Activation of Acid Sphingomyelinase by Interleukin-1 (IL-1) Requires the IL-1 Receptor Accessory Protein*

The cytokine interleukin-1 (IL-1) plays an important role in inflammation and regulation of immune responses, but the mechanisms of its signal transduction and cell activation processes are incompletely understood. Ceramide generated by sphingomyelinases (SMases) is known to function as an important second messenger molecule in the signaling pathway of IL-1 and tumor necrosis factor. To investigate the activation of SMases by IL-1, we used an IL-1 receptor type I (IL-1RI)-positive EL4 thymoma cell line, which is defective in IL-1R accessory protein (IL-1RAcP) expression. In this cell line (EL4D6/76), tumor necrosis factor induced ligand/receptor internalization, NFκB nuclear translocation, IL-2 production, and the activation of neutral (N)-SMase and acid (A)-SMase. In contrast, stimulation with IL-1 resulted only in the activation of N-SMase whereas ligand/receptor internalization, NFκB translocation, IL-2 production, and activation of A-SMase were not detected. Transfection of this functionally defective EL4D6/76 with IL-1RAcP cDNA restored these functions. These data suggest that A-SMase activity is strongly linked with the internalization of IL-1RI mediated by IL-1RAcP and that A-SMase and N-SMase are activated by different pathways.

Interleukin-1 (IL-1) 1 and tumor necrosis factor (TNF) belong to a group of pro-inflammatory cytokines with overlapping biological activities, which might be brought about by common signaling mechanisms (1)(2)(3). In the past few years, several groups have reported the involvement of sphingomyelin breakdown in the signaling of IL-1 and TNF (4 -6). Ceramide generated by sphingomyelinases (SMases) is an important second messenger molecule in signal transduction pathways of IL-1 (7,8), TNF (9), and CD28 (10). Ceramides appear to be involved in cell differentiation, apoptosis, and cell cycle arrest (11)(12)(13), e.g. ceramide was able to mimic interferon-␥ and TNF effects in the differentiation of the monocytic cell line HL60 (14). Different types of cell-permeable ceramides induced apoptosis in various cell systems (15,16). In cell cycle studies, C 6 -ceramides have been demonstrated to induce growth suppression by dephosphorylation of Rb (17,18). For IL-1␤, the involvement of sphingomyelin hydrolysis to ceramide and stimulation of a ceramideactivated protein kinase has been reported (19,20). Synthetic cell-permeable ceramides or exogenous SMase have been shown to bypass IL-1 receptor activation in EL4 cells and mimic biologic activities of this cytokine (8). The activity of ceramide-activated protein kinase is directed to c-Raf-1 and appears to be activated by TNF and IL-1. Other targets of downstream signaling processes of ceramides are ceramideactivated protein phosphatase (21,22) and protein kinase C (23). Additional events in the signaling cascade of ceramides are the phosphorylation of mitogen-activated protein kinase and activation of the c-Raf-1 kinase (24,25).
Binding of TNF to the 55-kDa TNF receptor activated two different types of SMases, a membrane-associated neutral (N)-SMase and an endosomally located acid (A)-SMase (9). Structure-function analyses of the p55 TNF receptor revealed that the SMases are activated independently through different cytoplasmatic domains of the receptor (26). Diacylglycerol (DAG) generated by a phosphatidylcholine-specific phospholipase C (PC-PLC) has been reported to serve as important factor of activation of A-SMase, which, through the generation of ceramide, is a co-factor for the activation of NFB (27). A key event in NFB activation is the rapid degradation of the inhibitory protein IB. In a cell-free system, SMase and synthetic ceramide could directly induce IB degradation, strongly indicating the involvement of SMase in NFB activation (28). On the other hand, N-SMase seems to exert its signaling capacity via proline-directed protein kinases, like ceramide-activated protein kinase and mitogen-activated protein kinase, which acts in turn on phospholipase A 2 (6).
IL-1 activity is represented by three structurally related molecules (3,29,30). IL-1␣ and IL-1␤ act in an agonistic manner and are internalized after binding to the receptor. The IL-1 receptor antagonist (IL-1Ra) blocks the binding of the agonists and inhibits the internalization of the receptor (31). Two types of receptors have been described with molecular masses of 85 kDa for the type I receptor (IL-1RI) and 65 kDa for the IL-1 type II receptor (IL-1 RII) (32), but binding to only IL-1RI has been shown to induce cell activation. IL-1RII does not trigger a signaling cascade and presumably inhibits IL-1 activity by acting as a decoy target for IL-1 (33,34). Binding of IL-1 to the IL-1RI leads to the association of serine/threonine kinases (35,36).
