NF-{kappa}B activation plays an important role in the IL-4-induced protection from apoptosis

José Zamorano, Ana L. Mora1, Mark Boothby1 and Achsah D. Keegan

Department of Immunology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA
1 Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN 37232, USA

Correspondence to: A. D. Keegan


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-4 alone protects cells from apoptosis by insulin receptor substrate (IRS)-dependent and -independent mechanisms. However, in vivocells are typically exposed to a number of signals at the same time. To determine the contribution of co-stimulatory signals to the regulation of apoptosis by IL-4, we first analyzed whether tumor necrosis factor (TNF)-{alpha}, which has been shown to inhibit the activation of IRS-1 by insulin, could modify IL-4 signaling and protection from apoptosis. We found that TNF-{alpha} cooperates with IL-4 in protecting 32D cells from factor withdrawal-induced apoptosis. This effect was independent of the expression of IRS-1, indicating that this cooperation is via an alternative anti-apoptotic pathway. Moreover, TNF-{alpha} had no effect on the activation of IRS-1 induced by IL-4. IL-4 enhanced TNF-{alpha}-induced activation of the transcription factor NF-{kappa}B. Interestingly, pharmacologic inhibition of NF-{kappa}B activation or protein synthesis resulted in the induction of cell death that could not be inhibited by IL-4, suggesting that IL-4 cooperates with NF-{kappa}B to signal protection from apoptosis. Supporting this hypothesis, IL-4 also increased NF-{kappa}B activation induced by anti-CD3 antibodies in primary T cells and protected them from apoptosis induced by receptor engagement. However, IL-4 was not able to inhibit apoptosis induced by anti-CD3 in T lymphocytes isolated from transgenic mice expressing a dominant-negative form of I{kappa}B{alpha} that prevents NF-{kappa}B activation. Thus, in addition to the previously identified IRS-1 pathway, IL-4-induced protection from apoptosis may also be mediated through cooperation with the NF-{kappa}B family of transcription factors.

Keywords: apoptosis, cytokines, T cells, transcription factors


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-4 is a cytokine that plays many roles in the regulation of the immune system (1). It is produced by activated T cells, basophils and mast cells, and promotes T cell differentiation towards the Th2 cell type (1, 2). In B lymphocytes, it participates in the processes of activation, proliferation and differentiation, promoting IgG1 and IgE production (3, 4). In addition, IL-4 has been shown to act as a survival factor by preventing apoptosis in a variety of cell types, including T cells, B cells and mast cells (5–9).

Responses to IL-4 are mediated by a cell-surface IL-4 receptor complex (IL-4R). This receptor consists of at least two different types. The type I receptor contains the IL-4R{alpha} chain and the common {gamma} chain (10). The type II receptor includes IL-4R{alpha} and the low-affinity receptor for IL-13 (IL-13R{alpha}1) (11). The binding of IL-4 to its receptor results in the activation of the Janus tyrosine kinases JAK1 and JAK3, and several cellular proteins including the insulin receptor substrates (IRS)-1 and -2, and the transcription factor STAT6 (12–16). IRS proteins have been shown to play a key role in IL-4-induced cell proliferation and protection from apoptosis (9, 15–17). The importance of IRS-1 as a general survival messenger was established by reports showing that IRS-1 also mediates protection from apoptosis transmitted by the insulin and leukocyte tyrosine kinase receptors (18, 19). In addition to IRS-1, IL-4 is able to activate an unidentified pathway to protect cells from apoptosis that likely does not involve the transcription factor STAT6 (9, 20).

During an immune challenge, cellular responses occur as a result of the interaction of a number of cell-surface molecules and secreted cytokines. In addition to inducing responses on its own, IL-4 cooperates with other extracellular signals that regulate responses in hematopoietic cells such as antigen receptors, bacterial lipopolysaccharide (LPS), tumor necrosis factor (TNF)-{alpha}, IL-1 and CD40 (1). The nature and mechanism of this cooperation can be quite complex. For example, several reports described scenarios of either cooperation or antagonism between TNF-{alpha} and IL-4. IL-4 was able to prevent TNF-{alpha}-induced cell death after helminthic infection while it potentiated the anti-proliferative effect of TNF-{alpha} in certain tumor cells (21, 22). TNF-{alpha} cooperates with IL-4 in B cell activation, and IgE and IgG4 production (25, 26). IL-4 inhibits E-selectin and enhances VCAM-1 expression induced by TNF-{alpha} (31, 32) by two different mechanisms. However, the role that TNF-{alpha} plays in IL-4-induced signal transduction and protection from apoptosis has not been addressed.

Stimulation of cells with LPS, IL-1 or TNF family members, or engagement of lymphocyte antigen receptors can induce NF-{kappa}B activation (27, 28) and act as accessory signals for IL-4-mediated responses. NF-{kappa}B proteins form dimers in the cytoplasm that are bound to I{kappa}B proteins. After stimulation, I{kappa}B proteins are phosphorylated and degraded via the proteosome pathway allowing the translocation of NF-{kappa}B complexes to the nucleus (27, 28). Although it has been reported that IL-4 alone does not promote the activation of NF-{kappa}B, several studies show that IL-4 can modify (enhance or inhibit) the activation of NF-{kappa}B induced by other agents (29–35). The participation of the NF-{kappa}B family of transcription factors in the regulation of apoptosis has been described extensively (36–39). Numerous anti-apoptotic genes have been found to be regulated by NF-{kappa}B (40–43). However, it is not known whether NF-{kappa}B activity is necessary for protection from apoptosis by IL-4.

In this study, we investigated whether two extracellular signals known to cooperate with IL-4 in the regulation of cellular responses could modify IL-4 signal transduction and its ability to protect cells from apoptosis. We found that IL-4 cooperates with TNF-{alpha} in 32D cells leading to enhanced protection from apoptosis and enhanced activation of NF-{kappa}B. In addition, we found that if NF-{kappa}B activation is blocked, IL-4 is no longer able to protect cells from apoptosis. Furthermore, we demonstrated that IL-4 enhances the activation of NF-{kappa}B mediated by anti-CD3 antibodies in primary T cells and this NF-{kappa}B activity is indispensable for IL-4 to signal protection from apoptosis induced by TCR complex engagement. These results suggest that in addition to IRS, IL-4 protects cells from apoptosis by a mechanism dependent on the transcription factor NF-{kappa}B.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells and reagents
The IL-3-dependent myeloid parental cell line 32D and 32D expressing IRS-1 (32D/IRS-1) as a result of transfection were maintained in RPMI 1640 culture medium supplemented with glutamine, penicillin, streptomycin, 5% FCS and 5% WEHI-3 conditioned medium. Transgenic mice expressing a dominant-negative form of I{kappa}B{alpha} that prevents NF-{kappa}B activation have been previously described (44). CD4+, CD8+ or total T lymphocytes were isolated from lymph nodes from wild-type or transgenic mice as reported (44). Recombinant murine IL-4 and TNF-{alpha} were purchased from R & D Systems (Minneapolis, MN), cycloheximide (CHX) and gliotoxin were obtained from Sigma (St Louis, MO), MG132 from Biomol (Plymouth Meeting, PA), and 2C11 anti-CD3 mAb from PharMingen (San Diego, CA). Recombinant human TNF-{alpha} was a gift from Dr Yufang Shi (American Red Cross, Rockville, MD).

