottom). We next examined the susceptibility of T cells to Dex in vitro. Lymph node T cells were cultured with Dex at final concentrations of 0, 1008, 1007, and 1006 M for 32 hr and the viability of cells in each T cell subset was determined (Fig. 3). Single positive as well as DN lymph node T cells in nontransgenic lpr mice were susceptible to Dex. In contrast, CD4/ and DN T cells were resistant to Dex treatment in Tg-lpr/lpr mice. Overexpression of Bcl-2 protein in CD8/ cells did not significantly help survival of these cells to Dex treatment. These cells express high levels of Bcl-2 in nontransgenic mice (13), and additional overexpression of this protein by the transgene appears ineffective in the prevention of apoptosis of these cells. T cells from Tggld/gld mice showed similar results (data not shown). Augmentation of Lymphoproliferation in Tg-lpr/lpr and -gld/gld Mice The number of T cells in the lymph nodes and the spleens of nontransgenic and transgenic lpr and gld mice was determined at 2, 4, and 6 months of age. The onset of lymphadenopathy in gld mice was detected as early as 2 months of age with the increase in the number of DN T cells in the lymph nodes of nontransgenic gld mice (1.7 1 106) (Table 2). In Tg-gld/gld mice, the number of lymph node T cells was even higher than that in nontransgenic gld mice. In addition, the propor-
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FIG. 2. Apoptosis and survival of T cells in Tg-lpr/lpr mice after culture. (Top) Nylon wool nonadherent lymph node cells from ///, Tg-///, lpr/lpr, and Tg-lpr/lpr mice (6 months of age) were cultured for 3 hr. The recovered cells were stained with PI and their DNA content was analyzed using flow cytometry. Each dot represents the ratio (%) of cells containing DNA below G0/G1 peaks in an individual mouse. Horizontal bars indicate the mean values of each group. (Bottom) Nylon wool nonadherent lymph node cells from an individual mouse (4 months of age) were cultured for up to 4 days. At the end of culture, the number of viable cells was counted using trypan blue. The ratio of viable cells was determined as percentage of viable cell number at Time 0. Results are expressed as means / SEM of the triplicate cultures.
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FIG. 3. The survival of T cells in each subset after glucocorticoid treatment in vitro. Nylon wool nonadherent lymph node cells from 4month-old mice were cultured for 32 hr in the absence or the presence of Dex at the final concentrations indicated. At the end of culture, cells were recovered, and their viable cell number and the phenotype were determined as in Fig. 2. The ratio of surviving cells (%) was determined as the ratio of viable cell number after culture with and without Dex. Results are expressed as means of duplicate cultures.
of T cells in the lymph nodes of transgenic and nontransgenic lpr mice was reduced as previously reported in C3H-gld mice (22) (Table 2). In contrast, a huge increase in the T cell number was observed in the spleens of Tg-lpr/lpr mice, reaching Ç10-fold that of nontransgenic littermates (Table 3, Fig. 4). These observations were specific for lpr and gld mice, since the number of T cells in Tg-/// mice was not significantly increased when compared with nontransgenic mice at all ages tested (Tables 2 and 3). Autoantibody, Ig Levels, and Renal Damage Polyclonal activation of B cells in lpr and gld mice results in the elevation of serum Ig levels and autoantibody production. Since this is a T cell-dependent process, the increase in the accumulation of abnormal T cells might alter the degree of autoimmunity in Tg-lpr/ lpr and -gld/gld mice. To test this possibility, serum Ig and anti-dsDNA Ab levels in these mice were evaluated (Fig. 5). The production of serum IgM, IgG, and antidsDNA Ab in Tg-/// and -lpr/lpr mice was comparable to that of their nontransgenic littermates. Similar results were obtained from gld mice (data not shown). We next examined the serum creatinine levels to assess the extent of renal damage (Fig. 6). At both 4 and 6
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months of age, serum creatinine levels in Tg-lpr/lpr were higher than in their nontransgenic littermates, suggesting the presence of renal damage. DISCUSSION The expression of the bcl-2 transgene was detected in Ç20% of T cells in our bcl-2 transgenic mice by flow cytometric analysis. The remaining Ç80% of T cells may not express the transgenic Bcl-2 protein or may express it at levels undetectable by our staining method. The cause of this heterogeneity in transgenic Bcl-2 expression remains unclear. It might derive from the differences in the expression of the transgene among T cell subpopulations or the differentiation/activation status of T cells. We were unable to find any correlation between the expression levels of transgenic Bcl-2 and of T cell subpopulation markers including CD44 (Pgp-1) and MEL14 (data not shown). In transgenic lpr and gld mice, the proportion of peripheral T cells expressing the transgenic Bcl-2 protein increased with age reaching Ç50 to 60% at 6 months of age. This increase was not observed in /// mice and may be due to the prolonged survival and accumulation
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TABLE 2 The Size and the T Cell Composition of Lymph Nodes Age (months) Mice Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN
///
Tg-///
gld/gld
Tg-gld/gld
lpr/lpr
Tg-lpr/lpr
2
4
6
NDa 6.9 { 0.6c 2.9 { 0.4 0.2 { 0.0 ND 7.5 { 1.5 3.2 { 0.6 0.2 { 0.0 ND 6.8 { 0.8 4.1 { 0.5 1.7 { 0.3 ND 8.7 { 0.8 5.5 { 0.6 3.7 { 0.5 ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND 1089.9 { 191.2 31.8 { 5.1 12.4 { 2.2 323.1 { 67.2 1393.0 { 251.1 70.1 { 13.5 23.4 { 4.8 653.8 { 110.6 1482.5 { 309.9 67.8 { 12.3 19.2 { 2.7 634.9 { 137.1 2144.9 { 191.3 100.5 { 24.0 26.0 { 4.0 1212.8 { 257.9
86.4 { 2.2b 8.1 { 1.1 4.1 { 0.5 0.2 { 0.0 112.9 { 14.4 8.5 { 1.1 4.6 { 0.5 0.4 { 0.1 ND ND ND ND ND ND ND ND 1308.5 { 212.3 62.6 { 11.9 22.8 { 3.2 549.3 { 191.4 962.6 { 182.7 61.9 { 16.4 19.3 { 4.0 753.5 { 158.2
a
ND, not determined. The total weight (mg) of inguinal, axillary, and cervical lymph nodes (six nodes). Values are expressed as means { SEM of the data collected from three to nine mice. c Cells were stained in three colors with anti-TCRab, anti-CD4, and anti-CD8 mAbs, and the proportion of each subset was determined using a FACScan. The number of each T cell subset was calculated by multiplying the total number of cells within six nodes with the ratio of each subset and expressed as the mean (11006) { SEM. b
