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In Vivo Inhibition of NF-B in T-Lineage Cells Leads to a Dramatic Decrease in Cell Proliferation and Cytokine Production and to Increased Cell Apoptosis in Response to Mitogenic Stimuli, But Not to Abnormal Thymopoiesis¹

Valérie Ferreira^*, Nicolai Sidénius, Nadine Tarantino^*, Pascale Hubert^*, Lucienne Chatenoud, Francesco Blasi and Marie Körner^2,*

^* Laboratoire d’Immunologie Cellulaire et Tissulaire, Centre National de la Recherche Scientifique UMR 7627, Batiment Centre d’Etudes et de Recherches Virologiques et Immunologiques, Hôpital de la Pitié-Salpêtrière, Paris, France; Unit of Molecular Genetics, DIBIT, Hospital San Raffaele, Milan, Italy; and Institut National de la Santé et Research Médicale, Unit 25, Hôpital Necker Enfants Malades, Paris, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
To understand the role of NF-B complexes in T cell development and activation, we have generated transgenic mice in which RelA and c-Rel complexes were selectively inhibited in the T-lineage cells by specific expression of a trans-dominant form of IB. Transgene expression did not affect the thymic development, but led to lowered numbers of splenic T cells and to a dramatic decrease in the ex vivo proliferative response of splenic T lymphocytes. Analysis of IL-2 and IL-2R expression demonstrated that the perturbation of the proliferation response was not attributable to an abnormal expression of these genes. In contrast, expression of IL-4, IL-10, and IFN- was strongly inhibited in the transgenic T cells. The proliferative deficiency of the transgenic T cells was associated with an increased apoptosis. These results point out the involvement of NF-B/Rel family proteins in growth signaling pathways by either regulating proteins involved in the IL-2 signaling or by functionally interfering with the cell cycle progression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Prototypical NF-B (p50/RelA) is a ubiquitous eukaryotic transcription factor activated in T lymphocytes in response to TCR triggering or to cytokines such as TNF- and IL-1 (1, 2, 3). It is thought to play a critical role in the transcription of numerous genes involved in the immune response and inflammation (4). NF-B-responsive genes include genes coding for various regulatory cytokines, adhesion molecules, cell surface receptors, and immune modulators that are involved in T cell effector function (4). In thymocytes, NF-B was described to be activated in vivo in both mice and humans (2, 5). Immunohistochemical and immunofluorescence analyses of human thymocytes and thymic sections revealed a higher density of prototypical NF-B and c-Rel/p50 proteins in the medullary thymocytes, but the prototypical NF-B was also detected in nuclei of some cortical thymocytes (6). In mice, constitutively activated NF-B was found in thymocytes from both embryos and newborn pups, but not in adult thymocytes (7, 8). These results suggested a role for NF-B in the complex maturation process that takes place in the thymus.

The prototypical NF-B is a heterodimer composed of RelA (a 65-kDa protein) and p50 (a 50-kDa protein, processed product of p105). It belongs to the NF-B/Rel family of dimeric transcription factors, which also includes c-Rel, RelB, and p52 (processed product of p100). All members of the family share a highly conserved amino-terminal sequence called the Rel homology domain, which is critical for nuclear translocation, protein-protein interactions, and sequence-specific DNA binding (9). NF-B complexes are localized typically to the cytoplasm, where they bind to inhibitory cytoplasmic proteins called IBs (3, 10). Several members of the IB regulatory family have been characterized, including IB, IBß, IB, IB, the two NF-B protein precursors p105 and p100, and Bcl3 (11). In T cells, the major NF-B dimer that is activated during T cell activation is RelA/p50, but c-Rel/p50 is also present (4). Studies of IB-deficient mice demonstrated that the dominant IB regulator of NF-B/Rel in T cells is IB (12), the product of the MAD3 gene (13). During normal T cell activation, IB is rapidly degraded via the ubiquitin-proteasome pathway, permitting the nuclear import of NF-B (14, 15). The molecular mechanism resulting in IB proteolysis is complex and not completely elucidated. However, at least two post-translational covalent modifications have been reported to be essential for its degradation. The first post-translational event resulting from T cell activation is phosphorylation of two serines at positions 32 and 36 by a recently identified group of IB kinases (16, 17, 18, 19). This initial phosphorylation is a prerequisite for the second modification of IB, which is the ubiquitination of lysines 21 and 22 (20). The ubiquitination of IB results in its rapid degradation by the 26S proteasome (21). Substitution of the two serines 32 and 36 by alanine residues protects IB from ubiquitination and proteasome-mediated proteolysis (22). Consequently, if expressed in sufficient amounts, the double-mutated IB prevents NF-B activation in transiently or stably transfected cells (22, 23, 24).