Apoptosis assays
The percentage of apoptotic cells was determined by staining nuclear DNA content with propidium iodide. After culture, cells were resuspended in 0.25 ml of propidium iodide solution (50 µg/ml propidium iodide, 0.1% sodium citrate, 0.1% NP-40 and 50 µg/ml RNase; Sigma) and incubated for 30 min at room temperature. DNA content was then analyzed by flow cytometer (FACScan; Becton Dickinson, Mountain View, CA). Apoptotic cells were defined as those with less than a 2N DNA content. In those experiments involving T cells, the percentage of apoptotic cells was determined using the TUNEL assay as described (44).

Analysis of IL-4R expression
After cell culture, cells were collected and incubated for 30 min with M1 antibody, a rat anti-mouse IL-4R{alpha} chain antibody. After washing, cells were incubated for an additional 30 min with a goat anti-rat antibody conjugated with phycoerythrin and analyzed by flow cytometry.

Immunoblotting
After stimulation, cell pellets were lysed in RIPA lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 50 mM NaF, 10 mM pyrophosphate, 1 mM PMSF and protease inhibitor cocktail) and clarified. To determine IRS-1 phosphorylation, cell lysates corresponding to 2x106 cells were separated on 7.5% SDS–PAGE before transfer to a PVDF membrane. The membranes were then probed with RC20, a monoclonal anti-phosphotyrosine antibody (Transduction Laboratories, Lexington, KY). The bound antibody was detected using enhanced chemiluminescence (Amersham, Arlington, IL). Afterwards, membranes were stripped and re-probed with an anti-IRS-1 rabbit polyclonal antibody (Upstate Biotechnology, Lake Placid, NY).

Electrophoretic mobility shift assay
Cells were starved for 3 h and then stimulated with the appropriate cytokine for the time indicated. After washing, cells were resuspended in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 5 mM NaF, 0.1 mM EDTA, 0.5 mM DTT, 1 mM PMSF and protease inhibitor cocktail) plus 0.5% NP-40 for 5 min on ice. Nuclei were pelleted and washed twice in buffer A. Later, nuclei were incubated for 30 min on ice in buffer B (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 5mM NaF, 0.5 mM DTT, 1mM PMSF, 25% glycerol and protease inhibitor cocktail) and clarified. Nuclear extract was measured using the BioRad protein assay and 1 µg of protein were incubated with 1 ng of 32P-labeled oligonucleotide in reaction buffer [40 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 20 mM HEPES, pH 7.9, 6% glycerol and 0.1 mg/ml poly(dI·dC)] for 20 min at room temperature. To determine STAT6 DNA-binding activity, we used the GAS sequence in the C{varepsilon} promoter (5'-CACTTCCCAAGAACAGA) (45). To determine NF-{kappa}B binding, we used a DNA probe derived from {kappa}B enhancer sequences in the IL-2R{alpha} promoter (5'-CAACGGCAGGGGAATTCCCCTCTCCTT) (44). In this case, the reaction buffer was supplemented with 1 mM DTT and 0.1% NP-40. Polyacrylamide gels (4.5%) containing 0.22 TBE were pre-run for 1 h at 200 V. After loading the samples, gels were run at 200 V for ~3 h. Afterward, gels were dried and exposed to film.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TNF-{alpha} enhances IL-4-induced protection from apoptosis
We have previously shown that IL-4 activates at least two different intracellular pathways to protect 32D cells from apoptosis, one of which is dependent on IRS-1 expression (9). These studies were performed in the absence of additional signals; however, cells are normally exposed to a complex environment in vivo. The aim of this study was to determine whether additional signals could modify IL-4 signaling and protection from apoptosis.

TNF-{alpha} has been shown to induce serine phosphorylation of IRS-1 that results in the inhibition of insulin receptor signaling (46, 47). Since IL-4 and insulin share the ability to signal IRS-1 activation (16), and this pathway is used in both cases to signal protection from apoptosis (9, 19), we examined whether TNF-{alpha} could modify IL-4 signaling protection from apoptosis. To this end, we employed the IL-3-dependent myeloid cell line 32D in which IL-3 withdrawal results in G1 arrest and cell death by apoptosis (48). These cells also lack IRS-proteins, and transfection of IRS-1 and -2 cDNA into 32D established their important role in cell signaling (9, 15–17, 19). IL-4 alone protected 32D cells expressing IRS-1 from apoptosis better than those lacking IRS-1 expression (9) (Fig. 1A and BGo) as expected. TNF-{alpha}, which alone had little effect on apoptosis, greatly enhanced the ability of IL-4 to protect 32D/IRS-1 cells from apoptosis induced by IL-3 withdrawal (Fig. 1AGo). The combination of TNF-{alpha} with only 0.3 ng/ml of IL-4 resulted in a complete inhibition of apoptosis and this combination was more effective than high doses of IL-4 alone. We also found that TNF-{alpha} cooperates with IL-4 in signaling protection from apoptosis in 32D cells lacking IRS-1 (Fig. 1BGo). 32D cells stimulated with IL-4 plus TNF-{alpha} showed ~50% less apoptotic cells than 32D cells stimulated with IL-4 alone at all concentrations tested. For example, the percentage of apoptotic cells decreased from 45% when stimulated with 1 ng/ml of IL-4 alone to 21% when stimulated in combination with TNF-{alpha}. High concentrations of IL-4 (3–10 ng/ml) in combination with TNF-{alpha} resulted in complete inhibition of apoptosis with only 5–7% of apoptotic cells. While IL-4 and TNF-{alpha} suppressed apoptosis, they had no effect on proliferation. Whether in the presence or absence of TNF-{alpha}, 32D cells did not proliferate in response to IL-4 (16 and data not shown).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1. TNF-{alpha} synergizes with IL-4 in protecting 32D cells from apoptosis. 32D cells expressing (A) or lacking (B) IRS-1 were cultured after IL-3 withdrawal for 24 h with various concentrations of IL-4 in the absence or presence of 10 ng/ml murine TNF-{alpha} (muTNF-{alpha}). Afterward, cells were collected and the percentage of apoptotic cells was determined by propidium iodide staining. Those cells with <2N DNA content were considered as apoptotic. (C) 32D cells were treated with various concentrations of IL-4 in the absence or presence of 10 ng/ml of human TNF-{alpha} (huTNF-{alpha}). The percentage of apoptotic cells was determined as above. These figures are representative of at least three similar experiments.

 
Human TNF-{alpha} also cooperated with IL-4 in protecting 32D cells from apoptosis (Fig. 1CGo). As with murine TNF-{alpha}, 32D cells stimulated with IL-4 plus human TNF-{alpha} showed ~50% less apoptotic cells than cells stimulated with IL-4 alone. Human TNF-{alpha} alone had little or no effect on apoptosis. Murine cells express two types of TNF-{alpha} receptors, TNF-R1 and TNF-R2, but only TNF-R1 is able to bind human TNF-{alpha} (49). Consequently, our data indicate that TNF-R1, which is able to signal cell death (50), can also transmit protective signals in conjunction with IL-4.

