A Novel Role for IκBζ in the Regulation of IFNγ Production

IκBζ is a novel member of the IκB family of NFκB regulators, which modulates NFκB activity in the nucleus, rather than controlling its nuclear translocation. IκBζ is specifically induced by IL-1β and several TLR ligands and positively regulates NFκB-mediated transcription of genes such as IL-6 and NGAL as an NFκB binding co-factor. We recently reported that the IL-1 family cytokines, IL-1β and IL-18, strongly synergize with TNFα for IFNγ production in KG-1 cells, whereas the same cytokines alone have minimal effects on IFNγ production. Given the striking similarities between the IL-1R and IL-18R signaling pathways we hypothesized that a common signaling event or gene product downstream of these receptors is responsible for the observed synergy. We investigated IκBζ protein expression in KG-1 cells upon stimulation with IL-1β, IL-18 and TNFα. Our results demonstrated that IL-18, as well as IL-1β, induced moderate IκBζ expression in KG-1 cells. However, TNFα synergized with IL-1β and IL-18, whereas by itself it had a minimal effect on IκBζ expression. NFκB inhibition resulted in decreased IL-1β/IL-18/TNFα-stimulated IFNγ release. Moreover, silencing of IκBζ expression led to a specific decrease in IFNγ production. Overall, our data suggests that IκBζ positively regulates NFκB-mediated IFNγ production in KG-1 cells.


Introduction
We previously showed that the Interleukin-1 (IL-1) family members, IL-1b and IL-18, synergize with tumor necrosis factor-a (TNFa) for interferon-c (IFNc) production in the human acute myeloid leukemic KG-1 cell line [1]. IL-1b and IL-18 signal via the Interleukin-1 receptor (IL-1R) and IL-18R, respectively, both of which belong to the IL-1R family and the interleukin-1R/Tolllike receptor (IL-1R/TLR) superfamily [2][3][4][5]. Members of the IL-1R/TLR family share a cytoplasmic domain known as the Toll/ interleukin-1 receptor (TIR) domain and recruit similar adaptor proteins, such as MyD88. Due to these and other similarities, the signaling pathways downstream of IL-1Rs and TLRs lead to similar outcomes, such as the activation of NFkB and MAPKs.
We recently showed that both, IL-1b and IL-18, synergize with TNFa for IFNc production in KG-1 cells [1]. Given the similarities between the IL-1R and IL-18R signaling pathways, we hypothesized that a common event downstream of these two receptors is crucial for the observed synergy between IL-1b/IL-18 and TNFa for IFNc production. Even though both, the IL-1R and IL-18R, belong to the IL-1R/TLR superfamily, and IkBf is specifically induced upon stimulation with several IL-1R/TLR ligands, IkBf expression has not been investigated in response to IL-18 stimulation. Therefore, we analyzed IkBf expression in KG-1 cells upon IL-18 and IL-1b stimulation, and the role of IkBf in IFNc production in response to combined IL-1b/IL-18 and TNFa stimulation. Our results indicate that stimulation with IL-1b and/ or IL-18 results in moderate levels of IkBf production, while TNFa has no effect. However, when combined with IL-1b or IL-18, TNFa strongly enhances IkBf protein expression. Moreover, NFkB inhibition, as well as silencing of IkBf expression, resulted in decreased IL-1b/IL-18/TNFa-induced IFNc production. Furthermore, IL-1R and IL-18R expression analysis indicated that the observed synergy may take place at the receptor level in the case of IL-18 and TNFa, but not IL-1b and TNFa combined stimulation. In summary, our findings indicate that stimulation with the IL-1 cytokines, IL-1b and IL-18, in combination with TNFa results in synergistic KG-1 IFNc production in an IkBf/ NFkB dependent manner.
KG-1 cells were plated at a final cell density of 10 6 /ml and incubated with test samples (monocyte conditioned media, 1/3 of the final volume) or recombinant proteins (10 ng/ml each). In selected experiments, neutralizing agents for IL-1b (IL-1b Ab -clone 2805, IL-1ra), IL-18 (IL-18 Ab -clone 125-2H, or IL-18R Ab -clone 70625), and/or TNFa (TNFa Ab -clone 2C8) were used at the indicated concentrations to neutralize the activities of rIL-18, rIL-1b and/or rTNFa, or the same endogenous cytokines present in the monocyte conditioned media, prior to incubation with KG-1 cells. Alternatively, the neutralizing agents, IL-1ra and IL-18R Ab, were used to neutralize IL-18 or IL-1 receptors prior to addition of rIL-18, rIL-1b, or monocyte conditioned media, to the KG-1 cells. KG-1 supernatants and cells were harvested at various time points for subsequent ELISA and Western blot analysis. In selected experiments, cells were treated with the NFkB inhibitor, JSH-23 (30 mM) (an NFkB nuclear translocation inhibitor), in combination with rIL-1b, rIL-18 and rTNFa for 24 h. KG-1 cells were harvested at 12 h for subsequent nuclear extraction and Western blot analysis. KG-1 supernatants were harvested at 24 h for IL-6 and IFNc ELISA.