of T cells overexpressing the Bcl-2 protein in lpr and gld mice. In previous studies, thymocytes in bcl-2 transgenic mice were shown to be resistant to glucocorticoid-, anti-CD3 mAb-, and X-irradiation-induced apoptosis (16, 23, 24). The effects of the bcl-2 overexpression on peripheral T cells was difficult to evaluate in these mice since mature T cells in normal mice express high levels of endogenous Bcl-2 protein. We previously reported that the peripheral T cells in aged lpr and gld mutant mice express reduced levels of the Bcl-2 protein (13). The current study extends these observations and shows that the overexpression of the Bcl-2 protein can confer resistance to apoptosis in peripheral T cells of Fas/Fas ligand-defective mice. This implies that the accelerated apoptosis in T cells of these mutant mice is at least partly due to the reduced expression of the Bcl-2 protein. However, the effects of the bcl-2 transgene were not identical among T cell subsets. CD4/ and DN cells in bcl-2 transgenic lpr mice became resistant to glucocorticoid-induced apoptosis but not CD8/ cells (Fig. 3). This differential effect on T cell subsets is not due to the differences in expression of the
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transgenic Bcl-2 protein since the proportion of cells expressing the transgenic Bcl-2 protein was generally higher in CD8/ cells than in CD4/ cells. The data suggest that there are differences among T cell subsets in Bcl-2 dependency when these cells undergo apoptosis. The glucocorticoid-mediated apoptosis in lpr and gld CD8/ cells may be relatively independent of Bcl-2 protein. In support for this possibility, CD8/ cells in nontransgenic lpr and gld mice are sensitive to glucocorticoid-mediated apoptosis despite their high levels of Bcl-2 expression (13). Alternatively, the expression of bcl-2-associated apoptosis-related gene products such as Bax (25), Bcl-xs (26), Bad (27), and BAG-1 (28) may be altered in CD8/ cells by the overexpression of the transgenic Bcl-2 protein, so that these cells remain sensitive to apoptosis. It is of interest to determine the expression levels of these proteins in bcl-2 transgenic mice. Previous studies using /// mice indicated that the development of T cells was not significantly disturbed in bcl-2 transgenic mice overexpressing Bcl-2 in the T cell lineage (16, 23, 24). In agreement with these studies, thymic development in bcl-2 transgenic lpr and gld
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TABLE 3 The Size and T Cell Composition of Spleens Age (months) Mice Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN
///
Tg-///
gld/gld
Tg-gld/gld
lpr/lpr
Tg-lpr/lpr
2
4
6
NDa 20.6 { 2.1c 6.9 { 0.6 2.5 { 0.4 ND 20.4 { 2.7 8.9 { 1.2 2.1 { 0.4 ND 9.1 { 0.3 3.7 { 0.2 2.2 { 0.3 ND 11.4 { 1.2 4.9 { 0.8 4.0 { 0.9 ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND 390.3 { 43.6 22.8 { 6.2 9.4 { 2.9 161.4 { 82.7 554.0 { 38.3 57.0 { 13.4 28.2 { 5.9 407.0 { 165.0 616.1 { 74.6 28.2 { 3.8 13.5 { 3.5 129.9 { 41.7 1010.8 { 121.4 68.5 { 14.0 30.7 { 5.2 712.8 { 187.6
241.8 { 39.5b 28.8 { 9.6 8.3 { 1.2 4.5 { 1.4 267.4 { 55.9 20.8 { 3.0 8.4 { 1.1 4.8 { 0.8 ND ND ND ND ND ND ND ND 606.8 { 81.2 72.1 { 26.4 25.8 { 8.0 210.0 { 80.5 1456.3 { 299.8 160.6 { 44.3 59.8 { 12.1 2557.3 { 1112.8
a
ND, not determined. Mean (mg) { SEM for data from three to nine mice. c The phenotype of spleen cells was analyzed as described in footnote c to Table 2. Values represent absolute numbers of cells (11006) in each T cell subset (means { SEM). b
mice was indistinguishable from nontransgenic mice (data not shown). However, the number and the composition of T cells in the periphery were clearly altered in Fas/Fas ligand-defective mice by the overexpression of the Bcl-2 protein. In particular, the accumulation of DN T cells in the peripheral lymphoid organs was augmented in these mice. The effect was observed as early as 2 months of age when the T cells of abnormal phenotype begin to appear in the periphery. These cells appear to accumulate initially in the lymph nodes and
TABLE 4 The Increase in Abnormal T Cells in the Lymph Nodes of Tg-gld/gld Mice at 2 Months of Age T cell phenotype Micea
DN T (%)
CD4/ B220/ (%)
Thy-10 (%)
gld/gld Tg-gld/gld
13.1 { 1.4b 20.5 { 1.0
4.6 { 0.3c 6.3 { 0.1
0.8 { 0.7d 4.3 { 1.5
a
Three mice (2 months old) were analyzed for each group. The ratio (%) of DN cells among TCR ab/ cells (mean { SEM). c The ratio (%) of B220/ cells among CD4/ cells (mean { SEM). d The ratio (%) of Thy-10 cells in TCRab/ cells (mean { SEM). b
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later in the spleen. A dramatic increase in the number of DN T cells was observed in the spleens of 6-monthold transgenic lpr mice. The Fas-mediated pathway appears to play a critical role in the regulation or termination of the immune response by inducing apoptosis in activated mature lymphocytes (29–31). The defect in this pathway results in the accumulation of abnormal T cells in peripheral lymphoid organs as seen in lpr and gld mutant mice. Our study demonstrates that the overexpression of Bcl-2 and the defects in the Fas/Fas ligand pathway can work synergistically to augment the accumulation of abnormal T cells in the periphery. The overexpression of Bcl-2 alone does not have significant effects on the number and the composition of peripheral T cells as seen in bcl-2 transgenic /// mice. These studies suggest that the size of the peripheral T cell pool is not solely regulated by the Fas-mediated pathway. There appear to be other mechanisms which are dependent on Bcl-2. This novel pathway is not usually apparent in normal mice and might be more pronounced and work as a fail-safe mechanism of the Fasmediated apoptosis pathway. Despite the augmented lymphocyte accumulation, serum Ig levels and anti-DNA Ab levels were not increased, suggesting that the overexpression of Bcl-2 in
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FIG. 4. The increased number of peripheral T cells in Tg-lpr/lpr and -gld/gld mice. Each dot represents the number of T cells in the lymph nodes (A) and the spleen (B) in an individual mouse. Viable cell numbers were counted using trypan blue. These cells were stained with anti-TCR ab mAb and the number of T cells was determined for each individual mouse. The number of lymph node T cells represents a total of inguinal, axillary, and cervical lymph node T cells (six nodes).