Despite accumulating amounts of in vitro and in vivo data suggesting an important role for NF-B/Rel in immunity and inflammation, the full role of this family of proteins in vivo remained unclear. To better understand the function of these proteins, a series of gene knockout experiments was performed. Mice lacking p50 or c-Rel exhibited deficiencies of mature lymphocyte proliferation as well as increased susceptibility to certain pathogens, but T cell development remained normal (25, 26). Preliminary reports suggested that mice lacking p52 have a similar phenotype (27). Mice lacking RelB develop diffuse T lymphocyte-dependent inflammatory infiltrates and granulocytosis, and present a defect of dendritic cell development in lymphoid organs rather than an intrinsic defect of the T cell lineage (28, 29). Mice lacking RelA do not survive past the 15th day of gestation because of apoptosis of developing hepatocytes (30). Because of this, the development and functionality of T cells in the absence of RelA could not be examined directly. However, transplantation of RelA knockout embryonic stem cells into SCID mice resulted in normal T and B cell development, suggesting that RelA is dispensable for differentiation and maturation of lymphocytes (31). However, the potential functional redundancy among NF-B/Rel family members has to be considered, and this complicates the understanding of these gene disruption experiments (32, 33).

To circumvent the problem of functional redundancy of NF-B members in the T cell lineage and to further characterize the roles of the NF-B complexes in T cell development and T cell-dependent immune response, we (the present paper) and others (31, 34, 35) have generated transgenic mice in which RelA- and c-Rel-NF-B were selectively inhibited in the T-lineage cells. This was achieved by using a transgene representing a trans-dominant form of IB. While Ballard et al. used an N-terminal-deleted IB transgene, IB(N), we used a mutated IB, termed IB (32/36A), in which the two serines at positions 32 and 36 were substituted by alanines. The expression of the transgene was under lineage-specific control of the proximal lck promoter (36). This mutated IB inhibitor repressed constitutively the nuclear import and DNA binding of RelA- and c-Rel-NF-B complexes in stably transfected human cell lines as well as in transitory transfection experiments. In mice, our results demonstrated that IB (32/36A) constituted a powerful NF-B inhibitor in thymocytes and to a lesser extent in peripheral T cells by inhibiting both RelA- and c-Rel-NF-B activities. Several biological consequences of the IB (32/36A) transgene were similar to those observed with IB(N), such as 1) a decreased number of splenic T cells, especially the CD8⁺ subpopulation; 2) a dramatic decrease in the ex vivo proliferative response of T lymphocytes; and 3) increased apoptosis of T cells. In addition we show in this report the effect of IB (32/36A) on ex vivo production of a series of cytokines. While IL-4 and IL-10 cytokines were transcriptionally inhibited, the production of IL-2 and IL-2R remained normal in IB (32/36A) mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of transgenic mice expressing IB (32/36A)

Generation of human IB cDNA, substituted by alanines at positions 32 and 36 (IB (32/36A)), was described by Traenckner et al. (22) and was a gift from P. Baeuerle (Tularik, CA). The IB (32/36A) cDNA was cloned into the BamHI site of the p1017-lck plasmid containing the murine lck proximal promoter and the human growth hormone (hGH)³ minigene (37). Deletion of the termination codon from the IB (32/36A) cDNA resulted in a 3' end tag corresponding to 30 aa at the C-terminus of the resulting transgenic protein (see Fig. 1A). In vitro transcription and translation of the tagged transgene (TnT T7 quick coupled transcription/translation system, Promega) produced proteins presenting apparent m.w. of 58,000 and 45,000 in SDS-PAGE (data not shown). A 6.2-kb NotI fragment containing the lck promoter, the tagged IB (32/36A) cDNA, and hGH minigene from the plasmide p1017-IB (32/36A) was prepared by gel purification. The concentration of the purified linear DNA was adjusted to 3 µg/ml in injection buffer (0.25 mM EDTA and 5 mM Tris-HCl, pH 7.4) before microinjection. All techniques used in the generation of transgenic mice were performed essentially as described previously (38). Briefly, transgenic mice were generated by pronuclear microinjection of 0.5 days postcoitus embryos obtained from 6-wk-old superovulated C57B16xCBAF1 females mated with males of the same genotype. Injected eggs were implanted into pseudopregnant CD1 females. Three transgenic founder animals (5, 3, 23) were identified by PCR and confirmed by Southern blotting on genomic DNA obtained from small tail biopsies. All animals were obtained from Charles River Italy (Milan, Italy) and were kept under specific pathogen-free conditions. The restriction of IB (32/36A) transgene expression to the T cell lineage was confirmed by Western blotting using protein extracts from spleen, liver, brain, thymus, and purified T and B lymphocytes.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Expression of the IB (32/36A) transgene. A, The complete human IB cDNA (MAD3 cDNA) mutated at positions 32 and 36 was placed under the control of the mouse proximal lck promoter; hGH gene sequences were added to confer stability to the transcripts. Deletion of the termination codon of IB cDNA resulted in a 3' end tag corresponding to 30 aa at the C-terminus of the resulting transgenic protein. Generation of transgenic mice and Southern blot genotyping of tail DNA were performed as described in Materials and Methods. B, Equal amounts of cytoplasmic extracts (50 µg) prepared from transgenic (Tg) and control (NTg) thymocytes were analyzed by Western blotting using an mAb directed against the N-terminal domain of human IB. Positions of the wild type (WT IB) and transgenic (IB (32/36A)) IB proteins are indicated by arrows. C, Equal amounts of cytoplasmic extracts from transgenic thymocytes, splenic T cells, and B cells were analyzed by Western blotting. Only the major form of the transgenic IB is shown by an arrow. D, Thymocytes of transgenic (Tg) and control (NTg) mice were cultured for 1 h in the presence (+) or the absence (-) of PMA plus ionomycin. Ten micrograms of nuclear extracts were assayed for NF-B by EMSA (see Materials and Methods). The major inducible complex p50/RelA is indicated by an arrow.

