Tumor necrosis factor induces lipopolysaccharide tolerance in a human adenocarcinoma cell line mainly through the TNF p55 receptor.

This study demonstrates that lipopolysaccharide (LPS) mediates induction of transcription factor NFκB and activation of the cytomegalovirus (CMV) promoter-enhancer in the SW480 cell line. These cells do not express a functional membrane CD14. The LPS response in SW480 cells was weaker and markedly slower than the tumor necrosis factor (TNF) response. Pretreatment with TNF for 72 h inhibited both TNF, tumor necrosis factor receptor (TNFR) p55, TNFR p75, and LPS-mediated activation of nuclear factor -κB (NFκB), whereas pretreatment with LPS only inhibited the LPS response. TNFR p55 antibody pretreatment resulted in marked inhibition of the LPS response, while pretreatment with TNFR p75 antiserum only had a weak inhibitory effect. Flowcytometric analysis showed that LPS binding as well as expression of TNFR p55 and TNFR p75 were not affected by LPS or TNF pretreatment, indicating that the observed inhibition is not due to reduction of specific binding sites at the cell surface. The results suggest that LPS signaling in SW480 cells involves intracellular components which may be depleted or inactivated via TNFR p55, indicating that the LPS and TNFR p55 pathways overlap. We propose that TNFR p55 can mediate activation of NFκB and cytomegalovirus promoter-enhancer in SW480 cells via two distinct mechanisms, one which is activated only via TNFR p55 and leads to rapid activation of NFκB, and another which is overlapping with the LPS pathway.

This study demonstrates that lipopolysaccharide (LPS) mediates induction of transcription factor NFB and activation of the cytomegalovirus (CMV) promoterenhancer in the SW480 cell line. These cells do not express a functional membrane CD14. The LPS response in SW480 cells was weaker and markedly slower than the tumor necrosis factor (TNF) response. Pretreatment with TNF for 72 h inhibited both TNF, tumor necrosis factor receptor (TNFR) p55, TNFR p75, and LPS-mediated activation of nuclear factor -B (NFB), whereas pretreatment with LPS only inhibited the LPS response. TNFR p55 antibody pretreatment resulted in marked inhibition of the LPS response, while pretreatment with TNFR p75 antiserum only had a weak inhibitory effect. Flowcytometric analysis showed that LPS binding as well as expression of TNFR p55 and TNFR p75 were not affected by LPS or TNF pretreatment, indicating that the observed inhibition is not due to reduction of specific binding sites at the cell surface. The results suggest that LPS signaling in SW480 cells involves intracellular components which may be depleted or inactivated via TNFR p55, indicating that the LPS and TNFR p55 pathways overlap. We propose that TNFR p55 can mediate activation of NFB and cytomegalovirus promoter-enhancer in SW480 cells via two distinct mechanisms, one which is activated only via TNFR p55 and leads to rapid activation of NFB, and another which is overlapping with the LPS pathway.
A wide variety of other cell types are also affected by LPS, and some of these cells do not express membrane CD14. Thus, LPS has been reported to stimulate arachidonate metabolism and surface expression of adhesion molecules in endothelial cells, and it can induce aggregation of platelets, stimulate cytokine release from mast cells and fibroblasts, and lead to generation of chemotactic factors in epithelial cells (8). Although CD14 is not present on the plasma membrane of these cells, soluble (s)CD14 present in serum is essential for their stimulation by LPS (9,10).
Transcription factors activated by LPS include NFB (10, 11) and NF-IL6 (12,13). TNF is also known as a potent inducer of NFB as well as of other transcription factors including AP-1, NF-IL6, IRF-1, and NF-GMa (14). NFB belongs to the rel family of transcription factors which form a number of different hetero-and homodimers participating in the regulation of a large number of genes involved in the immune response (15). NFB proteins are constitutively expressed in the cytoplasm, bound to inhibitor IB, and are released and translocated to the nucleus upon phosphorylation and degradation of IB (16 -18). Most likely, more than one IB kinase is involved, and different stimuli lead to phosphorylation and degradation of different IB species. Thus, while IB-␣ is degraded both by TNF and LPS, IB-␤ is only degraded in response to LPS and IL-1, but not in response to TNF (19).