Recently an IL-1RI accessory protein (IL-1RAcP) was described which does not bind IL-1 but associates with and increases the affinity of IL-1RI (37). We have previously described an IL-1RI-positive subclone of EL4 cells, EL4D6/76, * This work was supported by grants from the Deutsche Forschungsgemeinschaft (to W. F. and M. K.) and by grants from the Bundesministerium fü r Bildung, Wissenschaft, Forschung und Technologie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶To whom correspondence should be addressed. which binds IL-1 with high affinity but does not respond with IL-1RI internalization or IL-2 production (38,39). This defect can be overcome by intracellular delivery of IL-1 (40) or by transfection with IL-1RAcP, which reconstituted the IL-1RI internalization and functional defects (41,42).
In the present study, therefore, we investigated the activation of SMase by different components of the IL-1R complex. Evidence is provided that IL-1RI internalization is required for the activation of the endosomal A-SMase. Ceramide produced by A-SMase provides an important signal for further downstream events like NFB activation or IL-2 production. The lack of A-SMase activation may thus explain the unresponsiveness of the IL-1RI internalization-defective cells.

EXPERIMENTAL PROCEDURES
Cell Culture and Biological Reagents-EL4 cells and corresponding transfectants were cultured in RPMI 1640 containing 10% fetal calf serum, 100 IU/ml penicillin, and 100 g/ml streptomycin at 37°C in air with 5% CO 2 . For stimulation, 2.5 ϫ 10 6 cells were seeded in 48-well plates at a density of 1 ϫ 10 6 cells/ml. Human (h) recombinant (r) IL-1␣ (rhIL-1␣) was kindly provided by Drs. A. Stern and P. Lomedico (Hoffmann-La Roche, Nutley, NJ). The specific activity was 5 ϫ 10 6 units/mg as determined by the lymphocyte activating factor assay and used at a concentration of 10 units/ml, representing a concentration of 150 pg/ml. Recombinant mouse (m) and human (h) TNF was a kind gift of Knoll AG (Ludwigshafen, Germany).
Cytokine Assay-IL-2 activity of the culture supernatants was quantified by enzyme-linked immunosorbent assay with the IL-2 Mini Kit (Biozol, Eching, Germany). The assay was performed according to the manufacturer's instructions. The IL-2 detection limit was Ն50 pg/ml.
Internalization Assay-To measure the internalization of 125 I-IL-1␣, 2 ϫ 10 6 cells were incubated for 4 h at 37°C or 4°C in 200 l of medium, pH 7.4, containing 500 pM 125 I-IL-1␣ (Amersham-Buchler, Braunschweig, Germany). Nonspecific binding was determined by adding a 100-fold excess of unlabeled rhIL-1␣. Cell surface-bound radioactivity was removed by washing the cells in medium, pH 3.0, for 2 min. Subsequently, the cells were centrifuged through a mixture of dibutyl phthalate and bis(2-ethylhexyl) phthalate (3:2) (Merck, Darmstadt, Germany). To determine the total cell-associated 125 I-IL-1␣, the cells were passed through the mixture of dibutyl phthalate and bis(2-ethylhexyl) phthalate without washing. Radioactivity in the cell pellets was measured using a ␥-counter.
For detection of internalized 125 I-TNF, 2 ϫ 10 6 cells were incubated for 1 h at 4°C with 1 ng/ml 125 I-TNF (recombinant TNF, NEN Life Science Products, specific activity 2160 kBq/g) to saturate cell surface receptors. Nonspecific binding was determined by adding an 200-fold excess of unlabeled TNF together with 125 I-TNF. After washing the cells three times in cold phosphate-buffered saline, temperature was shifted to 37°C to allow receptor internalization or kept at 4°C. To determine the amount of internalized 125 I-TNF receptor complexes, noninternalized ligand was removed by centrifuging (50 ϫ g) the cells through serial pH 3.0 gradients consisting of (a) 0.5 ml of culture medium supplemented with 20% Ficoll; (b) a second layer of 0.5 ml of 50 mM glycine-HCl, pH 3.0, 100 mM NaCl supplemented with 10% Ficoll; and (c) a third layer of 0.5 ml of culture medium containing 5% Ficoll. To determine the total amount of cell-associated 125 I-TNF, a second aliquot of cells was passed through a gradient, in which the second layer was replaced by phosphate-buffered saline, pH 7.3, containing 10% Ficoll. Radioactivity of the cell pellets was determined by counting in a ␥-counter.