Cells from thymus and spleen were prepared by crushing the organs in complete medium (RPMI 1640, 10% FCS, and standard concentrations of glutamine and antibiotics). After hypotonic lysis of erythrocytes, the splenic T cells were depleted of B cells by chromatography through nylon wool columns (39), followed by adsorption on pan-B Abs coupled to magnetic beads (B220, Dynal, Compiègne, France). The resulting population was systematically 95% T cells and 1% B cells as determined by flow cytometric analysis.

Western blotting and immunoprecipitation

Cytoplasmic extracts from thymocytes and splenocytes were prepared by hypotonic disruption of cells (5 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.1 mM PMSF, and 0.1 mM aprotinin). Proteins were resolved by 10% SDS-PAGE and immunoblotted on nylon membranes, and IB proteins were visualized using an mAb (MAD10B) specific for an N-terminal domain of IB (40), the HRP-coupled anti-mouse Ab, and the Amersham enhanced chemiluminescence visualization system kit (Arlington Heights, IL).

EMSAs

Nuclear proteins from either resting or 1-h PMA- plus ionomycin-treated thymocytes or purified splenic T cells were prepared as described previously (41). The DNA-protein reaction was performed as previously described (41) using 10 µg of nuclear protein extracts and ³²P 5' end-labeled oligonucleotide coding for the tandem B sequence from the HIV-long terminal repeat enhancer. The resulting protein-oligonucleotide complexes were resolved by 6% PAGE and visualized by autoradiography.

Cell proliferation assays

Purified splenic T cells (250,000 cells/well) were cultured in 96-well plates in triplicate with 250 ng/ml ionomycin plus 10 ng/ml PMA or with coated anti-CD3 145 2C11 Ab (0.5 µg/well) (42) alone or in the presence of anti-CD28 PV-I (6G) Ab (0.5 µg/well; provided by Dr. Carl June, National Cancer Institute, Bethesda, MD) for 48 or 72 h. Tritiated thymidine, (1 µCi) from ICN (Costa Mesa, CA) was added to the culture for an additional 12 h. Cultured cells were then harvested (Scatron, Tranby, Norway), and incorporated [³H]thymidine was analyzed by scintillation counting.

Quantitation of cytokine mRNA by RT-PCR

RNAs were prepared from purified splenic T cells before and after stimulation by ionomycin plus PMA or by coated 145 2C11 anti-CD3 Ab. Total RNA was isolated by the guanidine isothiocyanate method with minor modifications (43). T cells were examined for IL-2, IL-4, IL-10, IFN-, TNF-, and ß-actin transcripts by a semiquantitative reverse transcribed PCR. Commercial primer sets were provided by Stratagene (La Jolla, CA).

Quantitation of IL-2, IL-4, and IFN- by ELISA and CTLL-2 proliferation

Production of IL-2 by purified splenic T cells in response to mitogens was estimated using an IL-2-dependent T cell line, CTLL-2 (American Type Culture Collection, Manassas, VA). Purified splenic T cells were activated with mitogens (10 ng/ml PMA plus 250 ng/ml ionomycin or coated 145 2C11 anti-CD3 Ab alone (5 µg in 500 µl) or in association with CD28-specific PV-1 (6G) Ab (5 µg in 500 µl)) for 24–48 h. Supernatants from the T cells (10⁶ cells/ml) were harvested and tested for IL-2 by CTLL-2 proliferation as previously described (44). Production of IL-4 and IFN- was measured by ELISA using plates precoated with anti-mouse IL-4 and IFN- Abs according to the protocol described previously (44). IL-2, IL-4, and IFN- were quantified by comparison to standard proteins (Sigma). All tests were performed in triplicate. Production of IL-4 by EL4 cell clones was also monitored by ELISA.