TNF-{alpha} enhances STAT6 but not IRS-1 activation induced by IL-4
The above data suggest that the cooperation between TNF-{alpha} and IL-4 in regulating apoptosis is independent of IRS-1 expression. In fact, we observed that in contrast to its inhibitory effect on insulin signaling (47), treatment of 32D/IRS-1 cells with TNF-{alpha} did not alter tyrosine phosphorylation of IRS-1 in response to IL-4 (Fig. 2AGo). As expected, IL-4 increased tyrosine phosphorylation of IRS-1, but the levels of phosphorylation were not affected when cells were also cultured in the presence of TNF-{alpha}. In contrast, we found that TNF-{alpha} cooperated in the activation of the transcription factor STAT6 (Fig. 2BGo). Treatment with TNF-{alpha} alone had no effect on STAT6 activation; however, it enhanced the activity of this transcription factor induced by IL-4. After 4 h of stimulation, nuclear extracts prepared from cultures stimulated with IL-4 plus TNF-{alpha} showed higher STAT6 DNA-binding activity than those from cells stimulated with IL-4 alone. The cooperation of TNF-{alpha} with IL-4 was even greater after 12 h. Similar results were obtained when we analyzed the tyrosine phosphorylation of STAT6 (data not shown). TNF-{alpha} enhanced IL-4-induced STAT6 activation in 32D cells lacking IRS-1 (Fig. 2BGo) or expressing IRS-1 (data not shown). indicating that the enhancement of STAT6 activity by TNF-{alpha} is independent of IRS-1 expression.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 2. TNF-{alpha} enhances STAT6 but not IRS-1 activation induced by IL-4. (A) TNF-{alpha} does not inhibit tyrosine phosphorylation of IRS-1 by IL-4. (A-I) 32D/IRS-1 cells were cultured in medium alone or with 10 ng/ml of TNF-{alpha} for 4 h. Then 10 ng/ml of IL-4 was added for an additional 30 min. After stimulation, cell lysates were prepared, and the equivalent to 2x106 cells were separated by SDS–PAGE and immmunoblotted with an anti-phosphotyrosine antibody to detect tyrosine phosphorylated IRS-1. (A-II) Membrane from A-I was stripped and re-probed with anti-IRS-1 antibody to detect total protein. (B) TNF-{alpha} enhances the activation of STAT6 mediated by IL-4. 32D cells were cultured for 4 (left) or 12 (right) h in medium alone, or with 10 ng/ml of TNF-{alpha}, 10 ng/ml of IL-4 or a mixture of both cytokines. Afterward, nuclear extracts were prepared and the STAT6 DNA-binding activity was analyzed using the GAS sequence contained in the C{varepsilon} promoter. (C) TNF-{alpha} does not induce IL-4R{alpha} expression. 32D cells were cultured 4 (left) or 12 (right) h in the presence of nothing (solid line) or murine TNF-{alpha} (dotted line). Then cells were collected and the expression of the IL-4R{alpha} was analyzed using a rat anti-mouse IL-4R{alpha} chain antibody (M1) followed by a goat anti-rat antibody conjugated with phycoerythrin. As a negative control, cells were only labeled with goat anti-rat antibody conjugated with phycoerythrin (filled histograms).

 
In keeping with our results, it has been shown by Lugli et al. that treatment of HUVEC cells with TNF-{alpha} enhanced the activation of STAT6 in response to IL-4 (51). These investigators proposed that the augmentation of STAT6 activation could be correlated with an up-regulation of IL-4R{alpha} chain by TNF-{alpha}. However, this does not appear to be the case in 32D cells since the expression of IL-4R{alpha} in these cells was unaffected by TNF-{alpha} (Fig. 2CGo). The levels of IL-4R{alpha} were quite similar in untreated cells or cells treated with TNF-{alpha} for 4 or 12 h as analyzed by FACS.

IL-4 enhances NF-{kappa}B activation induced by TNF-{alpha}
The data presented thus far indicate that TNF-{alpha} is able to enhance IL-4 signaling. On the other hand, it was possible that IL-4 could also enhance intracellular pathways activated by TNF-{alpha}. In addition to cell death (50), TNF-R1 is able to signal NF-{kappa}B activation (37). The activation of NF-{kappa}B has been shown to have a protective role against apoptosis in different systems (36–39). In fact, NF-{kappa}B has been shown to block cell death signals induced by TNF-{alpha} (36, 38, 39, 52). Therefore, we analyzed the effect of IL-4 in the activation of NF-{kappa}B by TNF-{alpha}. The activation of 32D cells with TNF-{alpha} resulted in the nuclear translocation of active NF-{kappa}B complexes that bound to a consensus DNA probe derived from the IL-2R{alpha} promoter (Fig. 3Go). While in these 32D cell lysates the spectrum of shifted bands was complex, all bands were super-shifted with antibodies to p50 or p65 (data not shown). IL-4 alone had no effect on NF-{kappa}B activation, but it enhanced the DNA-binding activity of NF-{kappa}B induced by TNF-{alpha} after 4 and 12 h of stimulation. Significant enhancement was not observed with 0.5 h of stimulation. These data indicate that IL-4 cooperates with TNF-{alpha}-promoted signals that lead to the activation of NF-{kappa}B. A similar enhancement has been reported using CD40 or IL-1 stimulation in the presence of IL-4 (32, 35).



View larger version (90K):
[in this window]
[in a new window]
 
Fig. 3. IL-4 enhances the activation of NF-{kappa}B induced by TNF-{alpha}. 32D cells were cultured for 0.5, 4 or 12 h in medium alone or with TNF-{alpha} (10 ng/ml), IL-4 (10 ng/ml) or a mixture of IL-4 and TNF-{alpha}. Afterward, nuclear extracts were prepared and the NF-{kappa}B DNA-binding activity was analyzed using the consensus sequence derived from the IL-2R{alpha} promoter.

 
Inhibitors of NF-{kappa}B activation abrogate protection from apoptosis by IL-4
The ability of IL-4 to enhance NF-{kappa}B activation by TNF-{alpha} may explain the cooperation between IL-4 and TNF-{alpha} in signaling protection from apoptosis. Although TNF-{alpha} cooperated with IL-4 in STAT6 activation, a central role for this protein is unlikely since we and others have shown that STAT6 does not play an important role in apoptosis regulation (20, 45, 53, 54). A role for IRS-1 in this process is also unlikely since its activation by IL-4 is not modified by TNF-{alpha} and both cytokines cooperate in protecting cells lacking IRS-1 from apoptosis. Therefore, we tested whether NF-{kappa}B, which regulates apoptosis in numerous systems, plays a role in the regulation of apoptosis by IL-4. The protective effect of NF-{kappa}B is believed to be mediated by de novo synthesis of proteins regulated by this family of transcription factors (36–39). It is widely believed that the effect of proteasome inhibitors and CHX on apoptosis is through their ability to inhibit NF-{kappa}B activation and synthesis of target proteins (27, 28, 36–38, 40–43). Thus, the inhibition of either protein synthesis by CHX or NF-{kappa}B translocation by proteasome inhibitors has been shown to promote cell death by TNF-{alpha} (37, HREF="#R55">55). Consistent with these studies, we found that TNF-{alpha} induced cell death in 32D cells treated with the protein synthesis inhibitor CHX (Fig. 4AGo) and the proteasome inhibitor MG132 (Fig. 4BGo). In both cases, TNF-{alpha} quickly induced cell death and most cells were dead after 5 h of culture. At this time, TNF-{alpha} induced ~75% of apoptotic cells in combination with the higher doses of CHX and MG132 tested. IL-4 did not block this cell death promoted by TNF-{alpha}. In the presence of inhibitors, the percentage of apoptotic cells treated with TNF-{alpha} alone almost overlapped with the percentage of cells treated with TNF-{alpha} plus IL-4. In addition, a fungal metabolite that selectively inhibits degradation of I{kappa}B{alpha}, gliotoxin (55, 56), prevented the IL-4-induced protection from factor-withdrawal-induced death (Fig. 4CGo). In the presence of gliotoxin, TNF-{alpha} induced rapid cell death that IL-4 was not able to suppress. Similar results were obtained using 32D cells expressing IRS1.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. IL-4 does not protect 32D cells from apoptosis in the presence of agents that block NF-{kappa}B activation. (A) 32D cells were cultured for 5 h in the presence of various concentrations of CHX alone or in combination with IL-4, TNF-{alpha} or TNF-{alpha} plus IL-4 (10 ng/ml of each). (B) Cells were cultured for 45 min in the presence of the proteasome inhibitor MG132. Cells were then cultured with medium alone, IL-4, TNF-{alpha} or TNF-{alpha} plus IL-4 for 5 h. After stimulation, cells were collected and the percentage of apoptotic cells was determined as in Fig. 2Go. (C) Cells were treated as in B in the presence or absence of gliotoxin (1 µg/ml) as indicated. The cells were then cultured in the presence or absence of TNF and/or IL-4 as above. After 5 h of culture, the percentage of apoptotic cells was analyzed. These figures are representative of three different experiments.