Western blotting
KG-1 cells were lysed in a 60 mM Tris-HCl (pH 6.8), 2% SDS buffer. Total cell extracts were sonicated and spun (5 min; 10 000 rpm, R.T.) to remove cell debris. KG-1 nuclear/cytosolic extracts were also analyzed by Western blotting. Protein concentration in total, nuclear or cytosolic extracts was estimated using the Bio-Rad Dc protein Lowry assay (Bio-Rad). Samples were boiled in Laemmli's buffer for 5 min or heated at 70uC for 10 min in NuPAGE Sample Reducing Agent (Invitrogen). 10-40 mg of total protein were loaded per well on pre-cast 10% Tris-Glycine or 7% Tris-Acetate SDS-PAGE gels and transferred to a PVDF or nitrocellulose membrane. Membranes were blocked with 10% nonfat milk (Carnation, Nestle) in 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween for 1 h at R.T. The membranes were probed with the indicated primary Abs, followed by peroxidase-conjugated secondary antibodies. Protein bands were visualized by chemiluminescence (GE Healthcare).

ELISA
Sandwich ELISAs were used to measure cytokine release in the supernatants of KG-1 cells.

Flow Cytometry
KG-1 cells (10 6 /ml) were stimulated, or not, with the indicated combinations of rIL-1b, rIL-18 and rTNFa (10 ng/ml each) for 24 h. Cells were Fc-blocked by treating with 1 mg of human IgG/ 10 5 for 15 min at R.T. Cells (10 5 /25 ml*reaction) were transferred to a 5 ml tube. Phycoerythrin (PE)-conjugated anti-IL-18Ra or fluorescein (FITC)-conjugated anti-IL-1R1 reagent (10 ml of each per reaction) were added to the cells. Cells were incubated for 30 min at 4uC, washed twice with 16PBS and re-suspended in 16PBS (10 5 /200 ml) for flow cytometric analysis. As controls, cells were also treated with phycoerythrin-labeled murine IgG 1 and fluorescein-labeled goat IgG.
Nuclear/Cytosolic extraction KG-1 cells (10 6 /ml) were stimulated, or not, with rIL-1b, rIL-18, rTNFa (10 ng/ml each), or a combination of this cytokines for the indicated time points. Cells were washed twice in 16PBS and gently re-suspended in cold Buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM dithiothreitol [DTT]; 16Complete Mini protease inhibitor cocktail, Roche) at 400 ml/ 0.521610 6 cells. Cells were allowed to swell for 15 min. 10% Nonidet NP-40 was added to the solution (25 ml per 400 ml). Samples were vortexed for 10 sec and centrifuged for 30 sec (4uC, 13 200 rpm). Supernatants containing cytosolic contents were transferred to fresh tubes containing an equal volume of Buffer B (10 mM Tris-HCl, pH 7.5; 7 M urea; 1% SDS; 0.3 M NaAc; 20 mM EDTA) and stored immediately at 220uC. The pellets containing the nuclear contents were re-suspended in cold Buffer C (20 mM HEPES, pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM dithiothreitol (DTT); 16Complete Mini protease inhibitor cocktail, Roche) at 50 ml/0.521610 6 cells. The samples were vigorously shook for 15 min at 4uC on a shaking platform and then centrifuged for 5 min (4uC, 13 200 rpm). The supernatants with the nuclear contents were stored at 220uC. Nuclear and cytosolic extracts were subsequently analyzed for protein concentration using the Bio-Rad Dc protein Lowry assay (Bio-Rad). Nuclear and cytosolic extracts were then prepared for Western blot analysis.