Stable transfections of EL4 cells

The parental Th2 murine cell line, EL4 (American Type Culture Collection), was cultured in RPMI 1640 medium containing glutamine, antibiotics, 2-ME, and 10% FCS. Expression vector pCMV-IB (32/36A) was made by inserting MAD3 cDNA mutant (positions 32 and 36) into the XbaI/HindIII sites of the pcDNA3 vector (Invitrogen, San Diego, CA). Expression vector pCMV-IB (32/36A) tag was made by inserting the IB (32/36A)-tagged murine transgene into the HindIII site of the pcDNA3 vector. EL4 cells were transfected with pCMV-IB (32/36A), pCMV-IB (32/36A) tag, or the empty pcDNA3 vector by electroporation. G418-resistant cells were cloned by limiting dilution and were genotyped by Western blot using the mAb MAD 10B or by PCR using MAD3- and hGH-specific primers. The clones with stable integration of the IB (32/36A) transgene were grown in the presence of 1 mg/ml G418.

Luciferase (Luc) assays

The following vectors were used for B-dependent Luc assays. The 4p110-Luc and 4p575-Luc constructs (45), which contain 110 and 575 bp of the murine IL-4 promoter, were provided by Prof. W. Paul (National Institutes of Health, Bethesda, MD). Transient transfections of the EL4 cell clones with Luc vectors were performed by electroporation (Bio-Rad Gene Pulser, Bio-Rad, Richmond, CA) with 10 µg of plasmid DNA/5 x 10⁶ cells. Two hours after transfection, cells were split into two pools. One pool of cells was incubated in medium alone, and the other pool was incubated with 10 ng/ml PMA plus 250 ng/ml ionomycin for 24 h. Luc assays were performed using the Promega luciferase assay system (Madison, WI). Light emission was measured in a luminometer (Bio-Rad). The results were calculated as relative light units (light emission/background/milligrams of protein). Transfections experiments were repeated at least three times.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression and functionality of IB (32/36A) transgene

We have generated transgenic mice that integrated a tagged mutated form of IB under the control of the T cell lineage-specific lck proximal promoter and the hGH minigene (Fig. 1A). To check the expression of the transgene in T cells, we performed Western blot experiments using cytosolic protein extracts from thymocytes of three founder mice lines, Tg 3, Tg 23, and Tg 5 (Fig. 1B). The amounts of expressed transgenic IB in these animals correlated with integration copy number as determined by Southern blotting (data not shown). The mice from the Tg 5 line expressed the highest level of the transgenic IB protein (Fig. 1B, lane 5), whereas the Tg 3 mice produced the lowest amount of the transgene (lane 2). The major form of the transgene migrated as a 58-kDa protein. The minor form of the transgene appeared as a 45-kDa protein and was detectable mostly in the Tg 5 line. Both the 58- and 45-kDa forms were obtained by in vitro transcription/translation assay (not shown), indicating that they do not result from a recombination or splicing of the transgene in vivo. Specific Abs raised against the 30-aa tag also recognized both 58- and 45-kDa forms (not shown). Finally, immunoprecipitation of thymocyte protein extracts with an Ab specific for the carboxyl-terminal portion of the IB followed by Western blotting using an mAb specific for the amino-terminal region of IB revealed that both 58- and 45-kDa forms are composed of IB (not shown). These results suggest that the tagged mutated IB cDNA was expressed as a protein presenting two conformations. The preferential conformation in denaturing conditions had a m.w. of 58,000, but a minor form of 45,000 was also detected.

Consistent with the in vitro IB gene regulation studies (24, 46, 47), we observed that in the most highly expressing transgenic thymocytes (Tg 5) the level of the endogenous form of IB was reduced compared with that in the control mice thymocytes (Fig. 1B, lanes 4 and 5).

Since the lck promoter was initially described as being mostly active in thymocytes, we sought expression of the transgenic IB in peripheral T cells purified from spleens. Western blotting of the cytosolic extracts from purified T and B cell splenic populations clearly indicated that the transgene was also produced in splenic cells, but exclusively in the T cell lineage (Fig. 1C). Consistent with the in vivo transgenesis studies using lck proximal promoter (37), we observed that the transgene is less expressed in T cells than in thymocytes. This decrease in IB (32/36A) protein in T cells was correlated with an increment in endogenous IB expression (Fig. 1C, lanes 1 and 2). In addition, comparison between purified CD4⁺ and CD8⁺ T cells revealed that both populations expressed comparable levels of the transgenic IB (not shown).