the T lineage had little effect on the B cell compartment. However, the increase in serum creatinine levels was observed in transgenic lpr and gld mice, suggesting that these mice have renal damage. We suspect that the increased renal damage is not mediated by Ig, but may be associated with the increased accumulation of abnormal T cells in the periphery, since the autoantibody production and immune complex formation appear unchanged in transgenic lpr and gld mice. Several reports suggest that these T cells can be harmful. DN T cells accumulating in the lymph nodes of lpr and gld mice express perforin and can exert cytotoxic activity and may able to cause tissue damage (32). In addition, DN T cells appear to be able to play a role in autoim-
mune nephritis by inducing the expression of MHC class II and adhesion molecules on renal tubular cell epithelia (33). Histological studies in MRL-lpr mice with elevated serum creatinine levels showed the infiltration of mononuclear cells in the kidney and glomerulonephritis (34). Collectively, these studies suggest that there may be a novel Bcl-2-dependent pathway which regulates the size of the peripheral T cell pool in addition to the Fas/ Fas ligand pathway. This pathway becomes apparent when the Fas-mediated pathway is defective and may be important not only for the regulation of the peripheral T cell number but also for the prevention of tissue damage.
FIG. 5. Serum Ig and anti-dsDNA Ab levels. Sera were obtained from each mouse at the age indicated and the levels of total IgM, total IgG, and anti-dsDNA antibodies were determined by ELISA as described under Materials and Methods. The means / SEM of 3 to 11 samples are shown.
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FIG. 6. Serum creatinine levels in ///, lpr/lpr, and Tg-lpr/lpr mice. The levels of serum creatinine in 6-month-old mice were determined as described under Materials and Methods. The results are expressed as means / SEM in each group.
ACKNOWLEDGMENTS We thank Dr. Michael P. Madaio for helping us with the methods and reagents of the anti-dsDNA assay and Drs. Akihiko Yano and Gail Massey for critical reading of the manuscript. We also appreciate Drs. Tetsuo Hamada and Shingo Ichimiya for their contribution. This work was supported by grants from the National Institute of Health (AI36303), the American Cancer Society (IM644), and Lucille P. Markey Charitable Trust.
REFERENCES 1. Cohen, P. L., and Eisenberg, R. A., Annu. Rev. Immunol. 9, 243, 1991. 2. Yui, K., Wadsworth, S., Yellen, A., Hashimoto, Y., Kokai, Y., and Greene, M. I., Immunol. Rev. 104, 121, 1988. 3. Bhandoola, A., Yui, K., Siegel, R. M., Zerva, L., and Greene, M. I., Int. Rev. Immunol. 11, 231, 1994. 4. Watanabe-Fukunaga, R., Brannan, C. I., Copeland, N. G., Jenkins, N. A., and Nagata, S., Nature 356, 314, 1992. 5. Takahashi, T., Tanaka, M., Brannan, C. I., Jenkins, N. A., Copeland, N. G., Suda, T., and Nagata, S., Cell 76, 969, 1994. 6. Dhein, J., Walczak, H., Baumler, C., Debatin, K.-M., and Krammer, P. H., Nature 373, 438, 1995.
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7. Brunner, T., Mogil, R. J., LaFace, D., Yoo, N. J., Mahboubi, A., Echeverri, F., Martin, S. J., Force, W. R., Lynch, D. H., Ware, C. F., and Green, D. R., Nature 373, 441, 1995. 8. Ju, S.-T., Panka, D. J., Cui, H., Ettinger, R., El-Khatib, M., Sherr, D. H., Stanger, B. Z., and Marshak-Rothstein, A., Nature 373, 444, 1995. 9. Williams, G. T., Cell 65, 1097, 1991. 10. Schwartz, L. M., and Osborne, B. A., Immunol. Today 12, 582, 1993. 11. Korsmeyer, S. J., Immunol. Today 13, 285, 1992. 12. Houten, N. V., and Budd, R. C., J. Immunol. 149, 2513, 1992. 13. Tamura, A., and Yui, K., J. Immunol. 155, 499, 1995. 14. Hockenbery, D. M., Oltvai, Z. N., Yin, X.-M., Milliman, C. L., and Korsmeyer, S. J., Cell 75, 241, 1993. 15. Katsumata, M., Siegel, R. M., Louie, D. C., Miyashita, T., Tsujimoto, Y., Nowell, P. C., Greene, M. I., and Reed, J. C., Proc. Natl. Acad. Sci. USA 89, 11376, 1992. 16. Siegel, R. M., Katsumata, M., Miyashita, T., Louie, D. C., Greene, M. I., and Reed, J. C., Proc. Natl. Acad. Sci. USA 89, 7003, 1992. 17. Wu, J., Zhou, T., He, J., and Mountz, J. D., J. Exp. Med. 178, 461, 1993. 18. Yui, K., Bhandoola, A., Radic, M. Z., Komori, S., Katsumata, M., and Greene, M. I., Eur. J. Immunol. 22, 1693, 1992. 19. Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., Curr. Protocols Immunol. 1, 2.1.2, 1991. 20. Madaio, M. P., Hodder, S., Schwartz, R. S., and Stollar, B. D., J. Immunol. 132, 872, 1984. 21. Heinegard, D., and Tiderstrom, G., Clin. Chim. Acta 43, 305, 1973. 22. Roths, J. B., Murphy, E. D., and Eicher, E. M., J. Exp. Med. 159, 1, 1984. 23. Strasser, A., Harris, A. W., and Cory, S., Cell 67, 889, 1991. 24. Sentman, C. L., Shutter, J. R., Hockenbery, D., Kanagawa, O., and Korsmeyer, S. J., Cell 67, 879, 1991. 25. Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J., Cell 74, 609, 1993. 26. Boise, L. H., Gonzalez-Garcia, M., Postema, C. E., Ding, L., Lindsten, T., Turka, L. A., Mao, X., Nunez, G., and Thompson, C. B., Cell 74, 597, 1993. 27. Yang, E., Zha, J., Jockel, J., Boise, L. H., Thompson, C. B., and Korsmeyer, S. J., Cell 80, 285, 1995. 28. Takayama, S., Sato, T., Krajewski, S., Kochel, K., Irie, S., Millan, J. A., and Reed, J. C., Cell 80, 279, 1995. 29. Singer, G. G., and Abbas, A. K., Immunity 1, 366, 1994. 30. Ettinger, R., Wang, J. K. M., Bossu, P., Papas, K., Sidman, C. L., Abbas, A. K., and Marshak-Rothstein, A., J. Immunol. 152, 1557, 1994. 31. Vignaux, F., and Golstein, P., Eur. J. Immunol. 24, 923, 1994. 32. Jevnikar, A. M., Grusby, M. J., and Glimcher, L. H., J. Exp. Med. 179, 1137, 1994. 33. Hammond, D. M., Nagarkatti, P. S., Gote, L. R., Seth, A., Hassuneh, M. R., and Nagarkatti, M., J. Exp. Med. 178, 2225, 1993. 34. Diaz-Gallo, C., Jevnikar, A. M., Brennan, D. C., Florquin, S., Pacheco-Silva, A., and Kelley, V. R., Kidney Int. 42, 851, 1992.
