IL-2-induced tumor necrosis factor (TNF)-ß expression: further analysis in the IL-2 knockout model, and comparison with TNF-{alpha}, lymphotoxin-ß, TNFR1 and TNFR2 modulation

Jayagopala Reddy, Patricia Chastagner, Laurence Fiette1, Xinyuan Liu2 and Jacques Thèze

Unité d'Immunogénétique Cellulaire and
1 Unité d'Histopathologie, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
2 Shanghai Institute of Biochemistry, 320 Yue Yang Road, Shanghai 200031, China

Correspondence to: Correspondence to: J. Thèze


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-2 induces the stimulation of inflammatory and immune reactions, and the apoptosis of antigen-activated cells. However, the molecular basis of these pleiotropic functions is largely unknown. We have previously reported that IL-2 induces genes involved in cytoskeleton organization, oncogene regulation and transcriptional control. In an IL-2-dependent cell line, we have also shown that IL-2 induces tumor necrosis factor (TNF)-ß mRNA through the Jak–STAT pathway. Here, we first demonstrate in vitro that IL-2 induces mature and partially spliced TNF-ß mRNA in the splenocytes and lymph node cells of both IL-2–/– and IL-2+/– mice. Under the same experimental conditions, IL-2 is seen to induce TNF-{alpha} mRNA. mRNA expression is followed by semiquantitative RT-PCR and this analysis is then extended in vivo by studying different lymphoid organs from IL-2–/–animals. Strikingly, the expression of TNF-ß mRNA is noted to be extremely low in the spleens and lymph nodes of IL-2–/– mice. Similarly, TNF-{alpha}, lymphotoxin (LT)-ß, TNFR1 and TNFR2 mRNA levels are also low in the spleens of IL-2–/– animals, whereas IFN-{gamma} and IL-4 mRNA levels remain unaffected in these animals. The experimental values are significantly different (P <= 0.05) from those of control IL-2+/– animals. Western blot analysis of TNF-{alpha} expression confirmed and extended the results at the protein level. For the first time, we demonstrate that IL-2 directly or indirectly regulates genes of the TNF–TNFR family in secondary lymphoid organs. Furthermore, IL-2–/– animals in which thymopoiesis is unaffected show normal expression of these genes. Altogether, our data further define the pleiotropic effects of IL-2 at the molecular level.

Keywords: cytokines, cytokine receptors, knockout


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-2 is a pleiotropic cytokine predominantly produced by CD4+ T lymphocytes (1). It exerts its effects on various cell types such as T, B, NK cells and monocytes by binding to its receptors (24). IL-2 has numerous functions including survival, activation, growth and differentiation of immune cells. In addition, IL-2 has also been implicated in programmed cell death (5). To understand the molecular basis of the pleiotropic functions of IL-2, we previously characterized IL-2-inducible genes using a cDNA subtraction approach involving a cell line grown in IL-2 or IL-4. Of these genes, those coding for cytoskeleton proteins ({alpha}-tubulin, ß catenin), oncogene-regulating proteins [CTCF, Jun inhibition factor (JIF)-1] and transcriptional factors (E2F4, CREB, ZhX-1) were further studied in vivo using IL-2–/–animals (6). These genes are underexpressed in the spleens and lymph nodes of IL-2–/– mice, suggesting that they may play a role in determining the phenotype of IL-2–/– animals by influencing gene expression, oncogene regulation and cellular adhesion. As an example, abnormal lymphocyte activation and proliferation in IL-2–/– mice is followed by lymphocyte hyperplasia characterized by splenomegaly, lymphadenopathy and colitis depending on age, genetic background and breeding conditions (710). While it has been suggested that the above phenotype may occur due to a defect in the FAS-mediated death pathway (10,11) or a lack of CD4+CD25+ T cells (12), we proposed that faulty expression of multiple IL-2-inducible genes leads to the complex phenotype found in IL-2–/– mice (6). Hence it is essential to verify in vivo the role played by IL-2 in the induction of genes characterized as IL-2-inducible in vitro (13).

Recently, we used a suppression–subtraction hybridization approach to demonstrate that IL-2 also induces tumor necrosis factor (TNF)-ß in an IL-2-dependent CTLL-2 cell line via the Jak–STAT signal transduction pathway (14). TNF and TNFR belong to a large family of molecules of which TNF-{alpha}, TNF-ß [also designated as lymphotoxin (LT)-{alpha}], LT-ß, TNFR1 and TNFR2 have been extensively studied (15). As well as regulating cell proliferation and apoptosis (16,17), the TNF–TNFR system also plays an important role in the control of lymphoid organogenesis (18,19). Furthermore, TNF-{alpha} and TNF-ß induce MHC class I and class II antigens (20,21) as well as chemokines, and play a critical role in cell–cell interactions and lymphocyte trafficking (22,23). TNFs are also known to be involved in the defense against pathogens and in the induction of inflammatory and autoimmune diseases (15,24). TNF-ß enhances cytotoxic responses against normal and transformed cell types (2527). TNF would therefore appear to be essential in the regulation and maintenance of immune system homeostasis under normal and pathological conditions.