Small interfering RNA
KG-1 cells (2610 6 /ml) were nucleofected following the protocol for KG-1 cell nucleofection provided with the cell line nucleofector kit R from Amaxa (Gaithersburg, MD) with a mixture of 3 different small interfering RNA (siRNA) oligonucleotides against IkBf or 3 different scrambled siRNA oligonucleotides (3 mg per 2610 6 cells). After 2 h, cells were stimulated with a combination of rIL-1b, rIL-18 and rTNFa (10 ng/ml each). Cells and supernatants were harvested at 24 h for subsequent RNA and protein analysis (qPCR and Western blot, respectively).

Quantitative PCR (qPCR)
KG-1 cells (10 6 /ml) were lysed in TRIzol reagent (Invitrogen Life Technologies) and mRNA was extracted and converted to cDNA using the Thermoscript RT-PCR system (Invitrogen Life Technologies). qPCR was performed using specific primers for IFNc, IL-6 and IL-8. Values were normalized to two housekeeping genes, CAP-1 and GAPDH.

Statistical analysis
Data are presented mean6S.E.M. from$3 independent experiments. Comparisons were done by paired t-test with p,0.05 defined as statistically significant.
In this context, KG-1 cells were stimulated with rIL-1b, rIL-18 and rTNFa for various time points, with and without co-addition of IL-1ra or IL-18 Ab. Total cell extracts were analyzed for IkBf protein expression by Western blotting. Results indicated that both rIL-1b and rIL-18, but not rTNFa, induced IkBf expression in KG-1 cells (Fig. 1A-C). The lack of rTNFa-mediated IkBf expression was not due to lack of biological activity of rTNFa, as judged by the modulation (degradation and de novo protein synthesis) of IkBa expression upon rTNFa stimulation (Fig. 1C). The finding that IL-1b, as well as IL-18, both induce IkBf protein expression supports the hypothesis that IkBf may be the common factor downstream of the IL-1R and IL-18R that allows for synergy between IL-1 cytokines (IL-1b and IL-18) and TNFa for IFNc production in KG-1 cells.

TNFa enhances IL-1b/IL-18-mediated IkBf expression.
Stimulation with TNFa alone does not lead to IkBf expression ( Fig. 1C) [19][20][21]24]. However, TNFa has been shown to induce IkBf transcription to a greater extent than IL-1b and LPS [20]. In contrast, upon actinomycin D treatment, the half-life of ectopically expressed IkBf mRNA was prolonged with IL-1b and LPS, but unaffected by TNFa. Therefore, even though TNFa has strong IkBf transcriptional activity, IL-1/LPS may provide additional mRNA stabilization (absent with solo TNFa stimulation) leading to subsequent protein expression. Based on this information, we hypothesized that TNFa may enhance IL-1b/IL-18-mediated IkBf protein expression by providing strong transcriptional activation, even though by itself it does not lead to IkBf protein expression.
To test this hypothesis, KG-1 cells were stimulated with rTNFa alone and in combination with rIL-1b, rIL-18, or both, for 8 and 24 h. Total cell extracts were analyzed for IkBf protein expression by Western blotting. Recombinant TNFa enhanced rIL-1b-and rIL-18-mediated IkBf protein expression at both time points (Fig. 2). We then analyzed the kinetics of KG-1 IkBf protein expression in response to different combinations of rIL-1b, rIL-18 and rTNFa, with and without co-addition of IL-1ra, IL-18R Ab, TNFa Ab, or different combinations of these neutralizing agents. Interestingly, IkBf protein expression followed an oscillating pattern (Fig. 3), which is typical of IkB proteins, such as IkBa. Moreover, it was evident that the observed induction of IkBf protein upon stimulation with rTNFa combined with rIL-1b, rIL-18, or both, was in part due to rTNFa, since the induction was only partially suppressed with a TNFa neutralizing Ab (Fig. 3B and C). The remaining IkBf protein expression after TNFa neutralization was likely due to IL-1b and/or IL-18. As expected, neutralization with IL-1ra or IL-18R Ab, resulted in complete inhibition of IkBf expression, indicating that TNFa by itself has no IkBf inducing activity. Interestingly, rIL-1b, rIL-18 or the combination of these cytokines (at a dose of 10 ng/ml) results in minimal amounts of IFNc production by KG-1 cells, despite their ability to induce IkBf protein expression [1]. Therefore, the levels of IL-1b/IL-18-induced IkBf protein may either not be sufficient for significant IFNc production (in the absence of TNFa stimulation), or may require an additional TNFa-induced factor for activation of the IFNc promoter.  The conditioned media from LPS/ATP-stimulated monocytes induces IkBf expression in an IL-1bdependent, but IL-18-independent manner We have recently shown that the conditioned media from LPS/ ATP-treated monocytes induces IFNc release by KG-1 cells and that this induction is due to the synergistic effect of IL-1b and TNFa, and independent of IL-18 [1]. Herein, we incubated KG-1 cells with conditioned media from LPS/ATP-stimulated monocytes for various time points, with and without co-addition of IL-1ra, IL-18R Ab, TNFa Ab, or different combinations of these neutralizing agents, and analyzed IkBf protein expression. The monocyte conditioned media induced IkBf protein expression in an IL-1b-dependent, but IL-18-independent manner (Fig. 4). This finding correlates with our previous observation that endogenous IL-18 present in the conditioned media from LPS/ATPstimulated monocytes does not induce IFNc production by KG-1 cells [1]. The lack of IL-18 IFNc inducing activity in the supernatants of LPS/ATP-stimulated monocytes may be due to low levels of IL-18 being released or to IL-18 being bound to its biological inhibitor, IL-18BP [1].