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Inhibition of Apoptosis and Augmentation of Lymphoproliferation in bcl-2 Transgenic Fas/Fas Ligand-Defective Mice AKIHO TAMURA,* MAKOTO KATSUMATA, MARK I. GREENE,
AND
KATSUYUKI YUI†
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; *Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, 390 Japan; and †Department of Medical Zoology, Nagasaki University School of Medicine, Nagasaki, 852 Japan Received September 7, 1995; accepted November 5, 1995
Mice defective in Fas (CD95 or APO-1) or its ligand (lpr or gld mice) develop age-dependent lymphadenopathy and systemic autoimmune disease. T cells accumulating in the lymph nodes of these mice express reduced levels of Bcl-2 protein and are susceptible to spontaneous and glucocorticoid-induced apoptosis. We backcrossed bcl-2 transgenic mice to lpr and gld mice to homozygosity to determine the effects of Bcl2 overexpression. T cells in these mice were resistant to spontaneous and glucocorticoid-induced apoptosis in vitro. Moreover, the accumulation of CD40CD80 T cells in the lymph nodes and the spleens was augmented, suggesting that a Bcl-2-dependent mechanism regulating the number of T cells residing in the peripheral lymphoid organs in addition to the Fas-mediated pathway exists. q 1996 Academic Press, Inc.
INTRODUCTION Mice homozygous for the lpr or gld gene develop an age-related massive lymphadenopathy characterized by the accumulation of CD40CD80 (DN)1 T cells and systemic autoimmune disease and have been used as models for human systemic lupus erythematosus (1– 3). These lpr and gld loci encode genes for the Fas (CD95 or APO-1) (4) and Fas ligand (5), respectively. Both gene products are expressed on activated T cells, and the occupancy of the Fas receptor by the ligand can induce apoptosis in activated T or tumor cells. This receptor–ligand interaction can occur in an autocrine or paracrine fashion and may limit the expansion of T cells. In fact, activation-induced death of T cells appears to be mediated by this receptor–ligand system (6–8). Therefore, the defects in the Fas-mediated 1 Abbreviations used: Dex, dexamethasone; DN, CD40CD80; PI, propidium iodide; SEM, standard error of the mean; Tg-/// mice, bcl-2 transgenic mice backcrossed to C3H mice; Tg-lpr/lpr mice, bcl2 transgenic mice homozygous for the lpr locus; Tg-gld/gld mice, bcl2 transgenic mice homozygous for the gld locus.
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MATERIALS AND METHODS Animals The production and characterization of the bcl-2 transgenic mice have been described previously (15, 16). The transgene construct contained portions of
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0008-8749/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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apoptosis in activated T cells may lead to uncontrolled expansion of activated T cells resulting in their accumulation in the lymphoid organs as seen in lpr and gld mice. Apoptosis appears to play a critical role in T cell development and the maintenance of self-tolerance (9– 11), but its molecular basis is not fully understood. Despite the defects in the Fas-mediated apoptosis pathway, T cells accumulating in the lymph nodes of lpr and gld mice are susceptible to spontaneous apoptosis in vitro and to glucocorticoid-induced death (12, 13). We have recently shown that the expression of the Bcl2 protein, a repressor of apoptosis (11, 14), is reduced in DN and CD4/CD80 T cells accumulating in the lymph nodes of lpr and gld mice (13). We hypothesized that this reduction in Bcl-2 expression is involved in the accelerated apoptosis of accumulating T cells and may participate in the removal of these T cells in vivo. To test this possibility, we backcrossed bcl-2 transgenic mice to lpr and gld mice to homozygosity. As expected, T cells of bcl-2 transgenic lpr and gld mice were resistant to spontaneous and dexamethasone (Dex)-induced apoptosis. Furthermore, the number of accumulating T cells in the lymphoid organs of these mice was significantly increased mostly due to the increased DN T cells. However, the bcl-2 transgene had little effect on autoantibody production in these mice, while serum creatinine levels in the transgenic lpr and gld mice were higher than in their nontransgenic littermates. These results suggest the presence of a Bcl-2-dependent apoptosis pathway, in addition to the Fas system, that is important in the maintenance of lymphocyte homeostasis.