The effect of IL-2 on TNF-ß induction in the CTLL-2 cell line suggests that IL-2 may have a critical impact on the expression of the TNF family of molecules and therefore may be an important link in the cytokine network. This hypothesis was tested at different levels. The positive effect of IL-2 on TNF-ß mRNA expression was first confirmed in vitro using polyclonal lymphocytes from the lymph nodes and spleens of IL-2–/– and IL-2+/– mice. Our analysis was then extended in vivo to TNF-ß mRNA expression in the lymph nodes, spleens and thymuses of IL-2–/– animals. IL-2+/– animals were used as controls during this analysis. The expression of TNF-ß mRNA was found to be substantially reduced in the lymph nodes and spleens of IL-2–/– mice. By contrast, TNF-ß was expressed normally in the thymus of IL-2–/– mice. The expression of TNF-{alpha} was studied at the mRNA and protein levels. The expression of LT-ß, TNFR1 and TNFR2 mRNA was also analyzed both in vitro and in vivo. The results suggest that IL-2 also plays an important role in the in vivo regulation of these molecules. Altogether, the data obtained indicate that TNF-ß and molecules of the TNF and TNFR family follow the same pattern of expression we previously described for IL-2R{alpha}, ß-catenin, CTCF, JIF-1, E2F4, CREB, ZhX1 and nucleolin (6), and are highly susceptible to the effects of IL-2 in the secondary lymphoid organs, but not in the thymus. This provides an additional insight into the mechanisms explaining the pleiotropic effects of IL-2.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
The IL-2–/– and IL-2+/– mice have already been described (7). They were bred in the animal facilities of the Pasteur Institute and were genotyped by PCR (7). The mice were housed under conventional conditions and animals ~3 months old were used for the study.

Preparation of cells and flow cytometric analysis
Splenocytes were prepared from whole spleens after treatment with ammonium chloride to remove erythrocytes. Lymph node and thymus cells were used as single-cell suspensions.

Lymphocyte subsets were characterized by suspending 5x105 cells in 0.1 ml PBS (0.5% FCS and 0.02% sodium azide) then staining with FITC-labeled mAb for 30 min on ice and washing before analysis. When indicated the activation of the different cell subsets was measured by adding phycoerythrin (PE)-conjugated anti-CD69 mAb during the incubation period. Flow cytometry was performed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).

The following mAb were prepared in the Pasteur Institute, Department of Immunology: FITC-conjugated anti-CD3 mAb (clone 2 C113.4), FITC-conjugated anti-CD4 mAb (clone GK1.5), FITC-conjugated anti-CD8 mAb [clone ({alpha})53.6.72], FITC-conjugated anti-B220 mAb (clone Ra3B2), FITC-conjugated anti-NK1.1 mAb (clone PK136) and FITC-conjugated anti-Mac-1 mAb (clone M1/70). PE-labeled anti-CD69 mAb (clone H1.2F3) was obtained from PharMingen-Clinisciences (Montrouge, France).

Cell culture
Single-cell suspensions were prepared from spleens and from a pool of mesenteric, inguinal and popliteal lymph nodes taken from IL-2–/– and IL-2+/– mice. Briefly, the tissues were homogenized in HBSS containing 10 mM HEPES and 10% FCS. The cells were washed twice and then cultured in complete medium at a density of 1x106 cells/ml in 75 cm2 flasks. The complete medium consisted of RPMI 1640 medium, 5% FCS, 2 mM glutamine, 10 mM HEPES, 2 µM 2-mercaptoethanol, penicillin (1000 U/ml), streptomycin (1 mg/ml) and fungizone (250 ng/ml) with or without IL-2 (5 nM). An aliquot was taken from both the IL-2-stimulated and unstimulated spleen cell cultures after 0, 0.5, 6, 12 and 24 h, and after 0, 0.5 and 6 h from the lymph node cells. RNA extraction was followed by measurement of mRNA expression for TNF-ß, TNF-{alpha}, LT-ß, TNFR1 and TNFR2.

RNA extraction, DNase I treatment and cDNA synthesis
Total RNA was extracted from the spleens, lymph nodes and thymuses of both IL-2–/– and IL-2+/– mice using RNA PLUS reagent (Quantum Bioprobe, Montreuil, France). RNA (1 µg) from each sample was exposed to amplification grade DNase I (Gibco/BRL, Cergy Pontoise, France). The first-strand cDNA was synthesized from the DNase-treated RNA using oligo (dT)12–18 (25 ng/µl) by incubating the mixture at 70°C for 10 min followed by quick chilling on ice. The following were then added according to the manufacturer's instructions: 5xfirst-strand buffer, 0.1 M DTT, dNTP mix, Superscript II reverse transcriptase (Gibco/BRL) and RNAsin (2 U/µl) (Promega, Madison, WI) to a final volume of 20 µl. The mixture was sequentially incubated at 42°C for 60 min, 95°C for 8 min and cooled to 4°C in a Thermal Cycler (Perkin-Elmer Applied Biosystems, Norwalk, CT).

PCR
cDNA mixture (2 µl) was used for PCR amplifications using gene-specific primers (Table 1Go). Since the primers used for TNF-ß, TNF-{alpha}, LT-ß, IFN-{gamma} and IL-4 spanned the introns, the cDNA-derived PCR products could be distinguished from the genomic DNA-amplified fragments by their size. All the reactions were carried out at a primer concentration of 0.12 µM in 25 µl containing 25 mM TAPS–HCl (pH 9.3), 50 mM KCl, 2 mM MgCl2, 1 mM 2-mercaptoethanol, 10 mM each of dATP, dTTP, dGTP and dCTP, and 1.25 U cloned Taq DNA polymerase (Amersham Life Science, Cleveland, OH). The reactions were performed using a Thermal Cycler (Perkin Elmer Applied Biosystems) as previously described (6).


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Table 1. Primers used for PCR and Southern hybridization
 
After initial denaturation (94°C/3 min) the cycling for each gene was conducted as follows (indicated in the order of denaturation, annealing and extension): TNF-ß: 94°C/30 s, 53°C/30 s, 72°C/1 min; TNF-{alpha}: 94°C/1.5 min, 62°C/2 min and 72°C/2 min (28); LT-ß: 95°C/1 min, 52°C/1 min and 72°C/2 min (29); TNFR1: 94°C/1.5 min, 55°C/2 min and 72°C/2 min (30); TNFR2: 94°C/1 min, 62°C/1 min and 72°C/2 min (31); IFN-{gamma} and IL-4: 94°C/1 min, 60°C/1 min and 72°C/2 min (32); and ß-actin: 94°C/30 s, 59°C/20 s and 72°C/20 s. In addition, a final 7 min extension at 72°C was included at the end of 25 cycles for all the genes. The PCR products were resolved on 2.0% agarose gel electrophoresis, stained with ethidium bromide and photographed on 667 polaroid film using a UV transilluminator.

