NF-κB/Rel Participation in the Lymphokine-Dependent Proliferation of T Lymphoid Cells

Mice

IκBα(ΔN) Tg mice, in which expression of a trans-dominant inhibitor of NF-κB/Rel transcription factors is targeted specifically to the T lineage using the proximal lck promoter and cointegration of a CD2 locus control region, have been described previously (21). In selected experiments, these mice were crossed with mice in which the lck proximal promoter alone leads to T cell-specific expression of a transgene encoding a chimeric cytokine receptor in which the mouse IL-2Rβ extracellular and transmembrane domains are fused to a mouse IL-4Rα cytoplasmic tail (26). All mice were maintained in specific pathogen-free conditions using microisolator cages and were used at 6–8 wk of age in accordance with the federal and state government regulations after institutional approval.

Cell preparations

Single cell suspensions were prepared from thymus, spleen, and lymph nodes by crushing the organs in complete media (RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, and 0.1% penicillin-streptomycin), followed by hypotonic lysis of erythrocytes. Splenic and lymph node T lymphocytes were depleted of B cells by chromatography through nylon wool columns, as described previously (21). Briefly, single cell suspensions from pooled spleen and lymph nodes were loaded onto nylon wool columns preequlibrated with RPMI 1640 supplemented with 5% FBS at 37°C. After 45 min at 37°C, the nonadherent cells were eluted from the columns. The resultant population was <10% B220⁺ and 75–90% T cells, as determined by flow cytometry.

Proliferation assays

Thymocyte suspensions were counted and plated (5 × 10⁴ cells per 100 μl of media) in microtiter wells. Triplicate samples were cultured for 48 h at 37°C in the presence of PMA (50 ng/ml), ionomycin (1 μg/ml), IL-2 (10 ng/ml), IL-4 (10 ng/ml), IL-12 (10 ng/ml), PMA and IL-4, PMA and IL-2, PMA and IL-12, or PMA and ionomycin, as indicated. Tritiated thymidine (1 μCi in 100 μl of media) was added to each well for the final 8 h before determination of radioisotope incorporation into DNA. T lymphocyte preparations from spleen and lymph node, depleted of B cells as described above, were plated in microtiter wells previously incubated overnight with PBS or anti-CD3 mAb (10 μg/ml, clone 2C11; PharMingen), as indicated. Triplicate samples were then cultured (48 h at 37°C) in the presence of IL-2, IL-4, an activating mAb against CD28 (10 μg/ml, clone 37.51; PharMingen, San Diego, CA), anti-CD28 and IL-2, or anti-CD28 and IL-4, or as for thymocytes, as indicated; DNA synthesis was then quantified as above.

Gel mobility shift analyses

Total cell extracts were prepared from single cell suspensions of thymocytes or T cells using high-salt extraction in the presence of protease inhibitors, as previously described (21, 22). These extracts were then used for gel mobility shift assays of NF-κB/Rel proteins (21). The probe used was a double-stranded ³²P-labeled oligonucleotide modified from a κB enhancer sequence in the IL-2Rα promoter (κB-pd) (5′-CAACGGCAGGGGAATTCCCCTCTCCTT-3′). DNA-binding reaction mixtures (20 μl) contained 5 μg of nuclear extract, 2 μg double-stranded poly(dI-dC), and 10 μg BSA buffered in 20 mM HEPES (pH 7.9), 5% glycerol, 1 mM EDTA, 1% Nonidet P-40, and 5 mM DTT. Nucleoprotein complexes were then resolved on a native 5% polyacrylamide gel and visualized by autoradiography.

For detection of Stat5- and Stat6-binding activities, thymocytes or B cell-depleted T lymphocytes were cultured overnight at 37°C in the presence of PMA and ionomycin, or plate-bound anti-CD3 (10 μg/ml) and anti-CD28 (2.5 μg/ml), respectively. These cells were then rinsed once with RPMI medium without serum, cultured 1 h at 37°C in serum-free media, treated for 30 min with IL-2, IL-4, or medium alone, and then lysed in 0.5% Nonidet P-40, 50 mM Tris-Cl (pH 8), 0.1 mM EDTA, 150 mM NaCl, 100 mM Na₃VO₄, 50 mM NaF, 1 mM DTT, 0.4 mM PMSF, 3 mg/ml aprotinin, 1 μg/ml leupeptin, and 10% glycerol. Lysates were cleared of insoluble material by centrifugation at 15,000 × g for 5 min. For the EMSA, cell extracts (5 μg of total protein) were incubated with 1 μg of poly(dI-dC) for 30 min with ³²P-labeled double-stranded oligonucleotide probes containing Stat binding sites, then resolved by electrophoresis on nondenaturing 4.5% PAGE. The probes used contained a consensus binding site for Stat5 (upper strand, 5′-AGATTTCTAGGAATTCAATCC3′) (Santa Cruz Biotechnology, Santa Cruz, CA) or a Stat6 binding site from the mouse germline epsilon Ig heavy chain promoter (5′-AACTTCCCAAGAACAGA-3′) (27, 28).