TNFa upregulates IL-18R, not IL-1R expression
TNFa has been shown to upregulate expression of the IL-18R in KG-1 cells [51][52][53][54][55][56][57]. Therefore, TNFa may synergize with IL-1b in a similar manner for IkBf and IFNc production -by upregulating surface expression of the IL-1R. In order to explore this possibility, KG-1 cells were treated with the indicated combinations of rIL-1b, rIL-18 and rTNFa for 24 h. TNFa treatment resulted in upregulation of IL-18R, not IL-1R expression, as determined by flow cytometry (Fig. 5). Therefore, a signaling event(s) downstream of the IL-1R and IL-18R, rather that TNFa-mediated receptor upregulation, is likely to be crucial for the observed synergy between IL-1 cytokines and TNFa for IkBf and IFNc production. Moreover, the greater IFNc production in response to rIL-18 in combination with rTNFa, compared to rIL-1b in combination with rTNFa [1], may be explained by additional TNFa-mediated upregulation of the IL-18R.

IkBf protein localizes to the nucleus
IkBf protein has been shown to localize to the nucleus in most cell types [19,23,26,28,34]. However, IkBf has also been shown to localize to the cytoplasm in B cell rich regions of immune organs, such as lymphoid follicles in the spleen [28]. In order to confirm the cellular localization of IkBf protein in the KG-1 cell line, cells were stimulated with rIL-1b, rIL-18, rTNFa and different combinations of these cytokines for 8 h and harvested for cytosol and nuclear extraction. Results demonstrate that IkBf protein localizes predominantly to the nucleus of KG-1 cells (Fig. 6), consistent with its role as a co-factor for NFkB-mediated transcription.

NFkB inhibition leads to decreased IFNc and IL-6 release
IkBf has been shown to act as an NFkB binding co-factor by associating with the p50 NFkB subunit. Therefore, we decided to test whether IFNc release in KG-1 cells in response to IL-1b, IL-18 and TNFa combined stimulation is NFkB dependent. KG-1 cells were incubated with an inhibitor of NFkB nuclear  translocation followed by IL-1b, IL-18 and TNFa combined stimulation. Western blot analysis with nuclear extracts indicated a reduction in p50 and p65 nuclear localization, indicative of a decrease in NFkB activity (data not shown). Moreover, IL-6 and IFNc release were significantly reduced with NFkB inhibition (Fig. 7). Therefore, IkBf may regulate IFNc release in KG-1 cells in response to IL-1b, IL-18 and TNFa combined stimulation by acting as a co-factor for NFkB-mediated transcription.
In order to test this hypothesis, KG-1 cells were nucleofected with a mixture of 3 different small interfering RNA (siRNA) oligonucleotides against IkBf or 3 different scrambled siRNA oligonucleotides. Cells were then stimulated with a combination of rIL-1b, rIL-18 and rTNFa. Western blot analysis indicated a reduction in IkBf protein expression with anti-IkBf siRNA delivery (Fig. 8). In order to determine the effect of IkBf silencing on IFNc protein production, we measured IFNc mRNA levels and protein release in KG-1 cells upon rIL-1b, rIL-18 and rTNFa combined stimulation (Fig. 9). As a positive   control, we measured IL-6 mRNA and protein release since this cytokine has been shown to be positively regulated by IkBf [21,26,32,34,35]. As a negative control, we measured IL-8 mRNA and protein levels, which have been shown not to be regulated by IkBf [24,33]. Results indicated that the mRNA and protein levels of IFNc and IL-6, but not IL-8, were significantly reduced with anti-IkBf siRNA delivery (Fig. 9). These results implicate a role for IkBf as a positive regulator of IFNc production.