Southern hybridization
The PCR products were transferred onto Hybond-N+ membranes (Amersham Life Science) and hybridized with gene-specific probes (Table 1Go). The hybridization temperatures used were 58°C (TNF-ß and IL-4), 52°C (TNF-{alpha} and LT-ß), 50°C (IFN-{gamma}), 56°C (TNFR1 and TNFR2) and 68°C (ß-actin). For TNF-ß, TNF-{alpha}, LT-ß, IL-4, IFN-{gamma}, TNFR1 and TNFR2, the membranes were hybridized overnight using 20 pM of each of the corresponding [32P]dATP-radiolabeled oligonucleotides. [32P]dCTP-labeled cDNA fragments were used for the detection of ß-actin. The membranes were washed twice with 5xSSC and 0.1% SDS for all the genes except ß-actin for which 2xSSC and 0.1% SDS were used. The hybridized membranes were then exposed to Kodak storage phosphorus screens.

The radioactive signals corresponding to cytokine mRNA (e.g. TNF-ß) and ß-actin mRNA were measured using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). A semiquantitative analysis of cytokine mRNA expression was obtained by dividing the radioactive signals for cytokine mRNA by the radioactive signals for ß-actin mRNA and reporting the result as a ratio, i.e. TNF-ß/ß-actin (6,33). The effect of IL-2 in in vitro studies was then determined as the ratio between IL-2-stimulated and unstimulated ratios for a given cytokine, e.g. IL-2 effect for TNF-ß = TNF-ß/ß-actin ratio in IL-2-stimulated cells/TNF-ß/ß-actin ratio in unstimulated cells.

Western blot analysis
Lysates were prepared from spleens, lymph nodes and thymuses of IL-2–/– and IL-2+/– mice by addition of sample buffer (125 mmol/l Tris–HCI, pH 6.8, 2% SDS, 10% ß-mercaptoethanol and 0.01% bromophenol blue). The protein samples (50 µg) were subjected to electrophoresis under reducing conditions on a 10% SDS–PAGE and transferred onto Immobilon P nitrocellulose membranes (Millipore, Bedford, MA). Filters were blocked for 2 h at room temperature with blocking buffer (7% BSA, 0.1% Tween 20 and 1xPBS) and subsequently incubated for 1 h at room temperature in blocking buffer containing the anti-TNF-{alpha} mAb (clone TN3-19.12; Genzime, Cergy St Christophe, France). Membranes were washed and incubated for 1.5 h at room temperature in blocking buffer containing 1:1000 peroxidase-labeled goat anti-hamster IgG (Biosys, Compiègne, France) and extensively washed. An ECL Western blotting detection kit (Amersham, Little Chalfont, UK) was used according to the manufacturer's instructions and the filters were autoradiographed for a few seconds.

TNF-{alpha} protein expression levels was quantified by densitometry. ß-Actin on the same blots was measured using mAb (clone C-2, IgG1; Santa Cruz Biotechnology, Le Perray, France). Bands corresponding to the amount of TNF-{alpha} or ß-actin were measured using NIH Image software (National Institutes of Health, Bethesda, MD). The TNF-{alpha} signal was first normalized to that of ß-actin and the TNF-{alpha}/ß-actin ratio calculated. The IL-2 effect was calculated by dividing the TNF-{alpha}/ß-actin ratio obtained from IL-2–/– and IL-2+/– mice by the TNF-{alpha}/ß-actin ratio of IL-2–/– mice.

Statistics
In vitro experiments were performed to study the IL-2-induced expression of TNF–TNFR mRNA in spleen and lymph node cells cultured in the absence or the presence of IL-2. A representative experiment is shown for each analysis. Groups of five to nine mice were chosen for the in vivo studies. The ratios between TNF-ß, TNF-{alpha}, LT-ß, IFN-{gamma}, IL-4, TNFR1 and TNFR2 mRNA expression and ß-actin mRNA expression were analyzed statistically as described previously (6). Student's t-test was used to determine the significance of the results (P < 0.05 was considered significant). The mean ± SD of the ratios is shown for each group of animals.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of lymphocyte subsets in IL-2-deficient mice
Disruption of the IL-2 gene has no major effect on either thymocyte production or peripheral mature T cell seeding (7). Before comparing the expression of molecules in the TNF–TNFR family by IL-2–/– and IL-2+/– mice we verified the distribution of the various lymphocyte subsets among the lymph nodes, spleens and thymuses of these two mouse strains. Table 2Go shows the results obtained for the lymph nodes, with five animals being analyzed. Percentages of CD3, CD4 and CD8 lymphocytes were comparable between the IL-2–/– and IL-2+/– animals. Contrary to previously published results (34) we did not detect any difference in the percentage of B220+ lymphocytes expressed by the IL-2–/– and IL-2+/– mouse strains. This discrepancy might be explained by the difference in the genetic background of the animals used in the two studies. In the same manner as for the lymph nodes, the distribution of the different lymphocyte subsets in the spleens of IL-2–/– and IL-2+/– animals did not show any significant difference (data not shown). As far as the thymus is concerned, we confirmed our previous results indicating that all the major thymocyte subpopulations are expressed in IL-2–/– mice (13).


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Table 2. Expression of lymphocyte subsets in lymph nodes from IL-2-deficient mice
 
The activation status of the different lymphocyte subsets found in the lymph nodes taken from IL-2–/– and IL-2+/– mice was also analyzed. Table 2Go shows the results obtained by measuring the expression of the early activation marker CD69. All subsets from the lymph nodes of IL-2–/– mice were activated. A low but significant proportion of lymphocytes from IL-2+/– mice were also expressing CD69. Comparable results were obtained in spleen samples from IL-2–/– animals (data not known). Altogether, our data confirm over-activation of the different lymphocyte subsets in the absence of IL-2. Under these conditions, the activated lymphocytes should produce more cytokines unless IL-2 is required for their production.