Flow cytometric analysis

Single cell suspensions of thymocytes treated overnight with PMA (50 ng/ml), ionomycin (1 μg/ml), or T cells from spleen and lymph node treated with anti-CD3 (10 μg/ml) and anti-CD28 (2.5 μg/ml) were incubated with fluorochrome-conjugated or biotinylated Abs (PharMingen) against IL-2Rα/CD25 (FITC), γ-chain (biotin), IL-2Rβ/CD122, or IL-4Rα (biotin) at 4°C, as described (20). CD122 was detected by a three-step staining, including biotinylated goat anti-rat Ig and streptavidin-PE (PharMingen).

Immunoprecipitation and immunoblotting

Whole cell extracts obtained from thymocytes and T cells (pooled splenic and lymph node cells) were prepared as described above. These extracts were then subjected to immunoprecipitation with antisera against Stat5A, Stat5B (Zymed Laboratories, San Francisco, CA), insulin receptor substrate-1 (IRS-1; Santa Cruz Biotechnology), and IRS-2 (Upstate Biotechnology, Lake Placid, NY). The precipitates were washed in lysis buffer, solubilized in SDS sample buffer, resolved on 7.5% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes. The membranes were probed in accordance with manufacturer’s protocols using mAbs against phosphotyrosine (RC-20; Transduction Laboratories, Lexington, KY; and 4G10; Upstate Biotechnology), Stat5A, Stat5B, IRS-1, or IRS-2, as indicated. After rinsing with PBS-0.1% Tween, bound Abs were detected using enhanced chemiluminescence and secondary Abs, where indicated (NEN, Dupont, Boston, MA).

Isolation and analysis of RNA

Suspensions of thymocytes and B cell-depleted T cells were plated (2.5 × 10⁶ cells/ml) and cultured overnight at 37°C in the presence of PMA (50 ng/ml), ionomycin (1 μg/ml), IL-4 (10 ng/ml), PMA and IL-4, or PMA and ionomycin for thymocytes, or with IL-4 and anti-CD3 plus anti-CD28 for T cells. Total cellular RNA was isolated using TRIzol reagent according to the manufacturer’s instructions (Life Technologies, Bethesda, MD). After resolving RNAs by electrophoresis on formaldehyde-agarose gels, nucleic acids were transferred to Nylon membranes (Amersham, Arlington Heights, IL), fixed by UV cross-linking, then probed using cDNAs labeled by random hexamer priming and hybridization in 50% formamide, 0.2% SDS, 0.6 M NaCl, 4× Denhardt’s, 50 μg/ml sheared, denatured salmon sperm DNA, and 5% dextran sulfate at 42°C. The filters were washed in 2× SSC, 0.1% SDS at room temperature, then in 0.5× SSC, 0.1% SDS at 60°C. Equal loading of RNAs was verified by ethidium bromide staining and by hybridization with a rRNA oligonucleotide probe (29). Band intensities were quantified using a Fuji BAS 1000 phosphor imager.

Role of NF-κB signaling in IL-4-dependent proliferation of T lymphoid cells

We previously reported that thymocytes and T cells from mice expressing a constitutive repressor of NF-κB/Rel signaling (IκBα(ΔN)) proliferated poorly after mitogenic stimuli, and that addition of exogenous IL-2 did not reverse this abnormality (21). These data were interpreted as indicating the presence of a defect refractory to IL-2, but it was unclear whether receptor expression was diminished or whether activation of specific signaling pathways downstream from the IL-2R was affected by the inhibition of NF-κB. As outlined above, it seemed possible that the findings were due to an inability of activating signals to create a state of competence. To determine whether NF-κB/Rel signaling is required for responses of T lymphoid cells to any other lymphokine, we measured proliferative responses to IL-4 using thymocytes and T cells derived from IκBα(ΔN) Tg mice and their wild-type littermates (nontransgenic (NTg) mice). The proliferative response to IL-4 of PMA-treated thymocytes from IκBα(ΔN) Tg mice was one-tenth that of control cells (Fig. 1⇓A). A similar proliferative defect was observed using cells cultured in PMA and IL-2, compared with the 4-fold difference between NTg and Tg samples stimulated by PMA and ionomycin (21) (Fig. 1⇓A legend). As expected, thymocytes did not proliferate appreciably when treated with PMA, IL-2, or IL-4 alone. These data indicate that PMA was required to induce thymocyte competence to proliferate in response to IL-2 or IL-4, and that NF-κB/Rel signaling was essential for induction of this competent state.