Discussion
Regulation of IFNc gene transcription involves the action of many different transcripition factors including STATs, AP-1, GATA-3, NFAT, T-bet, Eomesodermin, NFkB, NFAT, T-bet, YY-1, DREAM, ERM and SMADs. In most cases, multiple signals synergize for IFNc production via induction of different transcription factors that act in concert to induce gene expression [58][59][60][61]. The combination of IL-12 and IL-18 is the most wellknown example of synergy between two cytokines for IFNc production in T cells, NK/NKT cells, B cells, macrophages and dendritic cells [62][63][64][65][66][67][68][69][70]. Synergy between IL-12 and IL-18 occurs not only at the transcription factor level via STAT4 and AP-1 activation, respectively, but also at the receptor level, with both cytokines upregulating cell surface expression of each other's receptors. Synergy for IFNc production has also been observed with the combination of receptor crosslinking and cytokine stimulation. As an example, the combination of LY49 activating receptor crosslinking and IL-12 or IL-18 synergistically enhance IFNc production in NK cells via the p38 MAP kinase and the ERK-dependent signal transduction pathways [71].
In general, IL-12 and IL-18 require each other for IFNc gene expression. However, at high doses (50 ng/ml), IL-18 alone can induce IFNc production in the human acute myeloid leukemic KG-1 cell line [1,24]. KG-1 cells have been widely used to study IL-18-mediated signaling events leading to IFNc expression. The responsiveness of KG-1 cells to IL-18 (in absence of IL-12) is partly due to constitutive expression of both chains of the IL-18R [72,73], whereas primary NK and T cells require IL-12 stimulation for expression of the binding chain of the IL-18R [70,[74][75][76][77].
NFkB has been shown to regulate the expression of many proinflammatory genes, IFNc being no exception. Two putative NFkB binding sites have been identified in the IFNc promoter region (kBB site and CD28RE) and one in the first intron (C3) [78]. The requirement for NFkB in IFNc gene expression appears to be contingent on the cell type and the specific stimulus. IL-18 signaling via the IL-18R leads to NFkB activation [78][79][80][81][82]. Moreover, stimulation of KG-1 cells with high doses (50 ng/ml) of IL-18 leads to IFNc production in an NFkB-dependent manner [78].
We have previously described a novel synergistic role for the members of the IL-1 family, IL-1b and IL-18, in combination with TNFa in IFNc production in KG-1 cells [1]. Importantly, at the dose of 10 ng/ml, the individual cytokines induced only minimal amounts of IFNc release. Given the striking similarities between the IL-1b and IL-18 signaling pathways, we proposed that a common factor downstream of the IL-1R and IL-18R is responsible for the observed synergy between IL-1b/IL-18 and TNFa. The latter is supported by the fact that induction of the IFNc promoter is generally mediated by multiple signals leading to activation of multiple transcription factors that synergistically induce IFNc gene expression [58][59][60][61].
The novel member of the IkB family of NFkB regulators, IkBf, is known to be induced by IL-1R/TLR ligands. Moreover, even though the TNFR signaling pathway shares some similarities with the IL-1R/TLR pathway, such as the use of TRAF adaptor molecules, TNF signaling alone does not result in IkBf protein expression [2][3][4][5][19][20][21][22][23][24]. Although IL-18 signals via a member of the IL-1R family, it has never been tested as an inducer of IkBf expression. We have shown for the first time that IL-18 stimulation also leads to IkBf protein expression in KG-1 cells.
IkBf has been shown to positively regulate NFkB-mediated transcription of secondary response genes such as IL-6 and NGAL, as a co-factor binding to the p50 NFkB subunit [21,24,26,32,33,35]. Moreover, NFkB has been shown to play an important role as a positive regulator of IFNc gene expression in thymocytes, peripheral blood T lymphocytes and KG-1 cells [66,78,83]. Therefore, we hypothesized that IkBf may be the common factor downstream of the IL-1R and IL-18R pathways, which allows for synergy between IL-1 cytokines and TNFa for IFNc production in KG-1 cells.