In vitro expression of TNF-ß, TNF-{alpha} and LT-ß mRNA by splenocytes from IL-2–/–and IL-2+/– mice stimulated with IL-2
IL-2 induced the expression of TNF-ß mRNA in splenocytes from both IL-2–/– and IL-2+/– mice (Fig. 1Go). Expression of TNF-ß showed two mRNA species (485 and 709 bp) corresponding to mature and a partially spliced (PS) mRNA respectively (Fig. 1AGo). The PS mRNA, containing intron 3, was distinguished from possible genomic DNA-amplified products by RNase and DNase treatments. The existence of PS TNF-ß mRNA exported into the cytoplasm as a mature message is unique (35). The biological role of this transcript is unknown. When stimulating splenocytes in vitro, IL-2 up-regulated TNF-ß mRNA expression in both IL-2–/– and IL-2+/– mice as early as 30 min post-stimulation (Fig. 1A and BGo). Both mature and PS TNF-ß mRNA followed the same time course response, peaking at 6 h post-stimulation (Fig. 1BGo). Of the two transcripts, mature mRNA levels remained consistently higher than those of PS TNF-ß mRNA. The effect of IL-2 on mature and PS TNF-ß mRNA calculated as described in Methods increased gradually, and peaked at 24 h in both IL-2–/– and IL-2+/– splenocytes (Fig. 1BGo).



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Fig. 1. In vitro effect of IL-2 on the expression of TNF-ß, TNF-{alpha} and LT-ß mRNA in splenocytes from IL-2–/– and IL-2+/– mice. (A) Splenocytes from IL-2–/– and IL-2+/– mice were cultured with or without IL-2 for 0–24 h. The time-course response for TNF-ß (mature mRNA, 485 bp; PS mRNA, 709 bp) and ß-actin mRNA was examined after 0, 0.5, 6, 12 and 24 h by RT-PCR. Amplified products were transferred onto Hybond-N+ membranes and hybridized with TNF-ß- and ß-actin-specific probes. The radioactive signals were recorded for TNF-ß and ß-actin mRNA using a PhosphorImager. (B) The time-course response for TNF-ß, TNF-{alpha} and LT-ß mRNA in both IL-2-stimulated and unstimulated cells was semiquantitatively analyzed by normalizing to ß-actin mRNA. TNF-ß/ß-actin, TNF-{alpha}/ß actin and LT-ß/ß actin ratios are reported. The IL-2 effect was then determined as a ratio between IL-2-stimulated and unstimulated ratios.

 
The expression of TNF-{alpha} and LT-ß mRNA was analyzed under similar experimental conditions (Fig. 1BGo). Time-course responses showed that TNF-{alpha} mRNA was up-regulated by IL-2 to peak 6 h after IL-2 stimulation in both IL-2–/– and IL-2+/– splenocytes. The effect of IL-2 on TNF-{alpha} mRNA expression reached a maximum at 24 h (Fig. 1BGo). When considering LT-ß mRNA, the response of splenocytes from IL-2–/– mice followed a similar pattern in the absence and the presence of IL-2. The response of splenocytes from IL-2+/– animals was decreased in unstimulated and IL-2-stimulated cultures (Fig. 1BGo). This suggests that IL-2 does not significantly effect the expression of LT-ß mRNA and that the overexpression noted in IL-2+/– splenocytes was not found in vitro, even in the presence of IL-2.

In vitro expression of TNF-ß, TNF-{alpha} and LT-ß mRNA by lymph node cells from IL-2–/– and IL-2+/– mice stimulated with IL-2
The expression of TNF-ß, TNF-{alpha} and LT-ß mRNA was studied 0, 0.5 and 6 h post-stimulation in lymph node cells from IL-2–/– and IL-2+/– mice cultured in the absence and the presence of IL-2. Since the induction of TNF-ß and TNF-{alpha} mRNA peaked after 6 h in the splenocytes, we chose this same time point for our studies in cultured lymph node cells.

IL-2 up-regulated TNF-ß mRNA expression in IL-2-stimulated lymph node cells. Marked induction of TNF-ß was noted after 6 h, particularly in cells from IL-2–/– animals (Fig. 2A and BGo). Since IL-2 also induced TNF-ß in lymph node cultures from IL-2+/– animals, a positive IL-2 effect was therefore observed in IL-2-stimulated cultures from both strains. As already seen in splenocytes, mature TNF-ß mRNA levels in lymph node cells were always more elevated than PS TNF-ß mRNA. When considering TNF-{alpha}, its mRNA expression was found to be up-regulated by IL-2 after 6 h of stimulation and the effect of IL-2 was more pronounced in IL-2-stimulated IL-2+/– than in IL-2–/– lymph node cells (Fig. 2BGo). As already seen in splenocytes, LT-ß mRNA levels did not show a response pattern suggestive of IL-2 induction (Fig. 2BGo).



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Fig. 2. In vitro effect of IL-2 on the expression of TNF-ß, TNF-{alpha} and LT-ß mRNA in lymph node cells from IL-2–/– and IL-2+/– mice. (A) Lymph node cells from IL-2–/– and IL-2+/– mice were cultured with or without IL-2 for 0 to 6 h. The time-course response for TNF-ß (mature mRNA, 485 bp; PS mRNA, 709 bp) and ß-actin mRNA was examined after 0, 0.5 and 6 h by RT-PCR. Amplified products were transferred onto Hybond-N+ membranes and hybridized with TNF-ß- and ß-actin-specific probes. The radioactive signals were recorded for TNF-ß and ß-actin mRNA using a PhosphorImager. (B) The time-course response for TNF-ß, TNF-{alpha} and LT-ß mRNA in both IL-2-stimulated and unstimulated cells was semiquantitatively analyzed by normalizing to ß-actin mRNA. TNF-ß/ß actin, TNF-{alpha}/ß actin and LT-ß/ß actin ratios are reported. The IL-2 effect was then determined as a ratio between IL-2-stimulated and unstimulated ratios.