TNF can induce NFB via two different receptors, TNFR p55 and TNFR p75 (20). In most cells, TNFR p55 is responsible for activation of NFB (21,22), and recent evidence indicates that TNFR p55 signaling involves ceramide (23,24). In some cell types, NFB may also be activated by TNFR p75 (20), but TNFR p75 signaling mechanisms are still poorly understood. Association of serine/threonine kinases with cytosolic domains of both TNFR p75 (25) and TNFR p55 (26,27) have been reported, and recently, two cytosolic proteins which specifically associate with TNFR p75 intracellular domains were identified and cloned (28). However, no intracellular pathways activated by any of these proteins have yet been identified, and it is not known whether they are involved in TNF-mediated activation of NFB.
LPS-mediated activation of NFB in monocytes is dependent on intracellular protein tyrosine kinase activity (29). Similarly, TNF-mediated activation of NFB in lysates of the monocytederived cell line U937 was inhibited by a tyrosine kinase inhibitor (30). Also, both LPS-and TNF-induced activation of NFB is inhibited by the antioxidant pyrrolidine dithiocarbamate (31,32) indicating that reactive oxygen intermediates play a role in both LPS-and TNF-mediated NFB activation.
So far, LPS signal transduction has been most extensively studied in cells expressing a functional membrane CD14. However, LPS effects on CD14 negative cell types may also contrib-ute to LPS-mediated pathology. In the present study, we show that LPS activates the transcription factor NFB and the cytomegalovirus (CMV) promoter-enhancer in SW480 human adenocarcinoma cells which do not have a functional membrane CD14. Since both TNFR p55 and TNFR p75 independently activate SW480 cells (20), we considered it important to compare the LPS signaling mechanism to the signal transduction pathways activated by the TNF receptors. The results demonstrate that TNF induces LPS tolerance in SW480 cells and that TNFR p55 and LPS may activate overlapping pathways.
Reagents-LPS from smooth Salmonella minnesota 6261 and from rough S. minnesota Re595 (Sigma) as well as S. minnesota lipid A diand monophosphate (Sigma) were solubilized in 0.9% NaCl at stock solutions of 2 mg/ml (LPS) or 1 mg/ml (lipid A). Recombinant (r)CD14 and (r)LBP were generously provided by Dr. M. Lichenstein and Dr. M. Zukowski, AMGEN (Thousand Oaks, CA) and were prepared as described (34). Human recombinant TNF, with specific activity 7.6 ϫ 10 7 units/mg protein, was generously supplied by Dr. Refaat Shalaby, Genentech Inc. (South San Francisco, CA). Anti-CD14 monoclonal antibody (mAb) 3C10 was obtained from a subclone of the hybridoma ATCC TIB 228 (American Type Culture Collection).
TNFR p75 antiserum (p75 AS) was generated by multiple injections of a rabbit with recombinant soluble TNFR p75 (20). The mAb htr-9 against TNFR p55 (35) was generously provided by Dr. M. Brockhaus, Hoffmann La-Roche Ltd. (Basel, Switzerland). Biotinylation of htr-9 and mAb utr-4 against TNFR p75 (35) was performed as described (36). The mAb 6H8 directed against a widely distributed 180-kDa membrane protein 2 was used as a control antibody. All mAbs were purified on a Sepharose goat anti-mouse IgG column (Zymed Laboratories Inc., South San Francisco, CA). Benzamidine (Sigma) was dissolved in 50% ethanol at 0.5 M and phenylmethylsulfonyl fluoride (Sigma) in isopropyl alcohol at 0.1 M.
␤-Galactosidase Assay-The ␤-galactosidase assay was performed essentially as described previously (20). Substrate conversion was measured as optical density (OD) at 570 nm. For pretreatment studies, cells were seeded out in A ϩ medium containing pretreatment reagents. After 72 h, the plates were washed three times in Hanks' buffered salt solution, and test reagents were added in A ϩ medium.