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FIGURE 1.

IκBα(ΔN) inhibition of the NF-κB/Rel pathway impairs the competence of T cells to respond to IL-4 growth signaling. A, Thymocytes from either NTg (□) or Tg (▪) IκBα(ΔN) mice were treated for 40 h with PMA (50 ng/ml), ionomycin (1 μg/ml), IL-2 (10 ng/ml), and IL-4 (10 ng/ml), as indicated. Cells were pulsed for an additional 8 h with tritiated thymidine and harvested for scintillation counting. The results are shown as the mean (±SEM) tritiated thymidine incorporation in three independent experiments with pooled thymocytes from six NTg and eight Tg mice in each experiment. Thymidine incorporation into these pooled thymocytes treated was 21,521 cpm (Tg) compared with 91,947 cpm (NTg) when treated with PMA and ionomycin. B, B cell-depleted T cells prepared from pooled lymph nodes and spleens of Tg and NTg mice were stimulated as indicated, using plate-bound anti-CD3 (10 μg/ml), agonistic Abs against CD28 (10 μg/ml), or PMA (50 ng/ml), each in the presence of IL-2 (10 ng/ml) or IL-4 (10 ng/ml). Tritiated thymidine incorporation was determined as in A. C, Gel mobility shift analysis of nuclear NF-κB/Rel proteins. Pooled thymocyte suspensions were prepared from six NTg and eight Tg mice and cultured overnight in the absence of stimulation, or in the presence of PMA or PMA plus ionomycin. Equal amounts of nuclear extracts from these cells were added to DNA-binding mixtures containing a ³²P-labeled κB probe. DNA-protein complexes were resolved on polyacrylamide gels and visualized by autoradiography.

To determine whether a similar requirement applies to mature T cells, proliferative responses to IL-2 and IL-4 were measured using B cell-depleted T lymphocytes from secondary lymphoid organs. Lymphokine-dependent proliferation of IκBα(ΔN) cells stimulated with PMA was again observed to be reduced by an order of magnitude (Fig. 1⇑B). T cell activation by anti-CD3 depends on IL-2 production and responsiveness. IκBα(ΔN) T cells exhibited a proliferative defect refractory to IL-4 as well as IL-2 after activation with anti-CD3 (Fig. 1⇑B), although the magnitude of the deficit was less dramatic. Costimulatory signaling by CD28 has been described as critical for the enhancement of T cell proliferation (30). To determine whether the observed decrease in proliferation was due to inadequate costimulation in these cultures and if signaling through the CD28 receptor could reverse the impairment of cytokine-dependent proliferation, we added an activating Ab against CD28 to the anti-CD3 stimulus. However, the presence of agonist Abs against CD28 did not reverse the failure of cells to proliferate normally in IL-2 or IL-4 (Fig. 1⇑B). We have previously shown that induction of NF-κB under biochemical conditions different from these (i.e., by anti-CD3/28 or by PMA plus ionomycin) is potently inhibited by the IκBα(ΔN) transgene (21, 31), but had not assayed PMA alone. To determine the effect of IκBα(ΔN) on the nuclear induction of NF-κB/Rel during PMA treatment, mobility shift assays were performed using nuclear extracts derived from NTg and Tg mice. These experiments demonstrated that PMA treatment alone was sufficient to induce high nuclear levels of NF-κB activity in cells from wild-type animals, but not IκBα(ΔN) Tg mice (Fig. 1⇑C). We conclude that T cell competence to proliferate in response to both growth signals, IL-2 and IL-4, depends on normal NF-κB signaling.