 
In vivo expression of TNF-ß mRNA in the spleens, lymph nodes and thymuses of IL-2–/– and IL-2+/– mice
Our in vitro experiments showed that polyclonal lymphocytes from the spleens or lymph nodes of IL-2–/– or IL-2+/– mice maintained a comparable capacity to induce TNF-ß mRNA synthesis after IL-2 stimulation. Our analysis was then extended to TNF-ß mRNA expression in vivo by studying spleens, lymph nodes and thymuses from both IL-2–/– and IL-2+/– mice. TNF-ß mRNA expression both in vivo and in vitro revealed mature and PS TNF-ß mRNA (Fig. 3AGo). As shown in Fig. 3Go(B), mature TNF-ß mRNA levels were far lower in the spleens of IL-2–/– than in the spleens of IL-2+/– mice (P = 0.00003). Mature TNF-ß mRNA was also expressed to a lesser extent in lymph nodes from IL-2–/– mice when compared to IL-2+/– mice (P = 0.038). By contrast, the thymus did not show any difference in the expression of mature TNF-ß mRNA between the two genotypes (Fig. 3BGo).



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Fig. 3. In vivo expression of TNF-ß mRNA in the spleens, lymph nodes and thymuses of IL-2–/– and IL-2+/– mice. (A) Total RNA was extracted from the spleens, lymph nodes and thymuses of IL-2–/– and IL-2+/– mice. The expression of TNF-ß mRNA (mature mRNA, 485 bp; PS mRNA, 709 bp) was examined by RT-PCR. Amplified products were transferred onto Hybond-N+ membranes and hybridized with a TNF-ß-specific probe. The radioactive signals were recorded for TNF-ß and ß-actin mRNA in spleens from IL-2–/– and IL-2+/– mice using a PhosphorImager. (B) mRNA expression for TNF-ß was semiquantitatively analyzed in spleens, lymph nodes and thymuses by normalizing to ß-actin mRNA. TNF-ß/ß-actin ratios are reported. Mean ± SD and P values are indicated.

 
PS TNF-ß mRNA was also studied in the spleens, lymph nodes and thymuses of IL-2–/– and IL-2+/– mice (Fig. 3A and BGo). Its expression may have been affected by the in vivo activation of lymphocytes from IL-2–/– mice. However, regardless of the organ or genotype studied, expression levels of PS TNF-ß mRNA were always lower than the levels of mature TNF-ß mRNA. The expression of PS TNF-ß mRNA was found to be reduced in the spleens and lymph nodes of IL-2–/– mice. However, the underexpression of PS TNF-ß mRNA in IL-2–/– mice was less significant than for mature TNF-ß mRNA. It may therefore be concluded that the in vivo expression of PS TNF-ß mRNA is less affected by IL-2 than that of mature TNF-ß mRNA.

Expression of TNF-{alpha}, LT-ß, IFN-{gamma} and IL-4 mRNA in spleens from IL-2–/– and IL-2+/– mice
mRNA expression for TNF-{alpha}, LT-ß, IL-4 and IFN-{gamma} was studied in vivo in the spleens of IL-2–/– and IL-2+/– mice (Fig. 4Go). TNF-{alpha} and LT-ß belong to the TNF family and were included as a comparison with TNF-ß expression. TNF-{alpha} mRNA was diminished in spleens, while LT-ß mRNA expression was very low in IL-2–/– (P = 0.077 and P = 0.027 respectively) compared to IL-2+/– animals. IFN-{gamma} and IL-4, the classical markers of Th1 and Th2 differentiation, were included as cytokine controls (36). We anticipated that IFN-{gamma} mRNA expression would be underexpressed in IL-2–/– mice since this cytokine has been reported to be induced by IL-2 in a murine lymphocyte cell line (CTLL) (37). By contrast, IL-4 levels were expected to be increased since overproduction of IgG1 and IgE isotypes has been observed in IL-2–/– mice (7). However, IFN-{gamma} mRNA expression was not significantly altered in IL-2 knockout animals (Fig. 4Go). Likewise, the expression of IL-4 mRNA did not differ significantly between IL-2–/– and IL-2+/– animals. Hence, IFN-{gamma} and IL-4 could be used as in vivo controls for IL-2-modulated genes such as TNF-ß, TNF-{alpha} and LT-ß.



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Fig. 4. Expression of TNF-{alpha}, LT-ß, IFN-{gamma} and IL-4 mRNA in the spleens of IL-2–/– and IL-2+/– mice. Total RNA was extracted from the spleens of IL-2–/– and IL-2+/– mice. mRNA expression for TNF-{alpha}, LT-ß, IFN-{gamma}, IL-4 and ß-actin was measured by semiquantitative RT-PCR. Amplified products were transferred onto Hybond-N+ membranes and hybridized with TNF-{alpha}, LT-ß, IFN-{gamma}, IL-4 and ß-actin-specific probes. The radioactive signals were recorded using a PhosphorImager. mRNA expression was analyzed by normalizing to ß-actin mRNA. TNF-{alpha}/ß-actin, LT-ß/ß-actin, IFN-{gamma}/ß-actin and IL-4/ß-actin ratios are reported. Mean ± SD and P values are indicated.

 
Thymuses from IL-2–/– and IL-2+/– mice were also studied for TNF-{alpha}, LT-ß and IL-4 mRNA expression. No significant differences were found between the two strains, suggesting that the thymic expression of genes of the TNF family, such as TNF-ß, TNF-{alpha} and LT-ß, is not affected by IL-2 (data not shown).