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the human bcl-2 gene and a t(14;18) breakpoint region with the human immunoglobulin heavy-chain enhancer. The founder mouse generated in an (SWR/J 1 SJL/J)F1 background was backcrossed to SWR/J mice for several generations. One of the lines of the transgenic mice which expresses the transgenic human Bcl-2 protein predominantly in the T lineage (line 2) (15, 16) was used in this study. This line of transgenic mice was backcrossed to C3H/HeJ (C3H)-lpr, -gld, or C3H/HeJ strains for two to six generations. The presence of the transgene in the offspring was screened by PCR as described (15). The homozygosity of the lpr locus was tested by Southern blot analysis of the BamHI-digested tail DNA using the HincII fragment of the mouse Fas cDNA probe as described (17). The homozygosity for the gld locus was determined by the presence of lymphadenopathy in all of their parental generation as described (18). In all experiments, nontransgenic and transgenic littermates were compared to evaluate the effect of the bcl-2 transgene expression. C3H, C3H-lpr, and -gld mice were originally purchased from The Jackson Laboratories (Bar Harbor, ME) and maintained in the animal facility of the University of Pennsylvania. Cell Preparation and Flow Cytometry Thymus, spleen, and lymph nodes (axillary, cervical, and inguinal) removed from each mouse were weighed. Single cell suspensions were prepared from each organ and the total viable cell numbers were counted using trypan blue. Lymph node T cells enriched using nylon wool columns contained ú96% TCR positive cells as measured by flow cytometry. Thymocytes (5 1 105) were stained with PE-conjugated rat anti-CD4 (Becton–Dickinson, Mountain View, CA) and FITC-conjugated rat anti-CD8 (Becton– Dickinson) mAb in PBS containing 0.5% BSA and 0.01% sodium azide for 30 min on ice, washed three times, and analyzed using a FACScan (Becton–Dickinson). Lymph node and spleen cells (5 1 105) were incubated with biotinylated anti-TCR ab mAb (Pharmingen, San Diego, CA) or biotinylated anti-B220 mAb (clone RA3-6B2, Gibco BRL, Gaithersburg, MD), washed, and stained with RED670-conjugated streptavidin (Gibco BRL) plus PE–anti-CD4 and FITC–antiCD8 mAb. These cells were also stained with FITCconjugated anti-mouse Ig Ab (Jackson Immunoresearch Lab., West Baltimore Pike, PA). Flow cytometric analysis of Bcl-2 expression was performed as described (13). Briefly, 1 1 106 cells were fixed with 1% paraformaldehyde in PBS (pH 7.2), permeabilized with 0.1% Triton X-100 in PBS, and incubated with hamster anti-human Bcl-2 (6C8; Pharmingen) or hamster antimouse Bcl-2 mAb (3F11; Pharmingen) followed by biotinylated anti-hamster Ig Ab (Jackson Immunoresearch Lab.). After washing these cells, unoccupied
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sites of the secondary antibody were blocked with 50 mg/ml rat IgG (Sigma, St. Louis, MO) for 10 min. Cells were stained with RED670 –streptavidin plus PE–antiCD4 and FITC–anti-CD8 mAb. All flow cytometric analyses were carried out using the LYSIS II software package (Becton–Dickinson). T Cell Survival and Apoptosis Assay Nylon wool nonadherent lymph node cells (2 1 106 cells/ml) were cultured in 24-well plates in RPMI 1640 medium supplemented with 10% FCS, 5 1 1005 M 2ME, 100 U/ml penicillin G sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere of 5% CO2 at 377C for 4 days. The number of viable cells was counted daily using trypan blue. At the end of culture, cells were stained with PE–anti-CD4 and FITC–antiCD8 mAb in addition to propidium iodide (PI, Sigma; 10 mg/ml) and analyzed using a FACScan. The survival of the lymph node T cells was also tested after culture in the presence of Dex. The apoptosis of T cells was determined as previously described (13). Briefly, T cells were fixed in 75% ethanol overnight at 0207C, washed, incubated in PBS containing PI (20 mg/ml) and DNasefree RNase (250 mg/ml; Sigma) for 30 min at room temperature, and analyzed using a FACScan. Serum Ig and Creatinine Levels Serum samples were obtained from the retroorbital plexus of each mouse and stored frozen until use. The total serum Ig levels were determined by ELISA (19). Flat-bottom 96-well microtiter plates (Immulon 4, Dynatech Laboratories, Chantilly, VA) were coated with goat anti-mouse IgG (g-chain specific) or goat antimouse IgM (m-chain specific) Ab (2 mg/ml; Sigma) overnight at room temperature and blocked with boratebuffered saline containing 0.05% Tween 20, 1 mM EDTA, 0.25% BSA, and 0.05% NaN3 for 1 hr. These plates were incubated with sera diluted in blocking buffer for 2 hr at room temperature and with alkaline phosphatase-conjugated anti-mouse Ig Ab (Sigma) overnight at 47C. Finally, p-nitrophenyl phosphate substrate (Sigma) was added and absorbance at 405 nm was measured using a plate reader. The detection of anti-dsDNA antibodies was performed as described (20). Briefly, Immulon microtiter plates were coated with poly-L-lysine (50 mg/ml, Sigma), S1 nucleasetreated calf thymus dsDNA (2.5 mg/ml), and poly-L-glutamine (50 mg/ml, Sigma). After blocking with PBS containing 3% BSA and 0.05% Tween 20, plates were incubated with a serial dilution of serum samples and the plate-bound serum Ig levels were determined. Serum creatinine levels were measured by a modified Jaffe reaction (21) using a calorimetric determination kit (Sigma) according to the manufacturer’s specifications.
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FIG. 1. The expression of human and mouse Bcl-2 protein in lymph node T cells of bcl-2 transgenic mice. Nylon wool nonadherent lymph node cells from ///, Tg-///, lpr/lpr, and Tg-lpr/lpr mice (all 6 months of age) were stained without the first antibody (0) or with anti-human Bcl-2 (human Bcl-2) or anti-mouse Bcl-2 (mouse Bcl-2) mAb and analyzed using a FACScan. Transgenic Bcl-2/ cells in bcl-2 transgenic mice were defined as cells expressing human Bcl-2 protein at levels higher than the control nontransgenic mice as shown in the middle panels.