Specific binding was calculated by subtracting nonspecific from total binding, and the amount of internalized 125 I-ligands was calculated as percent of specific binding determined at neutral pH.
Electrophoretic Mobility Shift Assay-Following stimulation of cells (5 ϫ 10 6 at 10 6 cells/ml density) for the times indicated in the figures, nuclear extracts were prepared according to Schreiber et al. (43). The protein concentration of the nuclear extracts was measured using a BCA assay (Pierce, Hamburg, Germany) with bovine serum albumin (Sigma, Deisenhofen, Germany) as standard protein. The doublestranded NFB specific oligonucleotide, containing two tandemly arranged NFB binding sites of HIV long terminal repeat enhancer (5Ј-ATCAGGGACTTTCCGCTGGGGACTTTCCG-3Ј) was end-labeled with [␥-32 P]ATP (Amersham-Buchler, Braunschweig, Germany) using T4 polynucleotide kinase (Boehringer Mannheim, Mannheim, Germany) and purified with Nick columns (Pharmacia, Freiburg, Germany). Nuclear extracts (10 g) were incubated for 15 min at room temperature in FIG. 1. A, IL-2 production by EL4 5D3 and EL4D6/76 cells. Cells were incubated in culture medium or stimulated with 10 ng/ml PMA, 10 ng/ml PMA ϩ 150 pg/ml rhIL-1␣, or 10 ng/ml PMA ϩ 100 ng/ml rmTNF. After 18 h, supernatants were collected and IL-2 production quantified by enzyme-linked immunosorbent assay. No IL-2 was detected after stimulation with 150 pg/ml rhIL-1␣ or 100 ng/ml rmTNF alone. Stimulation index ϭ PMAϩIL-1/PMA or PMAϩTNF/PMA. B, internalization of 125 I-IL-1␣ by EL4 5D3 and EL4D6/76 cells. Cells were incubated with 500 pM 125 I-IL-1␣ for 4 h at 37°C or 4°C. For determination of total cell-associated radioactivity, cells were centrifuged through an oil mixture (see "Experimental Procedures"). In a parallel reaction, surface-bound 125 I-IL-1␣ was removed by a pH 3 washing step and the internalized radioactivity was measured in the cell pellet after centrifugation through the oil mixture. C, internalization of 125 I-TNF in EL4 5D3 and EL4D6/76 cells. Cells were incubated in 1 ng/ml 125 I-TNF for 1 h at 4°C. After removing excess radioactivity, internalization of 125 I-TNF was allowed for 1 h at 37°C or 4°C. Subsequently, for determination of total cell-associated radioactivity, the cells were centrifuged through a pH 7.3 Ficoll gradient; for determination of internalized 125 I-TNF, cells were centrifuged through a pH 3.0 Ficoll gradient. 100% equals total specific cell-associated radioactivity.
binding buffer (5 mM HEPES, pH 7.8, 5 mM MgCl 2 , 50 mM KCl, 5 mM dithiothreitol, 10% glycerol, 50 mM poly(dI-dC), final volume 20 l). The 32 P-labeled double-stranded oligonucleotide was then added, and the reaction mixture was incubated for another 15 min. In competition experiments, an 200-fold excess of the unlabeled B oligonucleotide and Oct2A site (5Ј-GTACGGAGTATCCAGCTCCGTAGCATGCAAATCCT-CTGG-3Ј) was added. For supershift experiments, the binding reaction mix was incubated with the indicated amounts of antibodies for an additional 1 h. The samples were fractionated on a low ionic strength (0.25 ϫ TBE), 6% nondenaturing polyacrylamide gel, and the bands were detected by autoradiography.
Assays for Neutral and Acid SMase-EL4 cells (3 ϫ 10 6 ) in 1 ml of RPMI 1640 were stimulated in triplicate culture with 100 units/ml rhIL-1␣ or 100 ng/ml rmTNF-␣ or medium to determine basal activity for the times indicated in the figures. SMase activities were expressed as percent of basal SMase activities determined for each time point separately. At indicated times, treatment was stopped by immersion of the culture vials in a methanol-dry ice bath. Cells were centrifuged for 5 min at 4°C and washed with ice-cold phosphate-buffered saline. To measure neutral SMase, pellets were resuspended in a buffer containing 20 mM HEPES, pH 7. The produced radioactive phosphorylcholine was measured as described for neutral SMase assay.