In vitro and in vivo expression of TNFR1 and TNFR2 mRNA
Both TNF-ß and TNF-{alpha} homotrimers use TNFR1 and TNFR2 (15,38,39). To test whether IL-2 has any impact on TNFR expression, mRNA for TNFR1 and TNFR2 was evaluated in spleen and lymph node cells from either IL-2–/– or IL-2+/– animals, cultured in the absence or the presence of IL-2 (Fig. 5Go). IL-2 did not have an obvious effect on TNFR1 and TNFR2 mRNA expression in splenocytes since their expression was also increased in unstimulated cultures (Fig. 5AGo). The IL-2 effect measured during time-course experiments did not show any consistent increase, thus suggesting that TNFR1 and TNFR2 expression in vitro is unaffected by IL-2 (Fig. 5AGo). This was confirmed by studying lymph node cell cultures. Time-course responses to IL-2 were followed and the IL-2 effect calculated. The results obtained showed that IL-2 had little effect on TNFR1 and TNFR2 mRNA expression in these cultures, regardless of the genetic origin of the cells (IL-2–/– or IL-2+/–) (Fig. 5BGo).



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Fig. 5. In vitro effect of IL-2 on the expression of TNFR1 and TNFR2 mRNA in spleen and lymph node cells from IL-2–/– and IL-2+/– mice. (A) Splenocytes from IL-2–/– and IL-2+/– mice were cultured with or without IL-2. The time-course response for TNFR1 and TNFR2 was examined after 0, 0.5, 6, 12 and 24 h by RT-PCR. Amplified products were transferred onto Hybond-N+ membranes, and hybridized with TNFR1, TNFR2 and ß-actin-specific probes. The radioactive signals were recorded using a PhosphorImager. mRNA expression was semiquantitatively analyzed by normalizing to ß-actin mRNA. TNFR1/ß-actin and TNFR2/ß-actin ratios are reported. The effect of IL-2 was then determined as a ratio between IL-2-stimulated and unstimulated ratios. (B) Lymph node cells from IL-2–/– and IL-2+/– mice were cultured with or without IL-2. TNFR1 and TNFR2 mRNA expression was followed over 6 h. The effect of IL-2 on TNFR1 and TNFR2 expression was determined as in (A) and as discussed in Methods.

 
Since IL-2 had little effect on TNFR expression in IL-2-stimulated spleen or lymph node cell cultures, we anticipated that its expression would remain unaltered in different lymphoid organs from IL-2–/– mice. Hence, we verified the expression of TNFR1 and TNFR2 in lymph nodes, spleens and thymuses from IL-2–/– mice. The results were compared with those obtained using IL-2+/– mice. Contrary to our expectations, TNFR1 and TNFR2 mRNA expression was significantly reduced in spleens from IL-2–/– as compared with IL-2+/– mice (P = 0.0004 and P = 0.016 respectively) (Fig. 6Go). However, and in agreement with the results obtained with TNF-ß, TNF-{alpha} and LT-ß, the thymic expression of TNFR1 and TNFR2 mRNA was comparable in IL-2–/– and IL-2+/– animals (Fig. 6Go). Therefore, it may be concluded that IL-2 has an effect on the in vivo expression of TNFR, but that the pattern of regulation is complex since it mainly affects the expression of these two receptors in the spleen.



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Fig. 6. Expression of TNFR1 and TNFR2 mRNA in the spleens, lymph nodes and thymuses of IL-2–/– and IL-2+/– mice. Total RNA was extracted from the spleens, lymph nodes and thymuses of IL-2–/– and IL-2+/– mice, and mRNA expression for TNFR1, TNFR2 and ß-actin was examined by semiquantitative RT-PCR. Amplified products were transferred onto Hybond-N+ membranes and hybridized with TNFR1-, TNFR2- and ß-actin-specific probes. The radioactive signals were recorded using a PhosphorImager. mRNA expression for TNFR1 and TNFR2 was analyzed by normalizing to ß-actin signal. TNFR1/ß-actin and TNFR2/ß-actin ratios are reported. Mean ± SD and P values are indicated.

 
Expression of TNF-{alpha} in spleens, lymph nodes and thymuses taken from IL-2–/– and IL-2+/– mice
IL-2 modulation of mRNA from molecules of the TNF–TNFR family was studied by semi-quantitative PCR. To obtain more extensive results at the protein level we analyzed TNF-{alpha} expression using the Western blot technique since a reliable and well-characterized mAb against murine TNF-{alpha} was available (40). By contrast, the specificity and reliability of other immunological reagents against TNF-ß, LT-ß and TNFR remain questionable.

Figure 7Go shows the results obtained with hamster anti-murine TNF-{alpha} mAb (clone TN3-19.12). It has recently been recognized that this reagent reacts with both the immature (26 kDa) precursor and the mature (17 kDa) form of TNF-{alpha} (40). Studies on cell extracts from spleen, lymph node and thymus showed that mAb TN3-19.12 recognized only the immature form of TNF-{alpha} (Fig. 7AGo). Under these experimental conditions it was noted that TNF-{alpha} is clearly less expressed in the spleen and lymph nodes of IL-2–/– animals than in the same organs of IL-2+/– animals (Fig. 7BGo). Thymuses from both genotypes expressed similar amounts of TNF-{alpha}. These results therefore extend to a protein level the results previously seen by semi-quantitative PCR.