Selective decreases in IL-4-induced gene expression

It has been proposed that growth regulation and differentiation/regulation of gene expression represent mechanistically distinct effects of IL-2 and IL-4 signaling that are dependent on different portions of the cytokine receptors and different signaling pathways (32, 33). IL-4 does not activate NF-κB, but the finding that one downstream effect of cytokine receptor signaling, proliferation, was diminished in lymphoid cells whose NF-κB/Rel signaling is inhibited prompted us to investigate whether IL-4 induction of target gene transcription was normal. Few genes have been identified whose expression is IL-4 dependent in T cells, but IL-4 does increase IL-4Rα-chain expression (8) and transcription of the mouse IL-2Rβ-chain gene (34). Therefore, we performed Northern blot analyses to analyze the effect of IκBα(ΔN) on this measure of IL-4 signaling. Levels of IL-4Rα and IL-2Rβ mRNA were substantially diminished (0.3 × control) in cells from IκBα(ΔN) mice after IL-4 stimulation (Fig. 2⇓). We have observed a decrease in c-myc mRNA in Tg T cells after activation by anti-CD3 and anti-CD28 (unpublished observations), perhaps reflecting the presence of κB-binding sites in the c-myc promoter (35). Since this proto-oncogene promoter is a target of IL-4-mediated signaling that may be independent of the IL-4-inducible transcription factor Stat6 in a nonlymphoid cell line (36), we investigated the levels of c-myc mRNA expression. As shown in Fig. 2⇓, levels of c-myc mRNA expression observed before and after IL-4 stimulation of Tg T cells were little different from those of wild-type controls. Thus, the inhibitory effects of IκBα(ΔN) on IL-4 signaling pathways include decreased expression of selected target genes, including a key signaling subunit of the IL-4R.

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FIGURE 2.

Decreased expression of IL-4 target genes. Northern blot analysis was performed on RNAs derived from cultured lymphoid cells of Tg mice and littermate controls (WT). Thymocytes, or B cell-depleted T cell preparations, as indicated, were cultured in media with IL-4 (10 ng/ml), or without exogenous cytokine, as indicated. Samples of total cellular RNA (10 μg) were fractionated under denaturing conditions on formaldehyde-agarose gels, transferred to nylon membranes, and sequentially hybridized with radiolabeled IL-4Rα, IL-2Rβ, and c-myc cDNA probes, then with an rRNA probe. B, Quantification of the intensity of the T cell IL-4Rα band was performed using a phosphor imager within its linear range. Values plotted represent arbitrary units of signal intensity.

Activation of IL-4 signal transduction pathways in IκBα(ΔN) Tg mice

The IL-4Rα cytoplasmic tail lacks intrinsic tyrosine or serine/threonine kinase activity, but upon ligand binding, resident Janus tyrosine kinases (Jak) become activated (37, 38). These kinases are thought to induce phosphorylation of downstream substrates, which include conserved tyrosine residues in the IL-4Rα tail, IRS proteins IRS-1/2, and the IL-4-associated Stat6 (37, 38, 39). Recruitment of the IRS proteins to IL-4Rα in particular has been implicated as an essential step in IL-4-induced proliferation of certain cell lines (39, 40, 41). Thus, engagement of the IL-4 (but not IL-2) receptor activates a signaling pathway in which recruitment of IRS-1 and -2 to a distinct tyrosine-phosphorylated residue in IL-4Rα leads to increased tyrosine phosphorylation of the IRS proteins in cell lines, followed by recruitment of downstream signal transducers (37, 42). IRS proteins are indispensible for IL-4-induced mitogenesis in the hemopoietic cell line 32D, and thus a defect in IRS activation might lead to decreased IL-4-dependent proliferation of lymphoid cells (40, 41). To investigate whether IRS activation is altered in IκBα(ΔN) Tg cells compared with normal primary cells, we performed immunoprecipitations of IRS-1 or IRS-2, followed by antiphosphotyrosine immunoblotting analyses (Fig. 3⇓). When thymocytes were first activated, rinsed, and then stimulated, we discovered that the preactivation step induced tyrosine phosphorylation of IRS-2 independent of the addition of exogenous IL-4 (panel A). The amount of tyrosine phosphorylation in IκBα(ΔN) Tg cells was normal. To investigate further whether the inhibition of NF-κB affected the ability of IL-4 to influence the IRS-2 pathway, responses to IL-4 were tested in cells that had not undergone preactivation (B). Under these conditions, the primary cells demonstrated IL-4-dependent increases in IRS-2 phosphorylation that could not be mimicked by IL-2 and were unaffected by IκBα(ΔN). Short-term IL-4 treatment induced the tyrosine phosphorylation of IRS-1 in resting cells (not shown) and those that had been preactivated (C). In contrast to the results with IRS-2, the magnitude of IL-4-induced IRS-1 phosphorylation was moderately reduced in Tg cells compared with wild-type preparations. Inasmuch as control immunoblots revealed similar levels of IRS proteins in wild-type and Tg cells (Fig. 3⇓), we conclude that the inhibition of NF-κB by IκBα(ΔN) led to a diminution in the degree of tyrosine phosphorylation of IRS-1, but not IRS-2 in response to IL-4. Since IRS-2 appears be the predominant IRS protein in primary T lymphoid cells rather than IRS-1 (43), the degree of inhibition selective for IRS-1 seemed unlikely to explain fully the more substantial impairment of proliferation.