RESULTS Expression of the Transgenic Human Bcl-2 Protein in T Cell Subsets The transgenic mice used in this study express transgenic human Bcl-2 protein predominantly in cells of the T lineage (15, 16). The expression of transgenic Bcl-2 in T cell subsets was analyzed using flow cytometry to determine whether the transgene is expressed in all T cells (Fig. 1, Table 1). Permeabilization of the cells in staining Bcl-2 protein had little effect on the background staining and cell size and enabled us to evaluate the levels of the Bcl-2 expression in a heterogeneous population of lymphocytes (Fig. 1 and Ref. 13). The bcl-2 transgenic mice backcrossed to C3H (Tg-// /) expressed transgenic Bcl-2 protein in 10 to Ç30% of both CD4/ and CD8/ T cells and this ratio did not change significantly between 2 and 6 months of age. The bcl-2 transgenic mice backcrossed to C3H-gld (Tggld/gld) or C3H-lpr (Tg-lpr/lpr) mice also expressed the transgenic Bcl-2 protein in a similar proportion of T cells at a young age, but the proportion increased with age reaching to 49–62% at 6 months of age in DN
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T cells (Fig. 1, Table 1). In the thymus, 24–30% of thymocytes expressed human Bcl-2 in both Tg-/// and Tg-gld/gld mice (data not shown). The expression of endogenous mouse Bcl-2 protein appears not to be affected by the presence of transgenic human Bcl-2 protein (Fig. 1). The expression of mouse Bcl-2 in T cells of the transgenic mice was superimposable on that of nontransgenic mice and was reduced with age in transgenic lpr and gld mice, as we previously reported in C3H-lpr and -gld mice (13) (Fig. 1 and data not shown). Apoptosis of T Cells in Tg-lpr/pr and -gld/gld Mice T cells in lpr and gld mice show accelerated spontaneous apoptosis ex vivo (12). To determine whether transgenic Bcl-2 protein can prevent this accelerated apoptosis, spontaneous and glucocorticoid-induced apoptosis of T cells in Tg-lpr/lpr and -gld/gld mice was evaluated. First, T cells from bcl-2 transgenic and nontransgenic mice (at 6 months) were cultured for 3 hr, and the proportion of apoptotic cells was determined using flow cytometry (Fig. 2, top). As expected, the proportion of T cells undergoing apoptosis was signifi-
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TABLE 1 Expression of the Transgenic Human Bcl-2 Protein in T Cell Subsets Age (months) Mice Tg-/// Tg-gld/gld
Tg-lpr/lpr
T subset CD4/ CD8/ CD4/ CD8/ DN CD4/ CD8/ DN
2 18.1 28.5 15.8 22.6 24.5
{ 10.2a { 17.5 { 2.1 { 2.9 { 2.6 NDc ND ND
4
6
12.6b 20.2 20.2 { 5.3 27.1 { 3.5 33.6 { 5.6 15.5 { 0.4 29.5 { 2.6 30.5 { 2.5
10.8 { 1.8 23.4 { 0.2 29.8b 29.5 49.3 29.4 { 10.1 47.5 { 13.6 62.4 { 14.0
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tion of T cells with the DN, CD4/B220/, and Thy-10 phenotype, which is characteristic for the accumulating T cells in these mice, also increased (Tables 2–4). This increase in T cell number became clearer at 4 months of age (Table 2, Fig. 4). The increased T cell number and organ weight were also observed in the spleens of these mice (Table 3). At 6 months of age, the number
a The ratio (%) of human Bcl-2high cells in each lymph node T cell subset is shown. The values represent means { standard error of the mean (SEM) of the data collected from three to seven mice. b From one mouse. c ND, not determined.
cantly reduced in T cells of Tg-lpr/lpr mice (mean 6.9%), when compared with nontransgenic lpr mice (mean 12.4%). The ratio of T cells undergoing apoptosis did not reach the levels of control mice, probably because the transgenic Bcl-2 was expressed only in a subpopulation of T cells. In addition, the survival of T cells from Tg-lpr/lpr mice in a longer culture period (up to 4 days) was prolonged when compared with nontransgenic lpr T cells (Fig. 2, bottom). We next examined the susceptibility of T cells to Dex in vitro. Lymph node T cells were cultured with Dex at final concentrations of 0, 1008, 1007, and 1006 M for 32 hr and the viability of cells in each T cell subset was determined (Fig. 3). Single positive as well as DN lymph node T cells in nontransgenic lpr mice were susceptible to Dex. In contrast, CD4/ and DN T cells were resistant to Dex treatment in Tg-lpr/lpr mice. Overexpression of Bcl-2 protein in CD8/ cells did not significantly help survival of these cells to Dex treatment. These cells express high levels of Bcl-2 in nontransgenic mice (13), and additional overexpression of this protein by the transgene appears ineffective in the prevention of apoptosis of these cells. T cells from Tggld/gld mice showed similar results (data not shown). Augmentation of Lymphoproliferation in Tg-lpr/lpr and -gld/gld Mice The number of T cells in the lymph nodes and the spleens of nontransgenic and transgenic lpr and gld mice was determined at 2, 4, and 6 months of age. The onset of lymphadenopathy in gld mice was detected as early as 2 months of age with the increase in the number of DN T cells in the lymph nodes of nontransgenic gld mice (1.7 1 106) (Table 2). In Tg-gld/gld mice, the number of lymph node T cells was even higher than that in nontransgenic gld mice. In addition, the propor-
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FIG. 2. Apoptosis and survival of T cells in Tg-lpr/lpr mice after culture. (Top) Nylon wool nonadherent lymph node cells from ///, Tg-///, lpr/lpr, and Tg-lpr/lpr mice (6 months of age) were cultured for 3 hr. The recovered cells were stained with PI and their DNA content was analyzed using flow cytometry. Each dot represents the ratio (%) of cells containing DNA below G0/G1 peaks in an individual mouse. Horizontal bars indicate the mean values of each group. (Bottom) Nylon wool nonadherent lymph node cells from an individual mouse (4 months of age) were cultured for up to 4 days. At the end of culture, the number of viable cells was counted using trypan blue. The ratio of viable cells was determined as percentage of viable cell number at Time 0. Results are expressed as means / SEM of the triplicate cultures.