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Fig. 7. Expression of TNF-{alpha} in the spleens, lymph nodes and thymuses of IL-2–/– and IL-2+/– mice. Protein lysates were prepared from the spleens, lymph nodes and thymuses of IL-2–/– and IL-2+/– mice (three animals for each genotype). TNF-{alpha} protein and ß-actin expression were sequentially examined by Western blot. After electrophoretic migration the products were transferred to membranes and revealed by the corresponding mAb followed by staining with the specific peroxidase-labeled polyclonal antibodies. The signals obtained (ECL) were quantified using NIH Image software. The TNF-{alpha} signal was normalized to the ß-actin signal and the IL-2 effect was calculated as described in Methods.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
To progressively unravel the pleiotropic functions of IL-2, we began by conducting a study of IL-2-induced genes characterized using cDNA subtraction or suppression– subtraction hybridization approaches (6,14). In the present paper, we extend our previous studies concerning the regulation of IL-2-induced genes coding for proteins involved in cytoskeleton organization, oncogene regulation and transcriptional control to the TNF–TNFR family. Our recent observation concerning IL-2-regulation of TNF-ß expression was made using an IL-2-dependent cell line. Here, these results were first confirmed in vitro after culturing polyclonal lymphocytes in the presence or the absence of IL-2. Furthermore, and more importantly, we reported for the first time that TNF-ß mRNA is regulated by IL-2 in vivo. Similarly, we observed that TNF-{alpha}, LT-ß, TNFR1 and TNFR2 mRNA are underexpressed in IL-2–/– animals. Western blot analysis of TNF-{alpha} expression confirmed and extended these results at the protein level.

IL-2 induces TNF-ß mRNA in splenocytes and lymph node cells from IL-2–/– and IL-2+/– animals (Figs 1 and 2GoGo). These results support our previous observation that IL-2 induced TNF-ß in an IL-2-responsive cell line (14). Under the same experimental conditions, IL-2 also up-regulated TNF-{alpha}, although to a lesser extent (Figs 1 and 2GoGo). This result is comparable to that obtained after stimulating murine T cell lines by anti-CD3 or IL-2 (37,41). By contrast, IL-2 has no effect on LT-ß expression in vitro. Similarly, the effect of IL-2 on TNFR1 and TNFR2 expression in vitro was slight and probably not significant (Fig. 5Go). It has already been reported that IL-2 induces TNFR expression in normal human lymphocytes after 5–9 days of culture (42). Therefore, independent of species differences, long-term culturing may be required for IL-2-enhanced expression of TNFR in vitro.

The expression of TNF-ß in spleens, lymph nodes and thymuses of IL-2–/– mice was analyzed to test the hypothesis that TNF-ß could be underexpressed in vivo. As shown in Fig. 3Go, both mature and PS TNF-ß mRNA were present at extremely low levels in spleens from IL-2–/– mice compared to IL-2+/– mice (P = 0.00003). This was also the case in lymph nodes, but with a narrower difference (P = 0.038). It is worthy of note that our data obtained by analyzing lymphoid organs of naive animals in the absence of any known stimulation. As reported in Table 2Go, lymphocytes from IL-2–/– animals were overactivated compared to cells of IL-2+/– mice. Despite this strong activation, lymphocytes from IL-2–/– mice produced less TNF-ß mRNA than cells from IL-2+/– mice. This underlines the specific role of IL-2 in the modulation of the TNF-ß gene. Interestingly, the underexpression was noted solely in peripheral lymphoid organs, i.e. spleen and lymph nodes, but not in thymus (Fig. 3Go). We previously reported that IL-2R{alpha}, ß-catenin, CTCF, JIF-1 and nucleolin mRNA, while expressed normally in thymus, were found to be underexpressed in the secondary lymphoid organs of IL-2–/– mice (6,13). This indicates that IL-2-inducible genes, including TNF-ß, may not have any major impact on thymus differentiation which otherwise is normal in IL-2–/– mice. These results also suggest that IL-2-inducible genes in thymus may be regulated by other molecules. The possible involvement of IL-15 has already been excluded (13). It is worthy of note that lymphocyte hyperplasia occurs in peripheral lymphoid organs but not in the thymus of IL-2–/– mice. The underexpression of IL-2-induced genes and peripheral lymphocyte proliferation are therefore correlated, and this further supports the hypothesis that the families of genes we have characterized contribute toward the phenotype of IL-2–/– mice.

Although TNF-{alpha} and LT-ß belong to the same family as TNF-ß (15), the regulation of their expression is different. Comparisons of in vitro and in vivo findings suggest that IL-2 induces TNF-{alpha} mRNA and we have previously demonstrated that IL-2-induced expression of TNF-ß mRNA involves the Jak–STAT pathway of IL-2R signaling (14). It therefore remains to be determined whether this pathway in also involved in TNF-{alpha} expression. In our studies, LT-ß mRNA was not induced in vitro, yet its expression was diminished in spleens from IL-2–/– mice (Fig. 4Go). This indicates that LT-ß expression is affected by IL-2 in vivo but that the corresponding regulation pathway is not functioning in vitro. Similarly, the effect of IL-2 on TNFR was marginal in vitro and their expression was low in spleens from IL-2–/– mice, thus indicating that IL-2 may also control TNFR expression in vivo (Fig. 6Go). The fact that TNF-ß, TNF-{alpha} and LT-ß expression is diminished in vivo may contribute indirectly to the reduced expression of TNFR (43,44). We show as controls that the expression of IFN-{gamma} and IL-4 did not differ significantly between IL-2–/– and IL-2+/– mice (Fig. 4Go), implying that their expression is IL-2 independent. Furthermore, our results indicate that the lymphocyte activation found in IL-2–/– mice, as measured by activation markers such as CD69 and CD71 (13), does not systematically lead to cytokine overproduction.