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FIGURE 3.

Effect of IκBα(ΔN) transgene on cytokine-induced IRS-1 and IRS-2 phosphorylation. A, Activated thymocytes from control and Tg mice were generated by overnight culture in the presence of PMA (50 ng/ml) and ionomycin (1 μg/ml). These preactivated cells were rinsed, replated in complete medium, and then stimulated with IL-4 (10 ng/ml for 30 min), as indicated. B, Thymocytes from wild type and Tg mice were cultured for 4 h in medium alone, and then stimulated for 30 min with IL-2 (10 ng/ml) or IL-4 (10 ng/ml), as indicated. C, Activated thymocytes were prepared as in A. Cell lysates were subjected to immunoprecipitation using Abs against IRS-2 (A and B) or IRS-1 (C). Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with Abs to phosphotyrosine (4G10). Filters were subsequently stripped and reprobed with Abs against IRS-2 or IRS-1, as indicated.

In addition to IRS recruitment and activation, members of the Stat transcription factor family are induced by IL-4 treatment of activated T cells (37, 38, 44). Following IL-4-induced phosphorylation, cytosolic Stat6 proteins dimerize, translocate to the nucleus, and bind to consensus sequences in the promoter regions of IL-4-regulated genes. Stat6 activation represents a key pathway for IL-4-induced gene induction and differentiation (10, 11, 45), but it has been suggested that Stat6 also may contribute to the regulation of T cell proliferation by IL-4 (46). To determine the effect of IκBα(ΔN) on this IL-4 signaling pathway, we compared Stat6-binding activity in thymocytes and T cells from NTg and Tg mice. As shown in Fig. 4⇓, induction of nuclear Stat6 was 2-fold higher in wild-type cells as compared with IκBα(ΔN)-expressing cells. mRNA levels for IL-4Rα were substantially decreased due to defective NF-κB/Rel signaling (Fig. 2⇑), and this inhibitory effect was also observed at the cell surface (see below). Stat6 activation depends on the availability of IL-4Rα-associated phosphotyrosine residues in the cytoplasm (32, 44), so we infer that there is no apparent decrease in the biochemical efficiency of Stat6 activation per IL-4R molecule when the net induction of this transcription factor in the nucleus was diminished to a modest extent.

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FIGURE 4.

IL-4 induction of Stat6- and Stat5-binding activity in IκBα(ΔN) T cells. A, B cell-depleted lymphoid cells (T cells) from spleen and lymph nodes of wild-type and Tg mice were activated 16 h with plate-bound anti-CD3 and anti-CD28, harvested, rinsed, and recultured 30 min in complete media, alone or supplemented with IL-4 (10 ng/ml). B, Thymocytes of wild-type and Tg mice were activated 16 h with PMA (50 ng/ml) and ionomycin (1 μg/ml), harvested, rinsed, and then stimulated with IL-4 (10 ng/ml for 30 min). Whole cell extracts from these cells were then assayed for Stat6 by mobility shift analyses using an N4-spacer (Stat6-specific) oligonucleotide probe spanning nucleotides −122 to −104 of the mouse Ig heavy chain germline epsilon promoter (28 ). The identity of the indicated complexes was confirmed by supershift analyses using antiserum specific for the C terminus of Stat6 (black dot). C, Impairment of Stat5 activation by IL-4. B cell-depleted lymphoid cells (T cells) from spleen and lymph nodes of wild-type and Tg mice were activated and stimulated with IL-4, as is described in A. Whole cell extracts were then assayed for Stat5 by mobility shift analyses using an oligonucleotide probe that contains a Stat5 consensus sequence. The identity of the indicated complexes was confirmed by supershift analyses using antiserum specific for Stat5 (A/B) (black dot). Similar results were obtained with thymocytes.