Quantitative Band Shift Assays-Preparation of nuclear extracts and band shift analysis was performed essentially as described (20). Briefly, equal amounts of nuclear protein from each sample were incubated with 1 g of poly(dI-dC) (Pharmacia Fine Chemicals, Uppsala, Sweden), in binding buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.25 mg/ml bovine serum albumin, 2% Ficoll) (20 l final volume) for 10 min at room temperature. Then, 17 fmol (1-5 ϫ 10 4 counts/min) of end-labeled NFB-specific doublestranded oligonucleotide probe 5Ј-AGTTGAGGGGACTTTCCCAGG-3Ј (Promega Corp., Madison, WI) was added, and the mixture was incubated for another 15-20 min. The samples were applied on native polyacrylamide gels (7% acrylamide, 0.25 ϫ Tris borate-EDTA, 2.5% glycerol) and run at 100 V for 1 h and then at 130 V for 2-3 h, after which the gels were dried and exposed to x-ray film (X-Omat AR, Kodak, Rochester, NY) for 2-16 h. Specific band shifts were quantitated with a PhosphoImager (Molecular Dynamics, Sunnyvale, CA) by measuring the radioactivity within a rectangle enclosing the band shift in gel scans after exposures of 18 -24 h. Radioactivity counts, which are a 2 B. Naume, A. Sundan, and T. Espevik, unpublished results. function of both gel exposure times and of the specific activity of the probe, were termed "PhosphoImager units" and were used to compare the relative amounts of radioactivity in band shifts within one gel. In order to control that a similar amount of nuclear proteins was applied for each sample, we (i) performed band shift assays with 32 P-labeled OCT oligonucleotide (Promega) (5Ј-TGTCGAATGCAAATCACTAGAA-3Ј) which binds to constitutively expressed "octamer" proteins (37), or (ii) measured the intensity of nonspecific bands in the NFB band shift. All samples were analyzed in at least two band shift assays.
Flow Cytometric Analysis of TNFR mAb and FITC-LPS Binding-SW480/␤-gal cells were pretreated with 10 ng/ml TNF or with 0.1 g/ml Re595 LPS in A ϩ medium for 72 h before the cells were labeled with 10 g/ml of biotinylated (B)htr-9 or Butr-4 and 0.2 g/ml streptavidinphycoerythrin (Becton Dickinson, Mountain View, CA) as described previously (38). Binding of FITC-LPS was estimated by adding 10 g/ml FITC-labeled LPS from S. minnesota 6261 in PBS with 2.5% A ϩ serum (PBS-A ϩ buffer) to 1 ϫ 10 6 SW480/␤-gal cells for 40 min at 0 -4°C. Background fluorescence was estimated by adding 6.2 g/ml of FITCgoat anti-murine Ig (Becton Dikinson). After washing three times in PBS-A ϩ buffer, cells were analyzed in a FACScan flow cytometer (Becton Dickinson).

LPS Activates the CMV Promoter-enhancer in the Colon
Carcinoma Cell Line SW480 -Transcriptional activation of the CMV promoter-enhancer was measured in a reporter gene assay in SW480 cells stably transfected with a plasmid containing ␤-gal under control of the CMV promoter-enhancer (33). Treatment of SW480/␤-gal cells with LPS resulted in strong induction of ␤-gal activity (Fig. 1) which shows that LPS can activate intracellular signals leading to activation of the CMV promoter-enhancer in SW480 cells. S. minnesota LPS was found to be several orders of magnitude more potent than LPS from Escherichia coli or from Pseudomonas. S. minnesota mutant Re595 LPS gave rise to a higher maximal response than wild type 6261 LPS, while the lipid A part of S. minnesota LPS showed very little activity on its own (Fig. 1).
The LPS activity was strongly influenced by human serum which caused a dose-dependent enhancement of LPS mediated ␤-gal activity ( Fig. 2A). The presence of neutralizing anti-CD14 mAb 3C10 completely inhibited LPS-mediated induction of ␤-gal activity (Fig. 2B), and we found that recombinant (r)CD14 could enhance LPS activity in serum-free medium (Fig. 2C). Recombinant LPS-binding protein (LBP) had little or no enhancing effect on the LPS response (Fig. 2C), and LBP did not further potentiate the rCD14-mediated enhancement of the LPS response (Fig. 2C). Taken together, the data indicate that SW480 cells do not express a functional membrane CD14 and that soluble (s)CD14 in human serum is necessary for the LPS response. The LPS effect was not mediated by TNF or LT-␣ as neutralizing antibodies against TNF or LT-␣ did not inhibit the LPS response (data not shown).
LPS Induces Activation of Transcription Factor NFB at a Significantly Slower Rate than TNF-Nuclear extracts from SW480 cells stimulated with LPS or TNF were analyzed for trancription factor NFB. LPS was found to induce activation of NFB in a dose-dependent manner, and S. minnesota Re595 mutant LPS was more potent than wild type (6261) LPS (Fig.  3). The response maximum of LPS was markedly lower than that of TNF.