 
Even in the absence of TNF-{alpha} treatment, we found that 32D cells (and murine B cell lymphoma lines) are extremely sensitive to the inhibition of NF-{kappa}B. High concentrations of CHX and MG132 induced apoptosis within the 5-h time frame of the experiment that was not inhibited by IL-4 (Fig. 4A and BGo). Eventually, these concentrations of inhibitors would kill all the cells even in the presence of IL-4. In addition, other agents that block NF-{kappa}B activation such as gliotoxin, lactacystin, TPCK and the SN50 peptide (57) promoted cell death that could not be inhibited by IL-4 (Fig. 4CGo and data not shown). These results suggest that in 32D cells, IL-4 is not able to protect cells from factor-withdrawal-induced apoptosis or TNF-{alpha}-induced apoptosis when NF-{kappa}B activation is blocked.

IL-4 requires NF-{kappa}B to protect T cells from anti-CD3-induced apoptosis
The above data indicate that IL-4 can enhance NF-{kappa}B activity induced by TNF-{alpha} and may require the activation of this transcription factor to signal protection from apoptosis. To determine whether a similar requirement was evident in another cell type and to directly address the role of NF-{kappa}B activation, we analyzed the effect of IL-4 on apoptosis in T cells derived from normal mice or mice expressing a dominant inhibitory form of I{kappa}B{alpha} (I{kappa}B{alpha}-DN). Like TNF-{alpha}, TCR are able to signal both cell death and NF-{kappa}B activation (44, 53). In addition, NF-{kappa}B has also been shown to mediate a protective signal against cell death induced by TCR engagement (44). Transgenic mice expressing I{kappa}B{alpha}-DN that prevents the activation of multiple forms of NF-{kappa}B in the T cells has been previously described in detail (44). Among other characteristics, splenic T cells derived from these transgenic mice are more sensitive than wild-type cells to apoptosis induced by stimuli such as concanavalin A and anti-CD3 antibodies (44 and see below). We found that IL-4 was able to enhance the activation of NF-{kappa}B induced by anti-CD3 antibodies (Fig. 5Go). In wild-type T cells, anti-CD3 antibodies promoted the nuclear translocation of p50-relA and p50/c-rel complexes as previously described (44), and the DNA-binding activity of these complexes to an NF-{kappa}B-specific probe was greatly enhanced by IL-4. As expected, T cells expressing I{kappa}B{alpha}-DN did not exhibit a significant NF-{kappa}B activity after anti-CD3 stimulation (Fig. 5Go).



View larger version (69K):
[in this window]
[in a new window]
 
Fig. 5. IL-4 enhances anti-CD3-induced NF-{kappa}B activation. Purified T lymphocytes from wild-type mice (NTg) or transgenic mice expressing a dominant-negative form of I{kappa}B{alpha} ({Delta}N) were cultured for 16 h in the presence of medium alone, anti-CD3 or anti-CD3 plus IL-4. Afterward, nuclear extracts were prepared and NF-{kappa}B DNA-binding activity was analyzed using an NF-{kappa}B consensus sequence derived from the IL-2R{alpha} promoter.

 
We then directly tested whether NF-{kappa}B was important for IL-4-induced protection from apoptosis after stimulation through the TCR. To this end, we analyzed the ability of IL-4 to protect primary T cells isolated from transgenic mice expressing the dominant-negative form of I{kappa}B{alpha} from apoptosis. We found that IL-4 was able to protect CD4+ and CD8+ lymphocytes isolated from wild-type mice from anti-CD3-induced cell death (Fig. 6Go). After 24 h of culture, 34% of CD4+ and 36% of CD8+ stimulated with anti-CD3 antibody were dead, while only 18% of CD4+ and 10% of CD8+ cells were dead when stimulated with anti-CD3 plus IL-4. However, IL-4 was completely unable to protect CD4+ and CD8+ lymphocytes expressing the inhibitory form of I{kappa}B{alpha} from apoptosis induced by anti-CD3 antibodies. In transgenic lymphocytes, 50% of cells were dead after anti-CD3 stimulation and this percentage was not affected by IL-4, suggesting that IL-4 requires NF-{kappa}B to protect from anti-CD3-signaled cell death.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 6. IL-4 requires NF-{kappa}B activation to protect lymphocytes from anti-CD3-induced cell death. (A) Purified CD4+ and CD8+ lymphocytes from lymph nodes from wild-type mice (NTg) or transgenic mice expressing a dominant-negative form of I{kappa}B{alpha} ({Delta}N) were cultured overnight in the absence (open bars) or presence of IL-4 (filled bars). Afterward, cells were treated with immobilized anti-CD3 antibody and cultured for an additional 24 h. After culture, the percentage of apoptotic cells was determined as described Methods.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-4 acts as a survival factor in a number of different biological environments (5–9). IL-4 protects cells from apoptosis induced by receptor stimulation (6, 8), but it can also protect cells from spontaneous apoptosis in the absence of stimulation (5, 7). We have previously demonstrated that IL-4 can use IRS-dependent and -independent pathways to signal protection from apoptosis (9, 20). Herein, we show that IL-4 enhances the activation of the NF-{kappa}B family of transcription factors induced by TNF-{alpha} or TCR engagement and that IL-4 requires the activation of this transcription factor to protect cells from apoptosis induced by the engagement of these receptors. The immune response occurs as a result of multiple complex interactions between cell-surface molecules and soluble factors; these results demonstrate that IL-4 cooperates with other receptor-activated signaling pathways to protect cells from apoptosis.