In addition to the activation of Stat6 by IL-4, more recent work has established that IL-4 induces the tyrosine phosphorylation and nuclear translocation of Stat5 isoforms A and B in activated T cells, followed by increased expression of Stat5-dependent reporters (47, 48). Importantly, several groups have implicated Stat5 as a key link between the IL-4R and a proliferative response to this cytokine (48, 49, 50). We therefore assayed Stat5 induction after IL-4 treatment of activated cells from wild-type and IκBα(ΔN) mice (Fig. 4⇑C). In contrast to the relatively modest effect on IRS proteins and Stat6, Stat5 induction was greatly diminished in cells from the Tg mice as compared with controls. We conclude that interference with the NF-κB/Rel pathway led to a preferential inhibition of Stat5 induction as compared with Stat6.

IL-4R expression

The IL-4R consists of an IL-4Rα-chain, which heterodimerizes with a second subunit, the common γ-chain (γ_c), which is shared with the receptors for IL-2 and other members of the hemopoietin receptor superfamily (37, 51, 52, 53). Importantly, loss-of-function mutants and Ab-blocking studies indicate that γ_c plays a critical role in IL-4-induced proliferation (52, 53), and the induction of Stat5 by IL-4 in activated T cells appears to be due to interactions with Jak3 that is noncovalently associated with γ_c rather than IL-4Rα (47, 48). To investigate further how IκBα(ΔN) leads to diminished responses to IL-4, we measured the cell surface expression of IL-4R α and γ_c subunits. We observed a substantial reduction in IL-4Rα expression on Tg thymocytes and T cell lymphocytes compared with wild-type counterparts that had been rendered competent to proliferate (Fig. 5⇓). In contrast, expression of the γ_c subunit was normal. These data indicate that NF-κB/Rel proteins serve a regulatory role in the expression of IL-4Rα. Morever, decreased IL-4-induced proliferation correlated with a diminution in IL-4 signaling reflected most by Stat5 activation, and to a lesser extent by IRS-1 phosphorylation and Stat6 induction.

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FIGURE 5.

Decreased expression of IL-4Rα and normal levels of IL-2R in IκBα(ΔN) mice. Activated thymocytes (PMA plus ionomycin) and T cells (anti-CD3 plus anti-CD28) from Tg mice and their NTg littermates were stained with mAbs specific for IL-2Rα (CD25), IL-2Rβ (CD122), γ_c, IL-4Rα, CD3, and TCRαβ, and then analyzed by flow cytometry. The expression of the indicated surface molecules on wild-type and Tg mice was assessed by gating on TCRαβ⁺ thymocytes (A) or on CD3⁺ T cells (B) and then displaying the indicated histograms. Solid lines indicate specific staining, while dashed lines (—-) show the relevant control histogram. Of note, production of IL-2 by anti-CD3/28-activated IκBα(ΔN) T cells is essentially normal (31 ) so that differences in receptor internalization are unlikely to contribute to the similarity of receptor expression on wild-type and Tg T cells. Minor variability leading to the apparent presence of IL-2Rβ^high or γ_c^high subpopulations is unlikely to account for differences in proliferation in light of the high frequency of T cells that undergo blast transformation under identical conditions (31 ) and 5-bromo-2′-deoxyuridine-labeling indices of 12–17%/h for wild-type CD4 and CD8 T cells (A. L. Mora and M. Boothby, unpublished observations).

In transfected B lymphoma cells, the activation of some signaling pathways by IL-4 was influenced by the number of receptors expressed (33). Therefore, we explored whether a decreased pool of available IL-4Rα cytoplasmic tails in these primary cells is the mechanistic basis for the observed diminution in IL-4-induced proliferation. To do so, we sought to increase the pool of IL-4Rα tails by an approach independent of prior activation. We had previously characterized Tg mice in which the T lineage specifically expresses a chimera of mouse IL-2Rβ and the IL-4Rα cytoplasmic tail (26). Both thymocytes and T cells from these mice demonstrated inducibility of Stat6 by IL-2 and other evidence that these cytoplasmic tails functioned in an IL-4-specific manner (26). Accordingly, we crossed chimeric cytokine receptor Tg mice with those expressing the inhibitor of NF-κB signaling and performed proliferation assays measuring the response to IL-2, IL-4, or a combination of these cytokines (Fig. 6⇓). Strikingly, under no condition (IL-4 alone or IL-2 with IL-4) did the increased pool of IL-4Rα cytoplasmic tails in double-Tg mice promote an enhancement of proliferation above that obtained with IκBα(ΔN) single-Tg animals. These data suggest that a limiting pool of cytoplasmic tails is not the sole basis for a decreased response to IL-4.

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FIGURE 6.