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FIG. 3. The survival of T cells in each subset after glucocorticoid treatment in vitro. Nylon wool nonadherent lymph node cells from 4month-old mice were cultured for 32 hr in the absence or the presence of Dex at the final concentrations indicated. At the end of culture, cells were recovered, and their viable cell number and the phenotype were determined as in Fig. 2. The ratio of surviving cells (%) was determined as the ratio of viable cell number after culture with and without Dex. Results are expressed as means of duplicate cultures.
of T cells in the lymph nodes of transgenic and nontransgenic lpr mice was reduced as previously reported in C3H-gld mice (22) (Table 2). In contrast, a huge increase in the T cell number was observed in the spleens of Tg-lpr/lpr mice, reaching Ç10-fold that of nontransgenic littermates (Table 3, Fig. 4). These observations were specific for lpr and gld mice, since the number of T cells in Tg-/// mice was not significantly increased when compared with nontransgenic mice at all ages tested (Tables 2 and 3). Autoantibody, Ig Levels, and Renal Damage Polyclonal activation of B cells in lpr and gld mice results in the elevation of serum Ig levels and autoantibody production. Since this is a T cell-dependent process, the increase in the accumulation of abnormal T cells might alter the degree of autoimmunity in Tg-lpr/ lpr and -gld/gld mice. To test this possibility, serum Ig and anti-dsDNA Ab levels in these mice were evaluated (Fig. 5). The production of serum IgM, IgG, and antidsDNA Ab in Tg-/// and -lpr/lpr mice was comparable to that of their nontransgenic littermates. Similar results were obtained from gld mice (data not shown). We next examined the serum creatinine levels to assess the extent of renal damage (Fig. 6). At both 4 and 6
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months of age, serum creatinine levels in Tg-lpr/lpr were higher than in their nontransgenic littermates, suggesting the presence of renal damage. DISCUSSION The expression of the bcl-2 transgene was detected in Ç20% of T cells in our bcl-2 transgenic mice by flow cytometric analysis. The remaining Ç80% of T cells may not express the transgenic Bcl-2 protein or may express it at levels undetectable by our staining method. The cause of this heterogeneity in transgenic Bcl-2 expression remains unclear. It might derive from the differences in the expression of the transgene among T cell subpopulations or the differentiation/activation status of T cells. We were unable to find any correlation between the expression levels of transgenic Bcl-2 and of T cell subpopulation markers including CD44 (Pgp-1) and MEL14 (data not shown). In transgenic lpr and gld mice, the proportion of peripheral T cells expressing the transgenic Bcl-2 protein increased with age reaching Ç50 to 60% at 6 months of age. This increase was not observed in /// mice and may be due to the prolonged survival and accumulation
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TABLE 2 The Size and the T Cell Composition of Lymph Nodes Age (months) Mice Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN
///
Tg-///
gld/gld
Tg-gld/gld
lpr/lpr
Tg-lpr/lpr
2
4
6
NDa 6.9 { 0.6c 2.9 { 0.4 0.2 { 0.0 ND 7.5 { 1.5 3.2 { 0.6 0.2 { 0.0 ND 6.8 { 0.8 4.1 { 0.5 1.7 { 0.3 ND 8.7 { 0.8 5.5 { 0.6 3.7 { 0.5 ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND 1089.9 { 191.2 31.8 { 5.1 12.4 { 2.2 323.1 { 67.2 1393.0 { 251.1 70.1 { 13.5 23.4 { 4.8 653.8 { 110.6 1482.5 { 309.9 67.8 { 12.3 19.2 { 2.7 634.9 { 137.1 2144.9 { 191.3 100.5 { 24.0 26.0 { 4.0 1212.8 { 257.9
86.4 { 2.2b 8.1 { 1.1 4.1 { 0.5 0.2 { 0.0 112.9 { 14.4 8.5 { 1.1 4.6 { 0.5 0.4 { 0.1 ND ND ND ND ND ND ND ND 1308.5 { 212.3 62.6 { 11.9 22.8 { 3.2 549.3 { 191.4 962.6 { 182.7 61.9 { 16.4 19.3 { 4.0 753.5 { 158.2
a
ND, not determined. The total weight (mg) of inguinal, axillary, and cervical lymph nodes (six nodes). Values are expressed as means { SEM of the data collected from three to nine mice. c Cells were stained in three colors with anti-TCRab, anti-CD4, and anti-CD8 mAbs, and the proportion of each subset was determined using a FACScan. The number of each T cell subset was calculated by multiplying the total number of cells within six nodes with the ratio of each subset and expressed as the mean (11006) { SEM. b
of T cells overexpressing the Bcl-2 protein in lpr and gld mice. In previous studies, thymocytes in bcl-2 transgenic mice were shown to be resistant to glucocorticoid-, anti-CD3 mAb-, and X-irradiation-induced apoptosis (16, 23, 24). The effects of the bcl-2 overexpression on peripheral T cells was difficult to evaluate in these mice since mature T cells in normal mice express high levels of endogenous Bcl-2 protein. We previously reported that the peripheral T cells in aged lpr and gld mutant mice express reduced levels of the Bcl-2 protein (13). The current study extends these observations and shows that the overexpression of the Bcl-2 protein can confer resistance to apoptosis in peripheral T cells of Fas/Fas ligand-defective mice. This implies that the accelerated apoptosis in T cells of these mutant mice is at least partly due to the reduced expression of the Bcl-2 protein. However, the effects of the bcl-2 transgene were not identical among T cell subsets. CD4/ and DN cells in bcl-2 transgenic lpr mice became resistant to glucocorticoid-induced apoptosis but not CD8/ cells (Fig. 3). This differential effect on T cell subsets is not due to the differences in expression of the
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transgenic Bcl-2 protein since the proportion of cells expressing the transgenic Bcl-2 protein was generally higher in CD8/ cells than in CD4/ cells. The data suggest that there are differences among T cell subsets in Bcl-2 dependency when these cells undergo apoptosis. The glucocorticoid-mediated apoptosis in lpr and gld CD8/ cells may be relatively independent of Bcl-2 protein. In support for this possibility, CD8/ cells in nontransgenic lpr and gld mice are sensitive to glucocorticoid-mediated apoptosis despite their high levels of Bcl-2 expression (13). Alternatively, the expression of bcl-2-associated apoptosis-related gene products such as Bax (25), Bcl-xs (26), Bad (27), and BAG-1 (28) may be altered in CD8/ cells by the overexpression of the transgenic Bcl-2 protein, so that these cells remain sensitive to apoptosis. It is of interest to determine the expression levels of these proteins in bcl-2 transgenic mice. Previous studies using /// mice indicated that the development of T cells was not significantly disturbed in bcl-2 transgenic mice overexpressing Bcl-2 in the T cell lineage (16, 23, 24). In agreement with these studies, thymic development in bcl-2 transgenic lpr and gld