The finding that LPS and TNF both induce activation of  13-18), in A ϩ medium. The two bands marked with arrowheads represent nuclear proteins binding specifically to the NFB consensus sequence, as identified previously (20). The faster migrating complex ( ) is only weakly up-regulated and mainly contains NFB p50 as judged by supershift analysis. 3 The slower migrating complex ( ) contains both NFB p50 and p65 3 and is strongly up-regulated. B, OCT band shift analysis of the same extracts as in A. C, quantitation of relative radioactivity in the slower migrating, strongly up-regulated p50/p65 NFB complex ( ) from the band shift analysis shown in A, by PhosphoImager measurements (mean Ϯ S.D. of triplicate measurements of the same band shifts). Similar results were obtained by analysis of two other series of nuclear extracts from cells stimulated as above.
transcription factor NFB and activate the CMV promoterenhancer in SW480 cells, raises the question whether these agents mediate their effects through similar intracellular mechanisms. In order to compare the LPS and TNF responses, we first analyzed the kinetics of NFB activation for each of the stimuli. It was found that LPS activated NFB at a significantly slower rate than TNF (Fig. 4, A and B). Activation of NFB by TNF was clearly detectable at 10 min and reached a plateau level after approximately 45 min. However, LPS-induced NFB activation was only detected after 60 min and further increased during incubations of up to 120 min (Fig. 4, A  and B). Stimulation with LPS for more than 2 h did not significantly increase the amounts of activated NFB (data not shown). In the reporter gene assay, LPS required at least 6 h to yield maximal response, while the TNF response reached plateau levels after 4 h of incubation (Fig. 4C), indicating that LPS also activates the CMV promoter-enhancer at a slower rate than TNF. These results suggest that LPS activates NFB and CMV promoter-enhancer via mechanisms that are different from the TNF-activated mechanisms.
Pretreatment with TNF Inhibits LPS-induced Activation of NFB but LPS Pretreatment Does Not Inhibit TNFR p55 or TNFR p75 Responses-LPS-and TNF-induced NFB activation was further compared in a series of experiments where SW480 cells were pretreated for 72 h with either LPS, TNF, or agonistic antibodies against TNFR p55 or TNFR p75, followed by stimulation with LPS, TNF, or agonistic TNFR antibodies. Pretreatment of the cells for 72 h with TNF only marginally influenced the expression of TNFR p55 and TNFR p75 receptors (Fig. 5A). Furthermore, pretreatment of the cells with TNF or LPS did not affect the binding of FITC-LPS from S. minnesota (Fig. 5B).
Band shift analysis of nuclear extracts from pretreated cells stimulated with LPS, TNF, or with agonistic anti-TNFR antibodies are shown in Fig. 6. The data demonstrate that the LPS response is inhibited by pretreatment with either LPS or with TNF or agonistic TNFR antibodies (Fig. 6, A and B), while the TNF response is only inhibited by pretreatment with TNF or TNFR antibodies and not by LPS pretreatment (Fig. 6, C and  D). Activation of NFB by TNFR p55 mAb htr-9 was inhibited by pretreatment with htr-9 or TNF (Fig. 6, E and F), while the TNFR p75-mediated response was mainly inhibited by p75 AS and TNF pretreatment (Fig. 6, G and H). Taken together, the results suggest that pretreatment with a given stimulus leads to depletion or reduction of active intracellular components involved in the signal transduction pathway induced by that stimulant. In addition, pretreatment with agents like htr-9 or TNF may also reduce the level of active components involved in signal transduction by other agents like LPS.
In all experiments performed, inhibition of the LPS response by pretreatment with TNFR p55 mAb htr-9 was of a similar magnitude as inhibition by LPS pretreatment (Fig. 6, A and B), indicating that activation of NFB by LPS is dependent on cellular components which are reduced or inactivated by long term stimulation of TNFR p55. Pretreatment with TNFR p75 antiserum led to a weak but consistent inhibition of the LPS response (Fig. 6, A and B) and thus, components activated via TNFR p75 may also be involved in LPS signal transduction. However, neither TNFR p55-nor TNFR p75-mediated activation of NFB was inhibited by pretreatment with LPS (Fig. 6,  E-H), suggesting that long term stimulation with LPS does not lead to a reduction or inactivation of components involved in either of these pathways. In contrast to the lack of inhibition of the TNFR p75 response by LPS, pretreatment with TNFR p55 mAb htr-9 caused a low but reproducible inhibition of the TNFR p75 response. This inhibition, however, was markedly

FIG. 4. Kinetics of Re595 LPS-and TNF-mediated activation of NFB and CMV promoter-enhancer in SW480/␤-gal cells.