Failure of increased IL-4Rα cytoplasmic tails to enhance proliferation of IκBα(ΔN) cells. IκBα(ΔN) Tg mice were crossed with a Tg mice encoding a chimeric IL-2/IL-4 receptor to the T lineage (chimeric (Chi) IL-2/4R) (26 ). B cell-depleted T cells were prepared from pooled lymph nodes and spleens from wild-type, Chi IL-2/4R, IκBα(ΔN), and Chi IL-2/4R × IκBα(ΔN) double-Tg littermate mice. Cell preparations were stimulated for 48 h, as indicated, using plate-bound anti-CD3 (10 μg/ml), agonistic Abs against CD28 (10 μg/ml), or PMA (50 ng/ml), each in the presence of IL-2 (10 ng/ml) or IL-4 (10 ng/ml). Cells were pulsed for an additional 8 h with tritiated thymidine and harvested for scintillation counting. The results are shown as the mean (±SEM) of the stimulation indexes in four independent experiments. Stimulation index was calculated as the mean cpm stimulated [³H]thymidine incorporation divided by the arithmetic mean cpm unstimulated [³H]thymidine incorporation.

Defective Stat5A activation in IκBα(ΔN) T cells despite IL-2R expression

The functional IL-2R in mice is composed of three subunits: IL-2Rα, IL-2Rβ, and the γ_c (4, 5). In light of the finding that IL-4R expression on cells from IκBα(ΔN) mice was substantially decreased, we measured the cell surface expression of IL-2R subunits. The signal-transducing components of the IL-2R, IL-2Rβ, and γ_c were expressed at comparable levels in thymocytes and T cells from Tg and wild-type mice (Fig. 5⇑). The IL-2Rα subunit is critical for the binding of mouse IL-2 to its receptor (3, 4, 5) and for mouse IL-2R function (3, 12, 13), so that the proliferative defect of thymocytes could arise from a decrease in IL-2Rα expression. Despite reports that NF-κB/Rel proteins are potent trans-activators of the IL-2Rα promoter in transfected cells, IL-2Rα/CD25 expression on IκBα(ΔN) T cells was at most slightly lower than on wild-type cells, and we observed only a modest decrease in IL-2Rα expression on mature thymocytes (Fig. 5⇑). Since cell surface expression of the IL-2R subunits on T lymphoid cells appeared normal, the data indicate that decreased proliferation of IκBα(ΔN) reflects a failure to capacitate signal transduction by the receptor subunits. Although a subject on which there is disagreement, the nuclear induction of Stat5 has been reported to be a key event in IL-2-induced proliferation (49, 54, 55, 56). Accordingly, our data on IL-4-induced Stat5 raised the possibility that inhibition of Stat5 induction might represent a defect shared by IL-2R and IL-4R signaling when NF-κB was inhibited. To test this possibility, we measured activation of Stat5 by IL-2.

Like IL-4, IL-2 induces phosphorylation of Stat proteins expressed from related genes (Stat5A and Stat5B) (38). To determine whether the influence of IκBα(ΔN) on IL-2-induced proliferation could be correlated with phosphorylation of Stat5, we activated thymocytes and T cells from Tg and wild-type mice, followed by mobility shift assays using extracts of control and IL-2-treated cells. This activation step was designed to mimic the conditions of proliferation assays and promote induction of IL-2Rα. It also leads to the production of IL-2 by wild-type thymocytes and T cells. This IL-2 production is blocked by the Tg inhibition of NF-κB in thymocytes (21). The binding activity of DNA-protein complexes that comigrate with Stat5 was reduced in thymocytes (Fig. 7⇓A) and T cells (Fig. 7⇓B) from IκBα(ΔN) Tg mice. Endogenous production of IL-2 by wild-type but not Tg cells led to high basal levels of binding activity in the extracts from wild-type thymocytes, but not those from IκBα(ΔN) Tg mice, and even the addition of exogenous IL-2 was unable to induce substantial binding activity. The presence of Stat5 in this complex was validated using an antiserum specific for both Stat5A and B, which eliminated the Stat5 complex and created a supershifted band of slower mobility (Fig. 7⇓A). An independent Ab specific for Stat5A also supershifted the indicated complex, whereas a control serum did not (data not shown). Stat5 induction in T cells also was impaired (B). To understand better which form(s) of Stat5 contributed to the mobility shift activity, extracts were subjected to immunoprecipitation with antiserum against the individual isoforms of Stat5, Stat5A, and Stat5B, followed by immunoblotting with an antiphosphotyrosine Ab. As in A, endogenous IL-2 production by wild-type thymocytes led to a substantial level of Stat5A tyrosine phosphorylation that was not further increased by exogenous IL-2. In contrast, we observed far less phosphorylation of Stat5A in cells from IκBα(ΔN) mice as compared with controls, and the addition of exogenous IL-2 did not induce phosphoStat5A (Fig. 7⇓C). Although at lower signal intensity overall, the level of Stat5B phosphorylation in IL-2-treated cells from Tg animals was indistinguishable from wild-type samples (Fig. 7⇓C). This finding suggested that IL-2 induction of Stat5B homodimers and perhaps Stat5AB heterodimers was quantitatively less affected than that of Stat5A homodimers. While the decreased Stat5A phosphorylation might have been attributed to a requirement for NF-κB/Rel signaling in the induction of Stat5A protein levels, wild-type and IκBα(ΔN) mice expressed similar levels of total Stat5A and Stat5B protein (Fig. 7⇓C). We conclude that the presence of IκBα(ΔN) in Tg T lineage cells is associated with impaired phosphorylation and activation of Stat5A despite expression of all subunits of the IL-2R. Taken together with observations linking Stat5 to T cell proliferation (49, 54, 55, 56) through association with IL-2 but not Ag receptors (49), these findings suggest that the observed impairment in IL-2-inducible proliferation by IκBα(ΔN) T cells is associated with defective Stat5A activation.