To verify the functionality of the transgene, we estimated the nuclear translocation capacity of the RelA- and c-Rel-NF-B complexes in response to mitogenic activation by EMSA. A 1-h treatment with PMA plus ionomycin of isolated thymocytes from control and transgenic mice revealed that NF-B activation was inhibited in the three transgenic lines (Fig. 1D). The efficiency of NF-B inhibition was dose dependent on the IB (32/36A) expression, since in the low transgene producer (Tg 3) NF-B activity was only reduced, whereas in the two other lines (5 and 23), which produce higher amounts of the transgene, no mitogen-induced NF-B activity was detected. In splenic T lymphocytes, we observed that the inhibition of NF-B translocation was less potent compared with that in corresponding thymocytes (not shown). This result correlated with the amount of expression of the IB transgene (Fig. 1C) and indicated that in peripheral T lymphocytes, the amount of the transgenic IB was not sufficient for complete NF-B inhibition. Supershift experiments identified the residual NF-B activity as being RelA-p50 dimer (not shown). This approach did not find c-Rel in splenic T cells from control and IB (32/36A) mice. Thus, we cannot exclude that small amounts of c-Rel activity remain in the transgenic T cells.

The inhibition of NF-B activity was due to a direct interaction of the 58-kDa transgene with NF-B proteins, as revealed by immunoprecipitation assays of proteins extracted from transgenic thymocytes with RelA- and c-Rel-specific Abs (not shown).

Together, these results demonstrated that the transgenic IB, in its major 58-kDa conformation, efficiently inhibited NF-B activation in thymocytes by interacting with both RelA and c-Rel NF-B complexes and induced a partial inhibition of NF-B in peripheral T cells.

Biological effects of the IB (32/36A) transgene

IB (32/36A) transgenic mice lived and reproduced normally in the pathogen-free conditions. The thymic cellularities in adult animals were normal. The CD4/CD8-positive cell ratio and TCR, CD62L, CD45RB, and CD44 expressions appeared normal, confirming the results reported for IB(N) (data not shown) (34). These results indicated that despite efficient NF-B inhibition, T cell intrathymic proliferation and differentiation occurred normally in IB (32/36A) transgenic animals, similarly to that in IB(N) transgenic animals, and contradicted our initial previsions.

In contrast, analysis of splenocyte subsets confirmed an alteration of the B/T cell ratio (increased in the Tg animals) and of the CD4/CD8 T cell ratio (the numbers of CD8⁺ T lymphocytes were reduced relative to those of CD4⁺ cells) similar to that in IB(N) mice (not shown) (34). This was not due to a differential expression of the transgene in the CD4⁺ and CD8⁺ populations, since Western blot analysis indicated that the transgene was produced with comparable efficiencies in both T cell populations.

IB (32/36A) inhibits mitogen-induced T cell proliferation

To analyze whether the T cells expressing the IB (32/36A) transgene are able to respond normally to mitogenic signals, we realized ex vivo proliferation assays with T cells purified from control and transgenic littermate spleens and from thymocytes. A 48-h treatment of T cells with PMA plus ionomycin evidenced a strong decrease in the proliferative response in both line 3 and line 5 animals compared with that in their respective control littermates (not shown). The proliferative failure of splenic T cells was noticeably stronger in the 5 line. At 72 h of cell culture, the proliferative defect was even stronger in the 5 line, whereas it was decreased in the 3 line, suggesting that the amount of residual NF-B activity detected in the Tg 3 line in EMSA experiments was sufficient to rescue the proliferation failure. In contrast, thymocytes from the transgenic and control animals proliferated in a comparable manner (not shown). To check whether addition of IL-2 or of stimulants that result in stabilization of IL-2 could rescue the proliferative response in the 5 line T cells, we performed cell activation with immobilized CD3-specific Abs in the presence or the absence of exogenous IL-2 or with a combination of immobilized CD28-specific Abs. The results of one characteristic experiment are shown in Fig. 2. Clearly, neither CD28 costimulus nor exogenous IL-2 was able to rescue the proliferative response of splenic T cells from the Tg 5 mice, indicating that the lack of proliferation was not due to the lack of IL-2 production. This result confirmed the findings of Boothby et al. (34). Addition of other mitogenic cytokines, such as IL-7 and IL-4, also did not rescue the proliferative response (not shown).

View larger version (42K): [in this window] [in a new window]   FIGURE 2. Proliferation of IB (32/36A) splenic T cells. Splenic T cells from control (NTg) and line 5 transgenic mice (Tg5) were treated for 48 and 72 h with medium alone (black bars); with immobilized Abs specific for CD3 (open bars), for CD3 and for CD28 (dashed bars), or for CD3 plus IL-2 (pale gray bars); or with a combination of PMA and ionomycin (gray bars) at the concentrations indicated in Materials and Methods. Proliferation was measured by [3H]thymidine incorporation. The results are shown as the arithmetic mean of triplicate determinations for three control and three transgenic mice.

