INTRODUCTION

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 (1-3). 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 (11-13), 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, C₆-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 ceramide-activated 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 ceramide-activated 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 A₂ (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, 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

EL4 cells and corresponding transfectants were cultured in RPMI 1640 containing 10% fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 °C in air with 5% CO₂. For stimulation, 2.5 × 10⁶ cells were seeded in 48-well plates at a density of 1 × 10⁶ 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 × 10⁶ 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).

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 50 pg/ml.

To measure the internalization of ¹²⁵I-IL-1, 2 × 10⁶ cells were incubated for 4 h at 37 °C or 4 °C in 200 µl of medium, pH 7.4, containing 500 pM ¹²⁵I-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 ¹²⁵I-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 ¹²⁵I-TNF, 2 × 10⁶ cells were incubated for 1 h at 4 °C with 1 ng/ml ¹²⁵I-TNF (recombinant TNF, NEN Life Science Products, specific activity 2160 kBq/µg) to saturate cell surface receptors. Nonspecific binding was determined by adding an 200-fold excess of unlabeled TNF together with ¹²⁵I-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 ¹²⁵I-TNF receptor complexes, noninternalized ligand was removed by centrifuging (50 × 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 ¹²⁵I-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 ¹²⁵I-ligands was calculated as percent of specific binding determined at neutral pH.

Following stimulation of cells (5 × 10⁶ at 10⁶ 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 double-stranded NFB specific oligonucleotide, containing two tandemly arranged NFB binding sites of HIV long terminal repeat enhancer (5-ATCAGGGACTTTCCGCTGGGGACTTTCCG-3) was end-labeled with [-³²P]ATP (Amersham-Buchler, Braunschweig, Germany) using T4 polynucleotide kinase (Boehringer Mannheim, Mannheim, Germany) and purified with Nick columns (Pharmacia, Freiburg, Germany). Nuclear extracts (10 µg) were incubated for 15 min at room temperature in binding buffer (5 mM HEPES, pH 7.8, 5 mM MgCl₂, 50 mM KCl, 5 mM dithiothreitol, 10% glycerol, 50 mM poly(dI-dC), final volume 20 µl). The ³²P-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 (5-GTACGGAGTATCCAGCTCCGTAGCATGCAAATCCTCTGG-3) 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 × TBE), 6% nondenaturing polyacrylamide gel, and the bands were detected by autoradiography.

EL4 cells (3 × 10⁶) 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 7.4, 10 mM MgCl₂, 2 mM EDTA, 5 mM dithiothreitol, 0.1 mM Na₃VO₄, 0.1 mM Na₂MoO₄, 30 mM p-nitrophenyl phosphate, 10 mM -glycerophosphate, 750 µM ATP, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM pepstatin, 0.2% Triton X-100. After an incubation time of 5 min at 4 °C, the cells were homogenized by repeated squeezing of the cell suspension through an 18-gauge needle. Nuclei and cell debris were removed by centrifugation (800 × g), and the protein content of the supernatant containing cytosolic and membrane fractions was quantified by a BCA assay. Protein (30-50 µg) was incubated for 2 h at 37 °C in a buffer (50 µl final volume) containing 20 mM HEPES, pH 7.4, 1 mM MgCl₂, and 2.25 µl of [N-methyl-¹⁴C]sphingomyelin (final concentration 20 µM, K_m of N-SMase: 15.5 µM) (25 µCi/ml, specific activity 56.6 mCi/mmol, Amersham-Buchler). Phosphorylcholine was extracted with 800 µl chloroform:methanol (2:1, v/v) and 250 µl of H₂O. Radioactive phosphorylcholine generated from [N-methyl-¹⁴C]sphingomyelin was determined in the aqueous phase by scintillation counting. To measure acid SMase, the cells were washed and the pellet was resuspended in 200 µl of 0.2% Triton X-100 and incubated for 15 min at 4 °C. To prepare cellular lysates, cells were homogenized and centrifuged in a microcentrifuge at 14,000 rpm. Protein (30-50 µg) from the supernatant was incubated for 2 h at 37 °C in a buffer containing 250 mM sodium acetate, pH 5.0, 1 mM EDTA, and 2.25 µl of [N-methyl-¹⁴C]sphingomyelin (final concentration 20 µM, K_m of A-SMase: 14.5 µM). 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).

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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.

Activation of NFB in EL4 5D3 and EL4D6/76 induced by IL-1 (A) and TNF (B).

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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 A- and 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).

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IL-1 stimulates the increase of activities of N- and A-SMase in EL4 5D3 cells.

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Recently, we found that expression of IL-1RAcP in EL4D6/76 reconstituted IL-1 responsiveness with respect to internalization 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).

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Reconstitution of IL-1-mediated NFB activation in EL4D6/76 when transfected with IL-1RAcP.

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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 C₂- and C₈-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 (47-49). 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 produced by A-SMase, therefore, might represent the functional link between IL-1RI internalization and activation of NFB and IL-2 production.