It has been reported that TNF-ß-deficient mice show defects in lymphoid organogenesis (18,19). Since we observed that TNF-ß was extremely low in spleens from IL-2–/– mice, we anticipated that both TNF-ß–/– and IL-2–/– phenotypes would show common pathological features. IL-2–/– mice develop splenomegaly and polyadenomegaly due to the accumulation of blast-like T cells in spleen white pulp and in the paracortex of hypertrophied lymph nodes. The structure of these two secondary lymphoid organs is nevertheless maintained (8,9,45,46). By contrast, no such lesions have been encountered in TNF-ß–/– mice nor in mice deficient for receptors interacting with TNF-ß or TNF-{alpha} (18,19,47,48). TNF-ß–/– mice lack lymph nodes and Peyer's patches, and their spleens show a loss of B and T cell zone compartmentalization, reduced white pulp with no B cell follicles and no germinal centers (18,19,49,50). Since no apparent correlation could be found between IL-2–/– and TNF-ß–/– pathological features, this implies that low amounts of TNF-ß may be sufficient for the lymphoid organogenesis of secondary lymphoid organs in IL-2–/– mice. Alternatively, TNF-ß-related molecules may compensate for the decreased expression of TNF-ß in IL-2–/– mice. However, we did notice that IL-2–/– mice show perivascular mononuclear cell infiltration in several non-lymphoid organs (colon, rectum, liver, esophagus, kidney, pancreas, salivary glands and lungs) (personal observations) (8,9,45,46,51). Such a feature has also been reported in TNF-ß–/– (19) and LT-ß-R–/– mice (52). Therefore, it may be speculated that the underexpression of TNF-ß in IL-2–/– could play a role in the perivascular infiltration observed in this model.

IL-2–/– mice develop lymphocyte hyperplasia resulting from uncontrolled proliferation of lymphocytes in secondary lymphoid organs (810). Three possible mechanisms have been put forward to explain this phenomenon. (i) At the molecular level, it has been shown that IL-2 suppresses FLICE inhibitory protein (FLIP), the negative regulator of FAS and TNF-mediated death pathways. The absence of IL-2 results in increased levels of FLIP (11,53). In addition, lymphocytes from IL-2–/– mice show increased resistance to FAS-mediated cell death (10). Hence it is suggested that the cell accumulation may be the consequence of FLIP overexpression followed by a defect in FAS-mediated apoptosis. (ii) At the cellular level, a lack of CD4+CD25+ T cells has been implicated in the occurrence of colitis in IL-2–/– mice (12). CD4+CD25+ T cells have been proposed as `professional suppressor cells' and can suppress polyclonal T cell activation by inhibiting IL-2 production. They can also act by antigen-presenting cell-independent mechanisms in an antigen non-specific manner (54,55). They are believed to exert their suppressive function via the production of IL-10 (12). Their absence in IL-2–/– mice might also explain the uncontrolled lymphocyte activation. (iii) From our data, we suggest that the IL-2–/– phenotype is under multigenic control. We have previously identified new IL-2-inducible genes coding for cytoskeleton proteins ({alpha}-tubulin, ß catenin), oncogene-regulating proteins (CTCF, JIF-1), transcriptional factors (E2F4, CREB, ZhX-1) and IL-2R{alpha} (6,13). {alpha} and ß catenin provide a link between T cell surface-expressed cadherins and actin cytoskeleton filaments (6,56,57). The fact that these genes are underexpressed in IL-2–/– mice may lead to faulty adhesion and the release of lymphocytes from normal regulatory control by cell–cell contact. In addition, the underexpression of CTCF and JIF-1, which suppress c-myc and c-jun respectively, may provide an explanation for the lymphocyte proliferation observed in IL-2–/– animals. The low levels of transcriptional factors (E2F4, CREB, ZhX-1) may also induce faulty expression of genes critical to the control of lymphocyte physiology. Hence, we propose that regulatory dysfunction of multiple IL-2-inducible genes is responsible for lymphoproliferation in IL-2–/– mice (6).

In the context of our multigenic hypothesis, the role played by TNF-ß, TNF-{alpha} and LT-ß underexpression in vivo in IL-2–/– mice may also be important since TNF-ß exerts a potent antiproliferative effect on murine fibroblasts (58). TNF-ß and TNF-{alpha} are cytotoxic for normal T blasts, suggesting that TNF may be essential for down-modulation of antigen-driven T cell responses in vivo (26). TNF may therefore play a critical role in the equilibrium between cell proliferation and cell death, and in maintaining immunological homeostasis. Since IL-2–/– mice have a defect in the Fas-mediated death pathway and FLIP inhibits the death pathways mediated by both Fas–Fas ligand and TNF–TNFR (10,11,53), we verified that lymphocytes from IL-2–/– mice remain sensitive to TNF killing in vitro despite reduced expression of TNFR1 and TNFR2. Under the same experimental conditions, lymph node blast cells from IL-2–/– and IL-2+/– mice were killed by TNF-{alpha} (unpublished observations). Therefore, our results suggest that insufficient amounts of TNF may at least partially contribute to the mechanism leading to uncontrolled proliferation of lymphocytes in IL-2–/–animals. Fas ligand, which is also under the control of IL-2, may play a similar role (11).

We have used different cell lines and various technical approaches to characterize IL-2-induced genes belonging to different families. Our studies have shed light on the complex network of regulatory functions that are directly or indirectly controlled by IL-2. Furthermore, the pattern noted for the expression of these genes is always the same: they are underexpressed in the lymph nodes and spleens of IL-2–/– mice, whereas they are normally expressed in the thymus. The secondary lymphoid organs are the main sites of the abnormal lymphoproliferation found in IL-2 knockout animals. Therefore, the underexpression of IL-2-induced genes, including members of the TNF family, is correlated with the main immune disorders found in IL-2–/– animals. This further supports the notion that the pleiotropic effects of IL-2 are under multigenic control.


    Acknowledgments
 
Dr J. M. Cavaillon is gratefully acknowledged for his valuable advice during the course of this work. We thank M. Jones for his help in editing the English manuscript. We also thank C. Vanderbergh, F. Bourgade and X. Montagutelli for their assistance in breeding the animals. This work was supported by grants from the Institut Pasteur.


    Abbreviations
 
CREB cAMP responsive element-binding protein
CTCF CCCTC binding factor
FLIP FLICE inhibitory protein
JIF-1 Jun inhibitor factor-1
LT lymphotoxin
PE phycoerythrin
PS partially spliced
STAT signal transducers and activators of transcription
TNF tumor necrosis factor

    Notes
 
Transmitting editor: G. Doria

Received 16 June 2000, accepted 14 October 2000.


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 Introduction
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 Discussion
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