A, band shift analysis of nuclear extracts from cells treated for the indicated time points with either Re595 LPS (1 g/ml) or with TNF (10 ng/ml) in A ϩ medium. The two specific NFB complexes are indicated as in Fig. 3. B, PhosphoImager quantitation of radioactivity in the slower migrating p50/p65 NFB complex (mean Ϯ S.D. of triplicate measurements of the same band shifts). Similar results were obtained by analysis of two other series of nuclear extracts from cells stimulated as above. C, induction of ␤-galactosidase activity in cells treated for the indicated time points with either Re595 LPS (0.1 g/ml), TNF (10 ng/ml), or A ϩ medium. Results (mean values of duplicates) of a representative experiment are given. lower than the inhibition of the TNFR p75 response caused by p75 AS pretreatment (Fig. 6, G and H). The TNFR p55-mediated response was not inhibited by pretreatment with p75 AS in any of the experiments. Thus, it seems that TNFR p55induced activation of NFB does not depend on components activated via TNFR p75.
Pretreatment with TNFR p55 Agonistic Antibodies Can Also Suppress LPS-induced Transcriptional Activation of the CMV Promoter-enhancer-LPS-and TNFR-mediated activation of the CMV promoter-enhancer was compared by subjecting the cells to similar protocols of pretreatment and stimulation as described above. It was found that LPS-induced ␤-gal activity was markedly reduced after pretreatment with LPS or with the TNFR p55 mAb htr-9, while pretreatment with p75 AS had less inhibitory effect (Fig. 7A). The TNFR p55-mediated activity was almost completely abolished after pretreatment with TNFR p55 mAb htr-9 and very weakly inhibited by pretreat-ment with p75 AS, while pretreatment with LPS showed no inhibitory effect (Fig. 7B). Thus, pretreatment of the cells affected the subsequent activation of both the CMV promoterenhancer and NFB in a similar manner.

DISCUSSION
The present paper demonstrates that LPS can induce activation of transcription factor NFB, as well as activation of the CMV promoter-enhancer in the human adenocarcinoma cells SW480. These cells do not express a functional membrane CD14 because addition of LBP did not enhance the LPS effect under serum free conditions. Such lack of enhancement is a typical phenomenon in cells which do not express a functional membrane CD14 (39,40). Other CD14 negative cells where LPS has been found to mediate activation of NFB include the murine pre-B-cell line 70Z/3 (10) and endothelial cells (11). The LPS response in SW480 cells was strongly dependent on hu-FIG. 6. Activation of NFB in pretreated SW480/␤-gal cells by Re595 LPS, TNF, or agonistic TNFR antibodies. Cells were pretreated of for 72 h with either A ϩ medium alone, Re595 LPS (0.1 g/ml), TNF (1 ng/ml), TNFR p55 mAb htr-9 (10 g/ml) (35), or with TNFR p75 antiserum p75 AS (dil. 1/100) (20), and band shift analysis performed on nuclear extracts from cells stimulated for 2 h with different doses of Re595 LPS (A), TNF (C), htr-9 (E), or with p75 AS (G), followed by PhosphoImager quantitation of relative radioactivity in the slower migrating, p50/p65 containing complex B, D, F, and H (mean Ϯ S.D. of triplicate measurements of the same band shifts). Similar results were obtained by analysis of three other series of nuclear extracts from cells pretreated and stimulated as above. man serum and could be completely inhibited by neutralizing antibodies against CD14. Recombinant soluble (s)CD14 could only partly compensate for the human serum enhancing effect since the activity of LPS in the presence of rCD14 was markedly lower than LPS activity in the presence of 20% human serum. Thus, sCD14 is necessary but not sufficient for LPS activity, and other serum factors in addition to sCD14 are necessary for maximal LPS response in SW480 cells.