IB (32/36A) transgenic T cells produce normal amounts of IL-2 and IL-2R

IL-2 acts as the principal auto- and paracrine T cell growth factor. In vivo and in vitro studies have suggested a central role for NF-B/Rel proteins in the regulation of both IL-2 and the inducible IL-2R (24, 48, 49, 50). IL-2 and IL-2R are normally produced by T lymphocytes in response to activation signals such as TCR triggering or phorbol ester treatment in association with calcium influx inducers. In IB(N) mice IL-2 production was reduced consequent to NF-B inhibition (34).

To measure the capacity of the mitogen-activated line 5 and control T splenocytes to secrete IL-2 we collected their supernatants and tested their capacity to activate proliferation of the CTLL-2 cell line that proliferates in a dose-response manner to IL-2. As shown in Fig. 3, no significant differences in IL-2 production were observed between transgenic and nontransgenic T cells, indicating that IL-2 was secreted normally by IB (32/36A) transgenic mice. This result further indicated that the proliferative failure of the transgenic T cells was not due to the lack of IL-2 secretion and confirmed the results of Doi et al. (31), who showed that RelA-/- T cells produce IL-2, but present a reduced mitogenic response.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. IL-2 production by IB (32/36A) T cells. IL-2 production by splenic T cells from control mice (NTg; dashed bars) and transgenic mice (Tg5; black bars) was measured by a CTLL-2 proliferation assay. T cells were cultured in triplicate for 48 h in the presence of plate-bound anti-CD3 Abs alone or in combination with anti-CD28 Abs (see Materials and Methods). Supernatants from these cell cultures were assayed for IL-2 by CTLL-2 proliferation and were quantitated by comparison to CTLL-2 proliferation in medium containing known doses of IL-2 protein. Results represent the mean value of three separate experiments.

IB (32/36A) Tg T cells fail to produce IL-4, IL-10, and IFN-

To investigate the effect of NF-B inhibition on the induction of other cytokines, we measured the secretion of IFN-, which was shown to contain an intronic binding site for c-Rel (51) and the secretion of which by the Th1 subset of T cells parallels that of IL-2. A typical result obtained by ELISA demonstrated that IFN- production by Tg T cells was 10 times less than that by controls (Fig. 4A). This result was further confirmed by RT-PCR analysis (Fig. 4C). The RT-PCR measurement further confirmed that IL-2 induction was normal in Tg mice (Fig. 4C). Measurement of TNF- production by RT-PCR show no significant difference in mRNA levels between Tg and control mice, although this gene was previously described as possessing B sites in its promoter domains (52). Curiously, IL-4 and IL-10 production was inhibited as shown by RT-PCR and ELISA of IL-4 (Fig. 4, B and C). To determine whether the absence of IL-4 production was due to a down-regulation of the Th2 cell population or to a direct effect on IL-4 promoter, we analyzed IL-4 production and IL-4 promoter activation in EL4 cells (a murine Th2-type cell line). Stable transfection of EL4 cells with pcDNA3-IB (32/36A) vectors (tagged and untagged) resulted in the inhibition of IL-4 secretion (Fig. 5A). Transcription of the luciferase reporter gene driven by two murine IL-4 promoter constructs was also significantly inhibited in IB (32/36A) clones compared with that in the control clone, especially the construct containing sequences upstream from the P1 domain (Fig. 5B). These results suggest that NF-B is required for efficient transcription of the IL-4 gene. Since the CD44^high T cell population was normally detected in IB (32/36A) Tg mice, our results suggest that the lack of IL-4 production by these mice is due to a deficient activation of the IL-4 gene rather than to an underdevelopment of the Th2 subpopulation.

View larger version (40K): [in this window] [in a new window]   FIGURE 4. Cytokine production by IB (32/36A) T cells. IFN- (A) and IL-4 (B) production by splenic T cells from control mice (NTg; dashed bars) and transgenic mice (Tg5; black bars). T cells were cultured in triplicate for 48 h in the presence of plate-bound anti-CD3 alone or in combination with anti-CD28 Abs. Supernatants from these cultures were assayed by ELISA and were quantified by comparison to standard IFN- or IL-4 proteins. Results represent the mean value of three separate experiments. C, Analysis of cytokine mRNA productions in T cells from control mice (NTg) and transgenic mice (Tg 5) was performed by RT-PCR. RNA was isolated from T cells cultured for 4 h (IL-2, IL-4, IL-10, and IFN-) or 12 h (TNF-) with medium alone (1), anti-CD3 Ab (2), or PMA plus ionomycin (3). The RNA was analyzed by RT-PCR using primers specific for various genes, as indicated at the left. The ß-actin gene constituted the internal control.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. IL-4 production by IB (32/36A) EL4 cell line. A, Production of IL-4 protein after 48 h of PMA plus ionomycin stimulation was measured by ELISA in supernatants from control EL4 cells (EL4), EL4 IB (32/36A) tag cell clone (B8), and EL4 IB (32/36A) cell clones (13, 19) as described in Materials and Methods. The experiments were performed three times in triplicate. SDs represent <10% of the mean value. B, Cells were transfected with the IL-4 promoter Luc constructs 4p110-Luc or 4p575-Luc (see Materials and Methods). Luc activity was determined 24 h after PMA plus ionomycin treatment of control cells (EL4) and EL4 IB (32/36A) cell clones (13, 19). The results are expressed as the fold increase in Luc relative units in activated cells relative to control level activities. The relative Luc activities were calculated from six independent experiments. SDs are shown with vertical bars.