RESULTS
To investigate the role of the different SMases in IL-1 signal transduction and their activation via the IL-1RI complex, we used two sublines of the murine thymoma cells EL4, EL4 5D3 and EL4D6/76, which differ in their response to IL-1 (38 -40). On the cell surfaces, both lines express normal numbers of IL-1RI, which show comparable affinity to IL-1 (39). Cloning of the IL-1RI cDNA from both cell lines and subsequent sequencing revealed that the nucleotide sequences of the IL-1RI of both cell lines are identical and correspond to the published sequence (data not shown). As shown in Fig. 1A, after binding of IL-1 only EL4 5D3 but not EL4D6/76 reacted with increased IL-2 production in presence of the tumor promoter PMA (39). To investigate whether this defect of EL4D6/76 was restricted to IL-1 cells were stimulated with TNF and/or PMA. In contrast to IL-1, co-stimulation with TNF and PMA led to increased IL-2 production in both cell lines (Fig. 1A), indicating the specificity of the functional defect in EL4D6/76 cells. The higher TNF responsiveness of EL6D6/76 cells compared with EL4 5D3 cells may be explained by the fact that EL4D6/76 cells bind more TNF under our assay conditions (data not shown). No differences in IL-2 production were observed when the cells were stimulated with saturating concentrations of rmTNF or rhTNF, suggesting that activation of the IL-2 production occurred via the 55-kDa TNF receptor (data not shown).
This defect in IL-1 responsiveness was shown to correlate with a defect in internalization of receptor-bound IL-1. Only EL4 5D3 cells were able to internalize IL-1, whereas EL4D6/76 cells were deficient (Fig. 1B). To investigate whether this defect of EL4D6/76 cells is also specific for IL-1, we tested both cell lines for their capability to internalize TNF. Fig. 1C shows that TNF was internalized in both cell lines to the same extent. Activation of the IL-1RI by IL-1 leads to the rapid activation of the transcription factor NFB. To examine whether the functional defects in IL-1 responsiveness correlated with defective NFB activation, cells were stimulated with saturating concentrations of IL-1 and TNF. As shown in Fig. 2, TNF but not IL-1 was able to trigger the rapid activation of NFB in EL4D6/76 cells, whereas the IL-1-responder EL4 5D3 responded to both stimuli equally well. Taken together, these data show the defect of internalization and function is specific for IL-1RI. Furthermore, the binding of IL-1 to the IL-1RI is not sufficient for triggering the nuclear translocation of NFB.
Induction of SMase activity was shown to be a very early event after TNF receptor or IL-1R triggering. To address the question whether the activity of the N-and A-SMase is coupled to a functional receptor, cells were stimulated with TNF or IL-1 and the activities of the N-and A-SMase were measured. In IL-1-stimulated IL-1-responsive EL4 5D3 cells, the activity of the N-SMase peaked after 90 s (Fig. 3A), whereas the maximum of A-SMase activity was detected after 3 min (Fig. 3B). Interestingly, IL-1 stimulated N-SMase activity in IL-1-nonresponsive EL4D6/76 cells (Fig. 3C), whereas no increase of A-SMase activity was detected (Fig. 3D). Again, stimulation with TNF led to activation of both N-SMase and A-SMase in both sublines (Fig. 3, A-D). As we have shown before, both EL4 5D3 and EL4D6/76 cells were able to internalize and respond to TNF. These data suggested that IL-1R internalization and activation of A-SMase but not N-SMase are functionally coupled. The enzymatic activities of the crude preparations of Aand N-SMase were analyzed. The enzymes showed classical Michaelis-Menten kinetics with IL-1 not significantly affecting K m , but increasing V max of both SMases (Fig. 4).
Recently, we found that expression of IL-1RAcP in EL4D6/76 reconstituted IL-1 responsiveness with respect to internaliza-

FIG. 2. Activation of NFB in EL4 5D3 and EL4D6/76 induced by IL-1␣ (A) and TNF (B).