LPS stimulates activation of NFB at a markedly slower rate than TNF, indicating that the LPS and TNF signal transduction pathways are not identical. As shown earlier, the kinetics of NFB activation by agonistic antibodies against the TNFR p55 is identical to the TNF kinetics with maximal levels reached after ϳ45 min, while stimulation of TNFR p75 results in maximum activation after ϳ60 min (20). Thus, in order to reach maximum NFB activation, stimulation with LPS has to be continued for a significantly longer period of time than stimulation of any of the TNF receptors, indicating that the LPS signaling mechanism differs from the mechanisms employed by the two TNF receptors. Supershift analysis showed that stimulation of SW480 cells with LPS or with agonistic TNFR p55 or p75 antibodies resulted in activation of an identical pattern of NFB hetero-and homodimers. 3 Thus, SW480 is a cell system where the different pathways employed by LPS and the two TNF receptors can be compared, and where the question can be asked as to whether these pathways are independent or overlapping.
Comparison of LPS and TNF signal transduction pathways was performed by analyzing activation of NFB and the CMV promoter-enhancer in cells pretreated with LPS, TNF, or with agonistic antibodies against TNFRs. We found that pretreatment of the cells did not result in down-regulation of TNF receptors or reduction in the binding of LPS. Thus, it is likely that the observed inhibition of activation of NFB and CMV promoter-enhancer is due to intracellular effects of the pretreatment analogous to depletion of protein kinase C by long term treatment with the phorbol ester PMA. Such treatment renders cells unresponsive to subsequent activation of NFB by PMA, while the TNF response remains unaffected, indicating 3 A. Laegreid and L. Thommesen, unpublished observations.

FIG. 6-continued
that TNF does not depend on PMA-responsive protein kinase C for activation of NFB (41,42).
The results from the pretreatment experiments are in agreement with our previous results which indicated that TNFR p75 mediates NFB activation through a different signal transduc-tion mechanism than TNFR p55 (20). Thus, the TNFR p75mediated response is maximally inhibited by pretreatment with p75 AS while pretreatment with TNFR p55 mAb htr-9 led to a markedly lower reduction of the TNFR p75 response. Furthermore, lack of inhibition of the TNFR p75 response by LPS pretreatment suggests that the TNFR p75-activated signaling mechanism is independent of components activated by LPS. Thus, the TNFR p75 pathway leading to NFB activation is different from the LPS, as well as from the TNFR p55 pathway, although it appears to include intracellular components which are depleted or inactivated by long term stimulation of TNFR p55.
TNFR p55, which mediates rapid NFB activation, apparently employs a signaling mechanism which is independent of intracellular components activated by LPS or TNFR p75, since pretreatment with LPS or TNFR p75 AS did not inhibit the TNFR p55 response. On the other hand, the LPS response seems to be mediated by a pathway which is partly overlapping with the TNFR p55 pathway, since pretreatment with TNFR p55 mAb htr-9 inhibited the LPS response to a similar extent as the LPS pretreatment. The observation that TNFR p55 and LPS signaling pathways may be partly overlapping suggests that TNFR p55 employs more than one pathway leading to activation of NFB in SW480 cells. Thus, our results suggest that TNFR p55 may activate one pathway which mediates rapid activation of NFB and is independent of intracellular components activated by LPS or p75 AS, and another pathway which overlaps with the LPS signal transduction pathway.
The stage at which the LPS and TNFR p55 signaling pathways overlap may involve ceramide, a lipid messenger which participates in the activation of NFB in several cell lines including Jurkat (23), HL-60 (43), and SW480 (42). TNFR p55-mediated activation of NFB in Jurkat cells as well as 70Z/3 cells has been found to proceed by ceramide generated by an acidic sphingomyelinase (23,44), while TNFR p55-mediated activation of NFB in HL-60 cells is reported to involve a 97-kDa ceramide-activated protein kinase which is activated via ceramide generated by a neutral sphingomyelinase (43,45,46). Recently, LPS was found to stimulate ceramide-activated protein kinase in HL-60 cells directly, in the absence of detectable sphingomyelinase activity (47). A possible reason for this LPS activity may be structural similarities between LPS and ceramide (47). Thus, ceramide-activated protein kinase may be an intracellular component putatively involved in both LPSand TNFR p55-mediated NFB activation in SW480 cells.
Release of LPS during Gram-negative infections may induce high levels of circulating TNF which can lead to shock and death (48). Our finding that TNF pretreatment inhibits LPSinduced NFB activation may have important clinical implications as release of low TNF levels during Gram-negative infections could render cells resistant to subsequent LPS stimulation. This is supported by in vivo data showing that pretreatment of mice with TNF or IL-1 induces partial tolerance to LPS (49). Thus, release of low TNF levels during Gramnegative infections may have an important function in limiting harmful effects of LPS in vivo.