Interestingly, we observed that TNFa enhanced IL-1b/IL-18mediated IkBf protein expression, even though by itself it had not effect on IkBf protein expression. However, TNFa, IL-1b and LPS have all been shown to induce IkBf mRNA transcription in NIH3T3 and A549 cells [20]. Importantly, TNFa stimulation alone results in strong activation of the IkBf promoter without subsequent protein expression. Moreover, nuclear run-on analysis in NIH3T3 cells also indicates that TNFa is a stronger transcriptional activator of the IkBf gene, compared to IL-1b or LPS. Furthermore, decay analysis of ectopically expressed IkBf mRNA upon actinomycin D treatment, indicates that degradation of IkBf mRNA is delayed by IL-1b and LPS stimulation, but not by TNFa stimulation. Moreover, the N-terminal half, not the C-terminal half, of the IkBf ORF confers IL-1/LPS-mediated IkBf mRNA stabilization. Therefore, the specificity of IL-1/LPS stimulus for IkBf mRNA and protein induction is most likely at the posttranscriptional level and due to stabilization of IkBf mRNA. A cis-element in the N-terminal half of the IkBf gene appears to be crucial for this stabilization [20].
In support of the latter findings, the NGAL promoter has been shown to be specifically induced by IL-1b, not by TNFa [9,21,84,85]. NGAL promoter activity requires NFkB activation and an intact NFkB binding site [24]. Even though IL-1b and TNFa, both induce NFkB nuclear translocation and recruitment to the NGAL promoter, only IL-1b is able to induce NGAL expression. IkBf has been shown to be the co-factor which allows NFkB to mediate NGAL gene expression downstream of the IL-1R/TLR signaling pathway [24]. The latter was shown by IkBf over expression in A549 cells, which rescued TNFa-induced NGAL expression. The same may apply to expression of the IL-6 gene, which at least in the KG-1 cell line (data not shown) and other cell types [16][17][18], is specific to IL-1b, not TNFa stimulation, and to other genes specifically induced downstream of the IL-1R/TLR pathway. As an additional example, hBD2 is also stimulated by IL-1b, but not TNFa stimulation in human keratinocytes [9,84] and A549 cells [24]. Moreover, siRNA experiments have shown that IkBf is critical for IL-1b mediated hBD2 mRNA expression in A549 cells [24].
Based on this information, we conclude that while IL-1b and IL-18 may provide the signal(s) required for IkBf transcriptional activation, mRNA stabilization and subsequent IkBf protein expression, TNFa may enhance IL-1b/IL-18-mediated IkBf expression by providing strong transcriptional activation of the IkBf gene. Moreover, TNFa stimulation of KG-1 cells provides robust binding of the p50 and p65 NFkB subunits to EMSA probes containing the NFkB binding sites present in the IFNc promoter and first intron (data not shown), compared to weaker binding provided by IL-1b and IL-18. Therefore, robust NFkB activation provided by TNFa may also result in increased IFNc gene expression since IkBf regulates transcription as a co-factor for NFkB and KG-1 IFNc production in response to IL-1/TNFa combined stimulation is NFkB dependent (Fig. 7). Thus, the synergy between IL-1b/IL-18 and TNFa may be due to their combined effects on IkBf expression, as well as on NFkB activation. Alternatively, other transcription factors induced by TNFa may synergize with IkBf/NFkB for IFNc gene expression. Receptor expression analysis indicated that TNFa-mediated upregulation of IL-1/IL-18R expression did not account for the synergy between these cytokines for IkBf production.
In summary, we have shown a positive role for IkBf on IFNc production in response to IL-1b, IL-18 and TNFa combined stimulation in KG-1 cells. This regulation is most likely dependent on the ability of IkBf to regulate NFkB mediated transcription of the IFNc gene. This finding represents a new addition to the complex and continuously growing literature on the regulation of IFNc expression.