Cells were left untreated or incubated with 150 pg/ml IL-1␣ and 100 ng/ml TNF for the indicated times. Nuclear extracts were prepared, and NFB binding was analyzed by electrophoretic mobility shift assay using a 32 P-labeled NFB binding site from HIV long terminal repeat. tion of IL-1 and IL-2 secretion (41). To investigate whether activation of A-SMase is linked to a functionally competent IL-1 receptor that is capable of receptor internalization, we used transfectants of the IL-1-nonresponding line EL4 D6/76, which stably expressed the IL-1RAcP. The ability to activate A-SMase in EL4D6/76 cells was also reconstituted in IL-1RAcP-transfectants. As shown in Fig. 5, IL-1 did not stimulate A-SMase in EL4D6/76 cells. The corresponding IL-1RAcP transfectants, however, showed the typical A-SMase activation pattern with maximum activity at 3 min after IL-1 stimulation. A-SMase through production of ceramide provides an important cofactor for NFB activation. When the IL-1-responsive EL4 5D3 and IL-1-nonresponsive EL4D6/76 were stimulated with IL-1, NFB was activated in EL4 5D3 but not in the nontransfected EL4D6/76 (Fig. 6A). Four corresponding IL-1RAcP transfectants, however, clearly showed activated NFB after stimulation with IL-1 (Fig. 6A). The identity of NFB was confirmed in competition experiments with a 200-fold excess of unlabeled B oligonucleotides in two representative IL-1RAcP transfectants (Fig. 6B). In contrast, a 200-fold excess of cold Oct2A oligonucleotide had no inhibitory effect on the formation of the NFB complex. In supershift experiments, an anti-RelA antibody specifically inhibited the formation of the NFB complex. An anti-RelB antibody was not able to replace the complex, indicating the involvement of the p65 rather than p68 subunit in the formation of the NFB complex (Fig. 6B).

DISCUSSION
During the last few years, the importance of ceramides as second messenger molecules generated by the breakdown of sphingomyelin has become evident (5,6). Previous studies indicated that the TNF signal activates two forms of sphingomyelinases, a membrane-bound N-SMase and DAG-dependent endo/lysosomal A-SMase (27). These two forms are triggered independently from each other and lead into different signaling pathways (9). A-SMase has been identified as a candidate for NFB activation. Raising the pH of the endo-/lysosomal compartments with monensin or ammonium chloride resulted in a loss of A-SMase activity and NFB activation selectively; neither N-SMase activity nor PC-PLC was affected (27).
As IL-1 is another potent activator of SMases and NFB, we, therefore, investigated the activation of A-and N-SMase by IL-1 and the relation to different components of the IL-1RI in two sublines of the EL4 thymoma cell line. The subline EL4 5D3 can be activated by IL-1, whereas EL4D6/76 cannot be activated although high affinity IL-1 binding sites are present (39). The defects in internalization of the IL-1R complex, in activation of A-SMase, and in nuclear translocation of NFB were all shown to be specific for IL-1R-mediated stimulation, since the IL-1-nonresponsive EL4D6/76 cells readily responded to TNF stimulation.
IL-1, however, activated N-SMase and A-SMase differentially. N-SMase was activated in both the IL-1-responder and IL-1-nonresponder lines by IL-1 and thus did not correlate with activation of NFB and IL-2 production. In contrast, A-SMase was not activated in IL-1-nonresponsive EL4D6/76 cells by IL-1, although TNF was readily able to activate A-SMase. Therefore, in the IL-1-signaling cascade, N-SMase activation is not sufficient for NFB activation. Thus, ceramide per se might not be an activator of NFB but only ceramide generated by A-SMase in a distinct cellular compartment. The importance of compartmentalization is underlined by investigations of Liu et al. (44). They have shown that IL-1␤ stimulated DAG and ceramide production only in caveolae fractions of fibroblasts. DAG induced by IL-1 in other cellular fractions was not coupled to ceramide production. In our experiments which are not shown in this paper, incubation with C 2 -and C 8 -ceramides did not activate NFB in both cell lines. Therefore, since A-SMase appears to be required for IL-1-induced NFB activation and ceramide analogs do not induce this event, ceramide might be a necessary but not sufficient co-signal for NFB activation. We also found previously that exogenous sphingomyelinases or sphingosine were not able to co-stimulate IL-2 production in EL4 cells (45). Therefore, it might be possible that small amounts of A-SMase-derived ceramide in specialized compartments contribute to activation of NFB or IL-2 production. In A-SMase-deficient Niemann-Pick fibroblasts, however, NFB activation is induced by IL-1, indicating that A-SMase activity is not essential for NFB activation (46).