IL-4 and TNF-{alpha} synergize in the activation of STAT6 and NF-{kappa}B, and moreover they cooperate in protecting 32D cells from apoptosis. Previous studies demonstrated that treatment of 32D/IRS-1 with TNF-{alpha} resulted in serine phosphorylation of IRS-1; as a result, IRS-1 inhibited the tyrosine kinase activity of the insulin receptor (46, 47). For the IL-4R, we have detected no effect of TNF-{alpha} in the activation of IRS-1, but it enhances the activation of STAT6 induced by IL-4. The mechanisms underlying this cooperation are still not defined. However, they likely do not require IRS-1 since TNF-{alpha} also cooperates with IL-4 in 32D cells lacking IRS-1 in the activation of STAT6. One hypothesis is that TNF-{alpha} and IL-4 share a common signaling pathway that regulates the activation of this transcription factor. Supporting this proposal, Guo et al. showed that TNF-{alpha} itself was able to signal JAK1 and STAT6 activation in murine adipocytes (58). In 32D cells, however, we have not detected activation of STAT6 by TNF-{alpha} in the absence of IL-4. Another possibility is that TNF-{alpha} negatively regulates an inhibitor of STAT6 such as a tyrosine phosphatase or SOCS-1 (59, 60). This may explain why the cooperation of TNF-{alpha} with IL-4 in the regulation of STAT6 activity increased over time.

The NF-{kappa}B family of transcription factors controls the expression of genes that play a principal role in the homeostasis of the immune system including the regulation of apoptosis (27, 28). The ability of IL-4 to regulate NF-{kappa}B activity has been studied in several cell types (29–34). Most studies found that IL-4 alone cannot promote NF-{kappa}B activation, but several reports showed that IL-4 could actually inhibit NF-{kappa}B activation induced by LPS or IL-1 (29, 31, 33, 61). In contrast, a report indicated that the cooperation between IL-4 and CD-40 is necessary to induce NF-{kappa}B DNA-binding activity, IgE production and IL-6 production in human B cells (32). Another report revealed that IL-4 was able to enhance the basal activity of NF-{kappa}B in a B cell line or that induced by CD40 engagement (30). Herein, we show that IL-4 enhances the activity of NF-{kappa}B induced by TNF-{alpha} and anti-CD3 antibodies, raising the possibility that the regulation of NF-{kappa}B by IL-4 may be an important event in the cellular response to IL-4.

The mechanisms that IL-4 uses to regulate NF-{kappa}B activity are still undefined. The fact that IL-4 can modify NF-{kappa}B activation by different stimuli and in several cell types suggests that IL-4 regulates a key component in the activation of NF-{kappa}B. This may include the regulation of the I{kappa}B proteins. Indeed, it was recently shown that IL-4 induces Bcl-3 expression in a T cell line (62). In addition, IFN-{gamma} has been shown to enhance NF-{kappa}B activation induced by TNF-{alpha} by prolonging the loss of I{kappa} (63). However, we found that IL-4 did not augment I{kappa}B{alpha} or I{kappa} protein degradation or induce expression of Bcl-3 in 32D cells (J. Zamorano, unpublished data). This is consistent with other studies showing that the IL-4-induced inhibition of NF-{kappa}B activation by LPS in pre-B cells and macrophages was independent of the degradation of I{kappa}B proteins (29, 61). Therefore, the regulation of NF-{kappa}B by IL-4 may be due to mechanisms other than the proteolysis of members of the {kappa}B inhibitor family. Another possibility is that IL-4 may regulate the translocation of NF-{kappa}B complexes to the nucleus. Although we have not directly addressed this issue, Clarke et al. showed that IL-4 did not affect nuclear translocation of p50/p65 proteins induced by LPS (61). Several studies have shown that STAT6 can associate with members of the family of the NF-{kappa}B proteins (64, 65); however, we have not been able to detect STAT6 in NF-{kappa}B complexes in 32D cells using supershift experiments (data not shown).

TNF-{alpha} also cooperates with IL-4 in protecting cells from apoptosis. Although TNF-{alpha} enhances STAT6 activation induced by IL-4, a main role for this protein in this process is unlikely. We and others have shown that STAT6 does not play a protective role in the regulation of spontaneous apoptosis in T cells and 32D cells (20, 45, 54) or apoptosis induced by T cell stimulation (53). A role for NF-{kappa}B, whose activity is enhanced by IL-4, in the regulation of apoptosis by IL-4 seems most likely. TNF-{alpha} can signal both cell death and NF-{kappa}B activation, and in this case the activation of NF-{kappa}B is believed to block cell death signals (36–39). Thus, the inhibition of NF-{kappa}B activation or protein synthesis results in cell death signaling by TNF-{alpha}. In 32D cells, treatment with TNF-{alpha} alone had little effect on the cell survival, suggesting a balance between cell death and survival signals. The enhancement of NF-{kappa}B activity by IL-4 may result in a shift towards survival over death signals resulting in the suppression of cell death. The fact that IL-4 was not able to counteract the induction of cell death by TNF-{alpha} in the presence of proteasome or protein synthesis inhibitors or gliotoxin suggests that IL-4 requires a functional NF-{kappa}B pathway to signal protection from apoptosis. The cooperation between these cytokines may be especially significant in inflammatory responses. IL-4 and TNF-{alpha} production by mast cells and T cells may play an important role during inflammatory processes in asthma (67, 68). It will be interesting to determine whether their cooperative ability to suppress apoptosis plays a role in lung pathology by increasing the survival of cells involved in inflammation.

The results that we obtained in T cells strongly support the hypothesis that IL-4 can cooperate with NF-{kappa}B transcription factors to signal protection from apoptosis. Like TNF-{alpha}, anti-CD3 antibodies can signal cell death and NF-{kappa}B activation (44). It has also been shown that in primary T cells, NF-{kappa}B activity can block apoptotic signals delivered through the TCR complex (44). We found that IL-4 increased NF-{kappa}B activity induced by anti-CD3 antibodies and this was correlated with the ability of IL-4 to protect cells from apoptosis induced after T cell stimulation. Using transgenic lymphocytes expressing a dominant-negative form of I{kappa}B{alpha} that prevents NF-{kappa}B activation, we demonstrate that IL-4-regulated protection from apoptosis induced by anti-CD3 antibodies is dependent on NF-{kappa}B activity.

Over the last several years, it has become evident that in activated T cell blasts, but not resting T cells, IL-4 is able to induce the activation of both STAT5 and STAT6. Activation of STAT5 and STAT3 has been linked to the suppression of apoptosis in a number of cell types (69). Therefore, it may be possible for these STATs to play a role in the IL-4-mediated protection. However, in our hands, IL-4 does not induce the activation of STAT3 or STAT5 in 32D cells whether or not they have been treated with TNF-{alpha} (45 and data not shown). Furthermore, we have demonstrated that IL-4 signaling pathways are largely intact in T cell blasts derived from the I{kappa}B{alpha}-DN transgenic mice, including the tyrosine phosphorylation of IRS2, STAT6 and STAT5 (70). Since these pathways are intact, while the I{kappa}B{alpha}-DN T cells are not protected from apoptosis by IL-4, we do not believe the defect in protection is due to a loss of STAT5 activation. Rather, we favor the proposal that the inhibition of cell death by IL-4 requires the expression of NF-{kappa}B-regulated proteins.