Enhanced apoptosis of IB (32/36A) Tg T cells

NF-B/Rel proteins have been implicated in inhibition (53) and stimulation (53, 54, 55) of cell susceptibility to apoptosis. For example, RelA-deficient mice die from apoptosis of embryonic hepatocytes (30). We investigated whether the splenic T cell lymphopenia and the lack of proliferative response in ex vivo experiments might be due to an enhanced apoptotic susceptibility of the IB (32/36A) transgenic T cells. Cell death was measured by the use of the TUNEL assay (56). Compared with controls, we observed a 40% increase in the spontaneous apoptosis of line 5 T cells, and a 3 times higher apoptosis after 48 h of PMA plus ionomycin treatment (not shown). Thus, the inhibition of NF-B in the T cells led to an increased apoptosis in both activation and resting conditions and might explain in part the strong inhibition of the proliferative response of Tg T cells as well as the hypoplasticity of splenic T cells in vivo.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In an attempt to elucidate the functions of NF-B/Rel factors in the T cell lineage, we have developed a transgenic mouse model in which a dominant-negative mutated form of IB was overexpressed in thymocytes and in T cells from secondary lymphoid tissues. This T cell-targeted inhibition of NF-B has clear advantages and inconveniences over gene disruption experiments, since it allowed inhibition of two NF-B complexes (c-Rel- and RelA-p50) without inducing lethality. On the other hand, we found that, at least in mice over the age of 6 wk, the transgenic IB was incompletely inhibiting NF-B activities in the peripheral T cells. The efficiency of the transgenic IB was related to its expression level, and consequently, it was less potent in peripheral T cells than in thymocytes. Despite these differences in NF-B inhibition, the biological effects of the transgene were detectable only in mature T cells. Indeed, our observations basically confirmed those of Boothby et al. (34) and Doi et al. (31) that thymocyte cellularities and intrathymic differentiation are little, if at all, affected by inhibition of NF-B within T cells. In contrast to these results, Esslinger et al. reported a significant reduction in thymocyte cellularity as well as a spectacular deficiency of the mature single-positive thymocytes in mice transgenic for human IB (35). Further investigations are necessary to explain these differences in thymus phenotypes. Despite only a partial NF-B inhibition in splenic T cells, mature T cell subsets were further compromised. In situ splenic T cell lymphopenia and alteration of the CD4/CD8 ratio were observed, similar to the findings of Boothby et al. In animals transgenic for IBß, a strong T cell lymphopenia was also obtained (57). Together, these results indicate that normal NF-B activation is required for T cell homeostasis.

Ex vivo proliferation studies showed that IB (32/36A) transgenic T cells failed to proliferate in response to mitogenic stimuli, and that in contrast to T cells from c-Rel-deficient mice, addition of IL-2 to incubation medium did not restore proliferation despite normal expression of CD25 (25). Similar results were previously reported for IB(N) T cells (34), except that the IB(N) T cells failed to produce IL-2, while IB (32/36A) T cells produced normal amounts of this growth factor. This difference could be due to the fact that in IB (32/36A) T lymphocytes, NF-B activation was only partially inhibited. In every case, these results indicated that the defect in T cell proliferation and homeostasis is not attributable to a deficiency of IL-2 or its inducible receptor, but, rather, to a defect in IL-2 signal transduction. Thus, NF-B is involved in the control of molecular events downstream of IL-2R, but the mechanism and the potential gene targets of NF-B that are involved in IL-2 growth signaling remain to be identified.

Our results also confirmed that NF-B is critical for apoptotic protection of T cells (34). Fas ligand and antiapoptotic gene products such as Bcl-2 and Bcl-x have been reported to participate in the regulation of T cell apoptosis and should be examined (58, 59, 60, 61, 62, 63).