In addition to internalization of IL-1 and IL-2 production, we found that IL-1-stimulated A-SMase activity was also reconstituted by transfection in four independent stable transfectants of EL4D6/76 cells (Fig. 5). Simultaneously, the activation of NFB was restored, strongly supporting the existence of a link between A-SMase activity and NFB activation. Thus, A-SMase activity, in contrast to N-SMase activity, correlated with internalization of a functional IL-1RI complex. The data suggest that A-SMase activation requires a functional receptor complex that is capable of receptor internalization. The need of internalization for cytokine action is controversially discussed in the literature. Endocytosis is reported to play a critical role in TNF-induced gene expression and induction of cytolysis (47)(48)(49). In EL4 cells, IL-1 signaling and internalization correlate (38,39) and an intracellular activation loop of IL-1 seems to be operative in EL4 (40). On the other hand, in Jurkat cells, internalization and nuclear localization of IL-1 was not sufficient for activation of the IL-2 promoter (50). Andrieu et al. (51) suggest that cytokine-receptor internalization is not required for activation of the sphingomyelin pathway because they found similar degradation of sphingomyelin and generation of ceramide in cells when endocytosis was blocked by low temperature and hypertonicity. These data, however, do not exclude the requirement for the internalization of the receptor complex to activate A-SMase, as there was no distinction made between ceramide produced by A-or N-SMase. It is therefore possible that the increased ceramide level results from N-SMase activity, which in our experiments did not require IL-1R internalization.
A possible mechanism for the activation of A-SMase is indicated by data obtained from studies with caveolae fractions. The caveola is a membrane domain that can undergo an internalization cycle. Invagination of the membrane is followed by the formation of plasmalemmal vesicles, which provide an optimal microenvironment for activation of A-SMase. IL-1␤ stimulated the production of DAG in a caveola-rich membrane fraction of whole fibroblasts. This was followed by a degradation of sphingomyelin and a concomitant increase of ceramide. Additionally, A-SMase activity could be detected in the caveolae fractions (44). In TNF signaling, the activation of A-SMase by 1,2-DAG generated by membrane-located PC-PLC has been reported (27). Thus, activation of A-SMase by IL-1 may occur via co-internalization of 1,2-DAG with the caveolae-associated IL-1/IL-1RI complex, if PC-PLC activation occurs in close vicinity to the membrane receptors.
In conclusion, the present study shows that the IL-1-induced increase in A-SMase activity and concomitant activation of NFB are dependent on the presence of IL-1RAcP. Ceramide 1G6, EL4 10B5, and EL4 10G12) expressing IL-1RAcP mRNA were stimulated for the indicated periods of time with 1.5 ng/ml IL-1␣ or left untreated for control and A-SMase activity was assayed as described under "Experimental Procedures." A-SMase activity is expressed in percentage of control. The standard errors were always lower than 4% of the mean.
FIG. 6. Reconstitution of IL-1-mediated NFB activation in EL4D6/76 when transfected with IL-1RAcP. The NFB complex was confirmed in competition experiments and supershift analyses. A, EL4 5D3, EL4 D6/76, and stably IL-1RAcP transfected EL4D6/76 cells (EL4 1F4, EL4 1G6, EL4 10B5, and EL4 10G12) were either left untreated or stimulated for 1 h with 150 pg/ml IL-1. After the indicated periods of time, nuclear extracts were prepared and NFB binding activity was detected by EMSA using the 32 P-labeled NFB binding site from HIV long terminal repeat. B, two transfectants (EL4 10B5 and EL4 10G12) were left untreated or stimulated with 150 pg/ml IL-1 for 1 h before nuclear extracts were prepared. Again, NFB binding activity was detected by EMSA. Additionally, competition experiments with unlabeled B and Oct2A oligonucleotides, respectively, were performed. The nuclear extracts were incubated with the radiolabeled NFB binding oligonucleotide, and either a 200-fold excess of unlabeled B or Oct2A site before the reaction mix was separated by gel electrophoresis. In supershift experiments, 1 g of anti-RelA or anti-RelB antibody was added to the reaction mix for an additional 1 h before separation by gel electrophoresis. produced by A-SMase, therefore, might represent the functional link between IL-1RI internalization and activation of NFB and IL-2 production.