Over the last few years, numerous studies demonstrated the importance of NF-{kappa}B in the regulation of cell death. Although NF-{kappa}B can act as a pro-apoptotic factor (71), it is able to mediate protection from apoptosis in a variety of cell types and systems (36–39, 44, 52). It is believed that this effect of NF-{kappa}B is mediated by the de novo synthesis of proteins with anti-apoptotic activities. Depending on the cell type, the products of several NF-{kappa}B-regulated genes including IAPs, TRAFs, IEX-1L and Bcl-XL have been shown to regulate apoptotic processes (40–43). Although we have shown that IL-4 and other stimuli leading to NF-{kappa}B activation cooperate in the protection from apoptosis, we do not know the molecular mechanism. One possible mechanism is that an increase in NF-{kappa}B activity by IL-4 would result in the enhancement of transcription of survival genes such us those mentioned above. However, we have not been able to associate levels of expression of any of these proteins with the protection from apoptosis by IL-4 (66, and J. Zamorano and A. D. Keegan, personal observations). A second mechanism is that anti-apoptotic proteins regulated by NF-{kappa}B may belong to the machinery that IL-4 requires to signal protection from apoptosis. In this regard, Boothby's group has found that the induction of Bcl-XL, but not TRAFs or IAPs, by anti-CD3 antibodies is dependent on the activation of NF-{kappa}B (52). In addition, gene products regulated by NF-{kappa}B have been shown in other systems to regulate the activation of caspases (42), suggesting that some NF-{kappa}B targets might belong to the machinery activated by IL-4 to protect cells from apoptosis. At this time, we cannot discriminate between these mechanisms, and it is likely that both can occur.

Regardless of the mechanism, the data presented in this study show that the activation of NF-{kappa}B proteins by extracellular signals is an important component of the regulation of apoptosis by IL-4. IL-4 and NF-{kappa}B are also involved in other biological functions such as T and B cell activation, Ig production, and inflammation. To determine whether IL-4 also modifies the activation of NF-{kappa}B in these processes would be important to understand the mechanisms that IL-4 activates to regulate complex immune responses.


    Acknowledgments
 
We wish to thank Dr Jacalyn Pierce for the parental 32D cells, Dr Yufang Shi for the human TNF-{alpha}, and Dr David W. Scott and Dr Wendy Davidson for critical reading of the manuscript. This work was supported by US Public Health Service grants AI38985 and AI45662 (A. D. K.), and GM-42550 and a Leukemia Society of America Scholar's Award (M. B.).


    Abbreviations
 
CHX cycloheximide
IRS insulin receptor substrate
LPS lipopolysaccharide
TNF tumor necrosis factor