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FIGURE 7.

Defective induction of Stat5A in activated, IL-2-treated T lymphoid cells. A, Thymocytes from wild-type and Tg mice were activated overnight with PMA plus ionomycin and anti-CD28, harvested, rinsed, and recultured 30 min with media, alone or supplemented with IL-2 (10 ng/ml). B, B cell-depleted lymphoid cells (T cells) from spleen and lymph nodes of wild-type and Tg mice were activated 16 h with plate-bound anti-CD3 and anti-CD28, harvested, rinsed, and recultured 30 min in complete media, alone or supplemented with IL-2 (10 ng/ml). Whole cell extracts from these treated cells were then assayed for Stat5 by mobility shift analyses using an oligonucleotide probe that contains a Stat5 consensus sequence. The background of Stat5-binding activity in cells not treated with IL-2 is due to IL-2 release from the activated cells; this band was not present when using extracts of resting cells. The identity of the indicated complexes was confirmed in thymocyte preparations by supershift analyses using antiserum specific for Stat5 (A/B). C, Activated thymocytes (16-h culture in PMA and ionomycin) were recultured 30 min in complete media, alone or in the presence of mouse IL-2 (10 ng/ml). Whole cell extracts from these stimulated cells were subjected to immunoprecipitation using an anti-Stat5A or Stat5B antiserum. Immune precipitates were resolved by SDS-PAGE, then subjected to immunoblotting using an anti-phosphotyrosine Ab (RC-20). Following this procedure, these filters were then divided, stripped, separately reprobed using the anti-Stat5A or Stat5B antiserum, as indicated, repositioned, and exposed to x-ray film. The position of the Stat5A and Stat5B band is marked with an asterisk and a diamond, respectively.

The evidence that the IκB/NF-κB/Rel system can regulate receptor capacitation raises the question as to whether such a role is limited to cytokines whose receptor uses Jak1 and Jak3 to induce Stat5, or instead may be more general. To explore this issue, we measured the ability of IL-12 to enhance the proliferation of T lymphoid cells. PMA-primed thymocytes could not be costimulated by IL-12 (data not shown). As shown in Fig. 8⇓, IL-12-enhanced proliferation of PMA-primed T cells was inhibited by IκBα(ΔN), although there was no enhancement of the response to other proliferative stimuli. Thus, while the mechanism of this effect remains to be determined, the action of this cytokine through different Janus kinases (Jak2 and Tyk2) and Stat proteins (Stat3 and Stat4) suggests the potential for additional NF-κB-dependent mechanisms of receptor capacitation.

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FIGURE 8.

Decreased proliferation of PMA-treated cells costimulated with IL-12. B cell-depleted T cells were prepared from pooled lymph nodes and spleens from wild-type and Tg mice. Cell preparations were stimulated for 48 h, as indicated, using plate-bound anti-CD3 (10 μg/ml), agonistic Abs against CD28 (10 μg/ml), or PMA (50 ng/ml), each in the presence or absence of IL-12 (10 ng/ml). Cells were pulsed for an additional 8 h with tritiated thymidine and harvested for scintillation counting. The results are shown as the mean (±SEM) tritiated thymidine incorporation in three independent experiments.