IL-2 and CD25 are produced in T cells during the early stage of the G₁ phase of the cell cycle (64). Since the transgenic T cells produced IL-2 and CD25 normally, our observations indicated that partial inhibition of NF-B activities did not block the initial G₀-G₁ transition. However, [³H]thymidine incorporation was strongly inhibited (as was bromodeoxyuridine incorporation, not shown), indicating that full NF-B activation is required for T cell cycle G₁/S progression. Although little is known about NF-B involvement in cell cycle regulation, several reports demonstrated that NF-B activity can be regulated by factors involved in the cell cycle progression. For example, the interaction of NF-B and CDKs through the p300 and CREB-binding protein coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression (65, 66). NF-B could also be involved in the activity or transcription of proteins controlling directly the G₁/S transition. Indeed, Bash et al. reported that c-Rel expression leads to cell cycle arrest in G₁ by changing the activation and/or expression of E2F, Cdk2, p53, and p21^waf1 proteins in HeLa cells (67). IL-2-induced T cell proliferation was shown to be mediated by down-regulation of the cyclin-CDK inhibitor p27 (68). Thus, one might speculate that NF-B is involved in the G₁/S phase transition in T cells by either transcriptionally controlling or functionally interacting with the cell cycle molecular machinery.

Although normal production of IL-2 by IB (32/36A) cells indicated that the low amounts of NF-B activity in these cells were sufficient to induce some of the NF-B gene targets, the important biological consequences of partial NF-B inhibition (lack of proliferation, apoptosis) indicated that several genes were down-regulated. Among those genes, Th1 subset cytokines such as IFN-, and Th2 subset cytokines such as IL-4 and IL-10 were transcriptionally inhibited. The down-regulation of IL-4 by inhibition of NF-B activity was mimicked in vitro by stable transfection of EL-4 cells by IB (32/36A), indicating that the reduction of Th2 cytokine expression resulted from a direct effect on the transcription of this gene and not on a lack of the Th2 cell subtype. IL-4 down-regulation was surprising, since the murine IL-4 promoter is not known to possess high affinity B sites. The human IL-4 promoter was reported to be repressed by RelA-p50 through the P1 sequence, but this did not seem to be the case for the murine promoter (69). These results indicated that murine and human IL-4 genes are not regulated identically by NF-B factors. Our results obtained both in vitro (EL4 cells) and in vivo indicated that even partial inhibition of NF-B activities in peripheral T cells leads to down-regulation of the murine IL-4 gene, suggesting that NF-B is required for positive regulation of the IL-4 gene in mice. Furthermore, an increased NF-B activity obtained in NF-B2^-/- mice led to increased IL-4 and IL-10 production (70). Further studies are necessary to explain the mechanism of IL-4 gene regulation by NF-B. IL-10 possesses several B sites far upstream of its transcriptional start, but its transcriptional regulation by NF-B has never been reported (71). A previous study of TNF- gene regulation demonstrated that multimers of TNF- B sites could confer inducibility on a heterologous promoter in a B, but not in a T, cell line (72). Our data further demonstrated that NF-B is not required for TNF- induction in T lymphocytes. Finally, another gene known to be critically dependent on NF-B activation, namely IB, was down-regulated in IB (32/36A) T cells, at least in the mice line producing the highest amounts of the transgene. Besides IL-2, other genes known to possess B sites in their promoter region were not down-regulated in IB (32/36A) T cells. Thus, the attenuated nuclear import of RelA and c-Rel dimers in splenic T cells seems to differentially affect transcription of different target genes.

These results highlight our ignorance of the real mechanism of in vivo regulation of transcription through particular B sites in individual genes and suggest that many genes with B sites could be either NF-B independent or dose dependent. In addition, many genes could be indirectly regulated by the NF-B/Rel family by transcriptional control of other genes specific for transcription factors.

Although many other biological consequences of NF-B inhibition in T cells remain to be investigated, the results presented here demonstrated the critical role of NF-B transcription factors in normal T cell proliferation, homeostasis, and production of cytokines.

	Acknowledgments

 
We thank Catherine Amarger for helping with the graphical processing of the data. We are indebted to Dr. Leo Lee for providing B oligonucleotides, to Dr. Carl June for the anti CD28 PV-I (6Ga) Ab, to Dr. William Paul for IL-4 constructs, and to Dr. Ron Hay for IB-specific mAb. We thank Stéphanie Gaillard and Christophe Parizot for technical assistance. We especially thank Prof. Patrice Debré for constant encouragement.

	Footnotes

 
¹ This work was supported by European Community Grants BiotechII BIO₂-CT92-0130 and Biomed II BMH-CT97-2453 and by a grant from the Agence Nationale de Recherche sur le Sida.

² Address correspondence and reprint requests to Dr. M. Körner, Laboratoire d’Immunologie Cellulaire et Tissulaire, Centre National de la Recherche, UMR 7627, Bâtiment Centre d’Etudes et de Recherches Virologiques et Immunologiques, Hôpital de la Pitié-Salpêtrière, 83 blvd. de l’Hôpital, 75013 Paris, France. E-mail address:

³ Abbreviations used in this paper: hGH, human growth hormone; Luc, luciferase; CDK, cyclin-dependent kinase.

Received for publication August 20, 1998. Accepted for publication March 17, 1999.

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