    Notes
 
Transmitting editor: D.T. Fearon

Received 9 July 2001, accepted 28 August 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Paul, W. E. 1991. Interleukin 4: a prototypic immunoregulatory lymphokine. Blood 77:1859.[ISI][Medline]
  2. Seder, R. A., Paul, W. E., Davis, M. M. and De St Groth, B. F. 1992. The presence of interleukin-4 during in vitro priming determines the lymphokine-producing potential of CD4+ T-cells from T-cell receptor transgenic mice. J. Exp. Med. 179:1091.
  3. Coffman, R. L., Ohara, J., Bond, M. W., Carty, J., Zlotnick, A. and Paul, W. E. 1986. B cell stimulatory factor-1 enhances the IgE response of lipopolysaccharide-activated B cells. J. Immunol. 136:4538.[Abstract/Free Full Text]
  4. Vitetta, E. S., Ohara, J., Myers, C., Layton, J., Krammer, P. H. and Paul, W. E. 1985. Serological, biochemical, and functional identity of B-cell stimulatory factor-1 and B cell differentiation factor for IgG1. J. Exp. Med. 162:1726.[Abstract]
  5. Brunetti, M., Martelli, N., Colasante, A., Piantelli, M., Musiani, P. and Aiello, F. B. 1995. Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes. Blood 86:4199.[Abstract/Free Full Text]
  6. Foote, L. C., Howard, R. G., Marshak-Rothstein, A. and Rothstein, T. L. 1996. IL-4 induces Fas resistance in B cells. J. Immunol. 157:2749.[Abstract]
  7. Illera, V. A., Perandones, C. E., Stunz, L. L., Mower, D. A. and Ashman, R. F. 1993. Apoptosis in splenic B lymphocytes. Regulation by protein kinase C and IL-4. J. Immunol. 151:2965.[Abstract/Free Full Text]
  8. Parry, S. L., Hasbold, J., Holman, M. and Klaus, G. G. 1994. Hypercross-linking surface IgM or IgD receptors on mature B cells induces apoptosis that is reversed by costimulation with IL-4 and anti-CD40. J. Immunol. 152:2821.[Abstract/Free Full Text]
  9. Zamorano, J., Wang, H. Y., Wang, L.-M., Pierce, J. H. and Keegan, A. D. 1996. IL-4 protects cells from apoptosis via the insulin receptor substrate pathway and a second independent signaling pathway. J. Immunol. 157:4926.[Abstract]
  10. Russell, S. M., Keegan, A. D., Harada, N., Nakamura, Y., Noguchi, M., Leland, P., Friedmann, M. C., Miyajima, A., Puri, R. K., Paul, W. E. and Leonard, W. J. 1993. Interleukin-2 receptor {gamma} chain: a functional component of the interleukin-4 receptor. Science 262:1880.[ISI][Medline]
  11. Miloux B., Laurent, P., Bonnin, O., Lupker, J., Caput, D., Vita, N. and Ferrara, P. 1997. Cloning of the human IL-13R alpha1 chain and reconstitution with the IL4R alpha of a functional IL-4/IL-13 receptor complex. FEBS Lett. 401:163.[ISI][Medline]
  12. Hou J., Schindler, U., Henzel, W. J., Ho, T. C., Brasseur, M. and McKnight, S. L. 1994. An interleukin-4-induced transcription factor: IL-4 Stat. Science 265:1701.[ISI][Medline]
  13. Ihle, J. N. 1995. Cytokine receptor signalling. Nature 377:591.[ISI][Medline]
  14. Quelle, F. W., Shimoda, K., Thierfelder, W., Fischer, C., Kim, A., Ruben, S. M., Cleveland, J. L., Pierce, J. H., Keegan, A. D., Nelms, K., Paul, W. E. and Ihle, J. N. 1995. Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis. Mol. Cell. Biol. 15:3336.[Abstract]
  15. Sun, X. J., Wang, L.-M., Zhang, Y., Yenush, L., Myers, M. G., Glasheen, E., Lane, W. S., Pierce, J. H. and White, M. F. 1995. Role of IRS-2 in insulin and cytokine signaling. Nature 377:173.[ISI][Medline]
  16. Wang, L.-M., Myers, M. G., Jr, Sun, X.-J., Aaronson, S. A., White, M. and Pierce, J. H. 1993. IRS-1: essential for insulin-and IL-4-stimulated mitogenesis in hematopoietic cells. Science 261:1591.[ISI][Medline]
  17. Keegan, A. D., Nelms, K., White, M., Wang, L.-M., Pierce, J. H. and Paul, W. E. 1994. An IL-4 receptor region containing an insulin receptor motif is important for IL-4-mediated IRS-1 phosphorylation and cell growth. Cell 76:811.[ISI][Medline]
  18. Ueno, H., Sasaki, K., Kozutsumi, H., Miyagawa, K., Mitani, K., Yazaki, Y. and Hirai, H. 1996. Growth and survival signals transmitted via two distinct NPXY motifs within leukocyte tyrosine kinase, an insulin receptor-related tyrosine kinase. J. Biol. Chem. 271:27707.[Abstract/Free Full Text]
  19. Yenush, L., Zanella, C., Uchida, T., Bernal, D. and White, M. F. 1998. The pleckstrin homology and phosphotyrosine binding domains of insulin receptor substrate 1 mediate inhibition of apoptosis by insulin. Mol. Cell. Biol. 18:6784.[Abstract/Free Full Text]
  20. Zamorano, J. and Keegan, A. D. 1998. Regulation of apoptosis by tyrosine-containing domains of IL-4R{alpha}: Y497 and Y713, but not the STAT6-docking tyrosines, signal protection from apoptosis. J. Immunol. 161: 859.[Abstract/Free Full Text]
  21. Brunet, L. R., Finkelman, F. D., Cheever, A. W., Kopf, M. A. and Pearce, E. J. 1997. IL-4 protects against TNF-{alpha}-mediated cachexia and death during acute schistosomiasis. J. Immunol. 159:777.[Abstract]
  22. Totpal, K. and Aggarwal, B. B. 1991. Interleukin 4 potentiates the antiproliferative effects of tumor necrosis factor on various tumor cell lines. Cancer Res. 51:4266.[Abstract]
  23. Aversa, G., Punnonen, J. and de Vries, J. E. 1993. The 26-kD transmembrane form of tumor necrosis factor {alpha} on activated CD4+ T cell clones provides a costimulatory signal for human B cell activation. J. Exp. Med. 177:1575.[Abstract]
  24. Boussiotis, V. A., Nadler, L. M., Strominger, J. L. and Goldfeld, A. E. 1994. Tumor necrosis factor {alpha} is an autocrine growth factor for normal human B cells. Proc. Natl Acad. Sci. USA 91:7007.[Abstract]
  25. Barks, J. L., McQuillan, J. J. and Iademarco, M. F. 1997. TNF-{alpha} and IL-4 synergistically increase vascular cell adhesion molecule-1 expression in cultured vascular smooth muscle cells. J. Immunol. 159:4532.[Abstract]
  26. Bennett, B. L., Cruz, R., Lacson, R. G. and Manning, A. M. 1997. Interleukin-4 suppression of tumor necrosis factor {alpha}-stimulated E-selection gene transcription is mediated by STAT6 antagonism of NF-{kappa}B. J. Biol. Chem. 272:10212.[Abstract/Free Full Text]
  27. Baldwin, A. S., Jr. 1996. The NF-{kappa}B proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[ISI][Medline]
  28. Ghosh, S., May, M. J. and Kopp, E. B. 1998. NF-{kappa}B and Rel proteins: evolutionary conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[ISI][Medline]
  29. Clarke, C. J. P., Taylor-Fishwick, D. A., Hales, A., Chernajovsky, Y., Sugamura, K., Feldmann, M. and Foxwel, B. M. J. 1995. Interleukin-4 inhibits {kappa} light chain expression and NF-{kappa}B activation but not I{kappa}B{alpha} degradation in 70Z/3 murine pre-B cells. Eur. J. Immunol. 25:2961.[ISI][Medline]
  30. Delphin, S. and Stavnezer, J. 1995. Characterization of an interleukin 4 (IL-4) responsive region in the immunoglobulin heavy chain germline {varepsilon} promoter: Regulation by NF-IL-4, C/EBP family member and NF-{kappa}B/p50. J. Exp. Med. 181:181.[Abstract]
  31. Donnelly, R. P., Crofford, L. J., Freeman, S. L., Buras, J., Remmers, E., Wilder, R. L. and Fenton, M. J. 1993. Tissue-specific regulation of IL-6 production by IL-4. J. Immunol. 151:5603.[Abstract/Free Full Text]
  32. Jeppson, J. D., Patel, H. R., Sakata, N., Domenico, J., Terada, N. and Gelfand, E. W. 1998. Requirement for dual signals by anti-CD40 and IL-4 for the induction of nuclear factor-{kappa}B, IL-6, and IgE in human B lymphocytes. J. Immunol. 161:1738.[Abstract/Free Full Text]
  33. Takeshita, S., Gage, J. R., Kishimoto, T., Vredevoe, D. L. and Martinez-Maza, O. 1996. Differential regulation of IL-6 gene transcription and expression by IL-4 and IL-10 in human monocytic cell lines. J. Immunol. 156:2591.[Abstract]
  34. Wang, P., Wu, P., Siegel, J. I., Egan, R. W. and Billah, M. M. 1995. Interleukin (IL)-10 inhibits Nuclear Factor {kappa}B (NF-{kappa}B) activation in human monocytes. J. Biol. Chem. 270:9558.[Abstract/Free Full Text]
  35. Pindolia, D. R., Noth, C. J., Xu, Y. X., Janakiraman, N., Chapman, R. A. and Gautam, S. C. 1996. IL-4 upregulates IL-1-induced chemokine gene expression in bone marrow stromal cells by enhancing NF-{kappa}B activation. Hematopathol. Mol. Hematol. 10: 171–185.[ISI][Medline]
  36. Beg, A. A. and Baltimore, D. 1996. An essential role for NF-{kappa}B in preventing TNF-{alpha}-induced cell death. Science 274:782.[Abstract/Free Full Text]