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TABLE 3 The Size and T Cell Composition of Spleens Age (months) Mice Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN Weight CD4/ CD8/ DN
///
Tg-///
gld/gld
Tg-gld/gld
lpr/lpr
Tg-lpr/lpr
2
4
6
NDa 20.6 { 2.1c 6.9 { 0.6 2.5 { 0.4 ND 20.4 { 2.7 8.9 { 1.2 2.1 { 0.4 ND 9.1 { 0.3 3.7 { 0.2 2.2 { 0.3 ND 11.4 { 1.2 4.9 { 0.8 4.0 { 0.9 ND ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND 390.3 { 43.6 22.8 { 6.2 9.4 { 2.9 161.4 { 82.7 554.0 { 38.3 57.0 { 13.4 28.2 { 5.9 407.0 { 165.0 616.1 { 74.6 28.2 { 3.8 13.5 { 3.5 129.9 { 41.7 1010.8 { 121.4 68.5 { 14.0 30.7 { 5.2 712.8 { 187.6
241.8 { 39.5b 28.8 { 9.6 8.3 { 1.2 4.5 { 1.4 267.4 { 55.9 20.8 { 3.0 8.4 { 1.1 4.8 { 0.8 ND ND ND ND ND ND ND ND 606.8 { 81.2 72.1 { 26.4 25.8 { 8.0 210.0 { 80.5 1456.3 { 299.8 160.6 { 44.3 59.8 { 12.1 2557.3 { 1112.8
a
ND, not determined. Mean (mg) { SEM for data from three to nine mice. c The phenotype of spleen cells was analyzed as described in footnote c to Table 2. Values represent absolute numbers of cells (11006) in each T cell subset (means { SEM). b
mice was indistinguishable from nontransgenic mice (data not shown). However, the number and the composition of T cells in the periphery were clearly altered in Fas/Fas ligand-defective mice by the overexpression of the Bcl-2 protein. In particular, the accumulation of DN T cells in the peripheral lymphoid organs was augmented in these mice. The effect was observed as early as 2 months of age when the T cells of abnormal phenotype begin to appear in the periphery. These cells appear to accumulate initially in the lymph nodes and
TABLE 4 The Increase in Abnormal T Cells in the Lymph Nodes of Tg-gld/gld Mice at 2 Months of Age T cell phenotype Micea
DN T (%)
CD4/ B220/ (%)
Thy-10 (%)
gld/gld Tg-gld/gld
13.1 { 1.4b 20.5 { 1.0
4.6 { 0.3c 6.3 { 0.1
0.8 { 0.7d 4.3 { 1.5
a
Three mice (2 months old) were analyzed for each group. The ratio (%) of DN cells among TCR ab/ cells (mean { SEM). c The ratio (%) of B220/ cells among CD4/ cells (mean { SEM). d The ratio (%) of Thy-10 cells in TCRab/ cells (mean { SEM). b
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later in the spleen. A dramatic increase in the number of DN T cells was observed in the spleens of 6-monthold transgenic lpr mice. The Fas-mediated pathway appears to play a critical role in the regulation or termination of the immune response by inducing apoptosis in activated mature lymphocytes (29–31). The defect in this pathway results in the accumulation of abnormal T cells in peripheral lymphoid organs as seen in lpr and gld mutant mice. Our study demonstrates that the overexpression of Bcl-2 and the defects in the Fas/Fas ligand pathway can work synergistically to augment the accumulation of abnormal T cells in the periphery. The overexpression of Bcl-2 alone does not have significant effects on the number and the composition of peripheral T cells as seen in bcl-2 transgenic /// mice. These studies suggest that the size of the peripheral T cell pool is not solely regulated by the Fas-mediated pathway. There appear to be other mechanisms which are dependent on Bcl-2. This novel pathway is not usually apparent in normal mice and might be more pronounced and work as a fail-safe mechanism of the Fasmediated apoptosis pathway. Despite the augmented lymphocyte accumulation, serum Ig levels and anti-DNA Ab levels were not increased, suggesting that the overexpression of Bcl-2 in
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FIG. 4. The increased number of peripheral T cells in Tg-lpr/lpr and -gld/gld mice. Each dot represents the number of T cells in the lymph nodes (A) and the spleen (B) in an individual mouse. Viable cell numbers were counted using trypan blue. These cells were stained with anti-TCR ab mAb and the number of T cells was determined for each individual mouse. The number of lymph node T cells represents a total of inguinal, axillary, and cervical lymph node T cells (six nodes).
the T lineage had little effect on the B cell compartment. However, the increase in serum creatinine levels was observed in transgenic lpr and gld mice, suggesting that these mice have renal damage. We suspect that the increased renal damage is not mediated by Ig, but may be associated with the increased accumulation of abnormal T cells in the periphery, since the autoantibody production and immune complex formation appear unchanged in transgenic lpr and gld mice. Several reports suggest that these T cells can be harmful. DN T cells accumulating in the lymph nodes of lpr and gld mice express perforin and can exert cytotoxic activity and may able to cause tissue damage (32). In addition, DN T cells appear to be able to play a role in autoim-
mune nephritis by inducing the expression of MHC class II and adhesion molecules on renal tubular cell epithelia (33). Histological studies in MRL-lpr mice with elevated serum creatinine levels showed the infiltration of mononuclear cells in the kidney and glomerulonephritis (34). Collectively, these studies suggest that there may be a novel Bcl-2-dependent pathway which regulates the size of the peripheral T cell pool in addition to the Fas/ Fas ligand pathway. This pathway becomes apparent when the Fas-mediated pathway is defective and may be important not only for the regulation of the peripheral T cell number but also for the prevention of tissue damage.
FIG. 5. Serum Ig and anti-dsDNA Ab levels. Sera were obtained from each mouse at the age indicated and the levels of total IgM, total IgG, and anti-dsDNA antibodies were determined by ELISA as described under Materials and Methods. The means / SEM of 3 to 11 samples are shown.
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TAMURA ET AL.
FIG. 6. Serum creatinine levels in ///, lpr/lpr, and Tg-lpr/lpr mice. The levels of serum creatinine in 6-month-old mice were determined as described under Materials and Methods. The results are expressed as means / SEM in each group.
ACKNOWLEDGMENTS We thank Dr. Michael P. Madaio for helping us with the methods and reagents of the anti-dsDNA assay and Drs. Akihiko Yano and Gail Massey for critical reading of the manuscript. We also appreciate Drs. Tetsuo Hamada and Shingo Ichimiya for their contribution. This work was supported by grants from the National Institute of Health (AI36303), the American Cancer Society (IM644), and Lucille P. Markey Charitable Trust.
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