  37. Liu, Z., Hsu, H., Goeddel, D. and Karin, M. 1996. Dissection of TNF- receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-{kappa}B activation prevents cell death. Cell 87:565.[ISI][Medline]
  38. Van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R. and Verma, I. M. 1996. Suppression of TNF-{alpha}-induced apoptosis by NF-{kappa}B. Science 274:787.[Abstract/Free Full Text]
  39. Wang, C. Y., Mayo, M. W. and Baldwin, A. S., Jr. 1996. TNF-{alpha} and cancer therapy-induced apoptosis: potentiation by inhibition of NF-{kappa}B, 1996. Science 274:784.[Abstract/Free Full Text]
  40. Chu, Z. L., McKinsey, T. A., Liu, L., Gentry, J. J., Malim M. H. and Ballard, D. W. 1997. Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-{kappa}B control. Proc. Natl Acad. Sci. USA 94:10057.[Abstract/Free Full Text]
  41. Stehlik, C., de Martin, R., Kumabashiri, I., Schmid, J. A., Binder, B. R. and Lipp, J. 1998. Nuclear factor (NF)-{kappa}B-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor {alpha}-induced apoptosis. J. Exp. Med. 188:211.[Abstract/Free Full Text]
  42. Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V. and Baldwin, A. S., Jr. 1998. NF-{kappa}B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281:1680.[Abstract/Free Full Text]
  43. Wu, M. X., Ao, Z., Prasad, K. V. S., Wu, R. and Schlossman, S. F. 1998. IEX-1L, an apoptosis inhibitor involved in NF-{kappa}B-mediated cell survival. Science 281:998[Abstract/Free Full Text]
  44. Boothby, M. R., Mora, A. L., Scherer, D. C., Brockman, J. A. and Ballard, D. W. 1997. Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-{kappa}B. J. Exp. Med. 185:1897.[Abstract/Free Full Text]
  45. Zamorano, J., Wang, H. Y., Wang, R., Shi, Y., Longmore, G. D. and Keegan, A. D. 1998. Regulation of cell growth by IL-2: Role of STAT5 in protection from apoptosis but not in cell cycle progression. J. Immunol. 160:3502.[Abstract/Free Full Text]
  46. Hotamisligil, G. S., Peraldi, P., Budavari, A., Ellis, R., White, M. F. and Spiegelman, B. M. 1996. IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-{alpha} and obesity-induced insulin resistance. Science 271:665.[Abstract]
  47. Peraldi, P., Hotamisligil, G. S., uurman, W. A., White, M. F. and Spiegelman, B. M. 1996. Tumor necrosis factor (TNF-)-{alpha} inhibits insulin signaling through stimulation of the p55 TNF receptor and activation of sphingomyelinase. J. Biol. Chem. 271:13018.[Abstract/Free Full Text]
  48. Askew, D. S., Ashmon, R. A., Simmons, B. C. and Cleveland, J. L. 1991. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene 6:1915.[ISI][Medline]
  49. Lewis, M., Tartaglia, L. A., Lee, A., Bennett, G. L., Rice, G. C., Wong, G. H. W., Chen, E. Y. and Goeddel, D. V. 1991. Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc. Natl Acad. Sci. USA 88:2830.[Abstract]
  50. Tartaglia, L. A., Rothe, M., Hu, Y. F. and Goeddel, D. V. 1993. Tumor necrosis factor's cytotoxic activity is signaled by the p55 TNF receptor. Cell 73:213.[ISI][Medline]
  51. Lugli, S. M., Feng, N., Heim, M. H., Adam, M., Schnyder, B., Etter, H., Yamage, M., Eugster, H. P., Lutz, R. A., Zurawski, G. and Moser, R. 1997. Tumor necrosis factor {alpha} enhances the expression of the interleukin (IL)-4 receptor {alpha}-chain on endothelial cells increasing IL-4 or IL-13-induced Stat6 activation. J. Biol. Chem. 272:5487.[Abstract/Free Full Text]
  52. Lee, S. Y., Kaufman, D. R., Mora, A. L., Santana, A., Boothby, M. and Choi, Y. 1998. Stimulus-dependent synergism of the antiapoptotic tumor necrosis factor receptor-associated factor 2 (TRAF2) and nuclear factor {kappa}B pathways. J. Exp. Med. 188:1381.[Abstract/Free Full Text]
  53. Kaplan, M. H., Daniel, C., Schindler, U. and Grusby, M. J. 1998. Stat proteins control lymphocyte proliferation by regulating p27kip1 expression. Mol. Cell. Biol. 18:1996.[Abstract/Free Full Text]
  54. Vella, A., Teague, T. K., Ihle, J., Kappler, J. and Marrack, P. 1997. Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: stat6 is probably not required for the effect of IL-4. J. Exp. Med. 186:325.[Abstract/Free Full Text]
  55. Pahl, H. L., Kraus, B., Schultze-Osthoff, K., Decker, T., Traenckner, E. B., Vogt, M., Myers, C., Parks, T., Warring, P., Muhlbacher, A., Czernilofsky, A.-P. and Baeuerle, P. A, 1996. The immunosuppressive fungal metabolite gliotoxin specifically inhibits transcription factor NF-{kappa}B. J. Exp. Med. 183:1829.[Abstract]
  56. Herfarth, H., Brand, K., Rath, H. C., Rogler, G., Scholmerich, J. and Falk, W. 2000. Nuclear factor-kappa B activity and intestinal inflammation in dextran sulphate sodium (DSS)-induced colitis in mice is suppressed by gliotoxin. Clin. Exp. Immunol. 120:59.[ISI][Medline]
  57. Lin, Y. Z., Yao, S. Y., Veach, R. A., Torgerson, T. R. and Hawiger, J. 1995. Inhibition of nuclear translocation of transcription factor NF-{kappa}B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J. Biol. Chem. 270: 14255–14258.[Abstract/Free Full Text]
  58. Guo, D., Dunbar, J. D., Yang, C. H., Pfeffer, L. M. and Donner, D. B. 1998. Induction of Jak/STAT signaling by activation of the type 1 TNF receptor. J. Immunol. 160:2742.[Abstract/Free Full Text]
  59. Losman, J. A., Chen, X. P., Hilton, D. and Rothman, P. 1999. SOCS-1 is a potent inhibitor of IL-4 signal transduction. J. Immunol. 162:3770.[Abstract/Free Full Text]
  60. Dickensheets, H. L., Venkataraman, C., Schindler, U. and Donnelly, R. P. 1999. Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by inducing SOCS-1 gene expression. Proc. Natl Acad. Sci. USA 96:10800.[Abstract/Free Full Text]
  61. Clarke, C. J. P., Hales, A., Hunt, A. and Foxwell, B. M. J. 1998. IL-10-mediated suppression of TNF-{alpha} production is independent of its ability to inhibit NF-{kappa}B activity. Eur. J. Immunol. 28:1719.[ISI][Medline]
  62. Rebollo, A., DuMoutier, L., Renauld, J.-C., Zaballos, A., Ayllon, V. and Matrinez-A, C. 2000. Bcl-3 expression promotes cell survival following IL-4 deprivation and is controlled by AP-1 and AP-1-like transcription factors. Mol. and Cell. Biol. 20:3407.[Abstract/Free Full Text]
  63. Cheshire, J. L. and Baldwin, A. S., Jr. 1997. Synergistic activation of NF-{kappa}B by TNF-{alpha} and IFN{gamma} via enhanced I{kappa}B{alpha} degradation and de novo I{kappa}B{alpha} degradation. Mol Cell. Biol. 17:6746.[Abstract]
  64. Messner, B., Stutz, A. M., Albrecht, B., Peiritsch, S. and Woisetschlager, M. 1997. Cooperation of binding sites for STAT6 and NF-{kappa}B/rel in the IL-4-induced up-regulation of the human IgE germline promoter. J. Immunol. 159:3330.[Abstract]
  65. Shen, C. H. and Stavnezer, J. 1998. Interaction of Stat6 and NF-{kappa}B: direct association and synergistic activation of interleukin-4-induced transcription. Mol. Cell. Biol. 18:3395.[Abstract/Free Full Text]
  66. Aronica, M. A., Goenka, S. and Boothby, M. 2000. IL-4-dependent induction of Bcl-2 and Bcl-xL in activated T lymphocytes through a STAT6- and PI 3-kinase-independent pathway. Cytokine 12:578.[ISI][Medline]
  67. Bradding, P., Roberts, J. A., Britten, K. M., Montefort, S., Djukanovic, R., Mueller, R., Heusser, C. H., Howarth, P. H. and Holgate, S. T. 1994. Interleukin-4, -5, and -6 and tumor necrosis factor-alpha in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines. Am. J. Respir. Cell. Mol. Biol. 10:471.[Abstract]
  68. Gibbs, B. F., Arm, J. P., Gibson, K., Lee, T. H. and Pearce, F. L. 1997. Human lung mast cells release small amounts of interleukin-4 and tumour necrosis factor-alpha in response to stimulation by anti-IgE and stem cell factor. Eur. J. Pharmacol. 327:73.[ISI][Medline]
  69. Leonard, W. J. and O'Shea, J. J. 1998. Jaks and STATs: biological implications. Annu. Rev. Immunol. 16:293.[ISI][Medline]
  70. Mora, A., Youn, J. H., Keegan, A. D. and Boothby, M. R. 2001. NF{kappa}b/Rel participation in the lymphokine-dependent proliferation of T cells. J. Immunol. 166:2218.[Abstract/Free Full Text]
  71. Hettmann, T., Didonato, J., Karin, M. and Leiden, J. M. 1999. An essential role for nuclear factor {kappa}B in promoting double positive thymocyte apoptosis. J. Exp. Med. 189:145.[Abstract/Free Full Text]