Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0703343 on November 11, 2003

Published online before print November 11, 2003
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(Journal of Leukocyte Biology. 2004;75:324-331.)
© 2004 by Society for Leukocyte Biology

LPS resistance in monocytic cells caused by reverse signaling through transmembrane TNF (mTNF) is mediated by the MAPK/ERK pathway

Silvia Kirchner*, Simone Boldt{dagger}, Walter Kolch{dagger},{ddagger}, Silvia Haffner*, Seran Kazak*, Petra Janosch{dagger}, Ernst Holler*, Reinhard Andreesen* and Günther Eissner*,1

* Department of Hematology and Oncology, University of Regensburg, Germany; and
{dagger} The Beatson Institute for Cancer Research and the
{ddagger} University of Glasgow, Scotland, United Kingdom

1 Correspondence: Department of Hematology and Oncology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. E-mail: guenther.eissner{at}klinik.uni-regensburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transmembrane form of tumor necrosis factor (mTNF), expressed on activated monocytes (MO) and macrophages (M{Phi}), is able to induce apoptosis in human endothelial cells (EC). Apoptosis is mediated by two distinct mechanisms: direct cell contact and a yet-unidentified soluble protein, death factor X. In addition, mTNF acts as a receptor that transduces a "reverse signal" into MO/M{Phi} when bound to the TNF receptor on EC. Reverse signaling by mTNF confers resistance to bacterial lipopolysaccharide (LPS). Stimulation of reverse signaling by mTNF blocks the ability of MO/M{Phi} to produce death factor X and proinflammatory cytokines. We have investigated which signaling pathways are used by mTNF acting as receptor. Reverse signaling triggers two independent pathways that can be distinguished by protein kinase C (PKC) inhibitors. The suppression of LPS-induced death factor X is dependent on PKC, whereas the suppression of LPS-mediated cytokine release is not. LPS and reverse signaling stimulate the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. It is interesting that the activation of reverse signaling by mTNF renders MO/M{Phi} refractory to a subsequent activation of the MAPK/ERK pathway by LPS. Thus, reverse signaling achieves LPS resistance in monocytic cells through interference with key signal-transduction pathways.

Key Words: signal transduction • endotoxin • bidirectional cytokine signaling


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor (TNF) is critically involved in a variety of inflammatory disorders, including endothelial cell (EC) damage following allogeneic stem-cell transplantation [1 ]. It is important that only transmembrane TNF (mTNF) not soluble TNF (sTNF) causes EC apoptosis. mTNF can cause apoptosis of EC cells in an autocrine and paracrine manner. When EC are treated with a combination of bacterial lipopolysaccharide (LPS) and irradiation, the induced autocrine expression of mTNF can cause EC apoptosis [2 ]. In addition, LPS-treated monocytes (MO)/macrophages (M{Phi}) can induce apoptosis of EC, which requires direct contact between EC and MO/M{Phi}. This is also a result of the induction of mTNF expression on the MO/M{Phi} surface [3 ]. However, in addition, MO/M{Phi} also produce a soluble apoptosis factor(s), tentatively, death factor X. This can be shown by experiments where cell-free supernatants (SN) of MO/M{Phi} induce EC death [3 ]. Death factor X does not correspond to sTNF [3 ] or any other obvious cytokine candidates but rather is a novel proapoptotic protein. The identification and characterization of death factor X will be reported elsewhere (S. Boldt et al., manuscript in preparation). In the current manuscript, the production of death factor X is used as a read-out for the suppressive effects of reverse signaling by mTNF. The production of death factor X as well as of a number of cytokines can be suppressed by preincubation of MO/M{Phi} with agents that bind to mTNF. This indicates that the stimulation of mTNF as a receptor suppresses LPS effects and confers resistance against LPS [4 ].

This suppression is a result of reverse signaling, a phenomenon that is still largely uncharacterized, but has been observed for a number of transmembrane members of the TNF/nerve growth factor (NGF) superfamily. Reverse signaling has also been described for CD40L [5 ], CD30L [6 , 7 ], CD95L (FasL) [8 ], CD137L [9 , 10 ], Ox40L [11 ], and mTNF itself [4 , 12 , 13 ]. However, these reports are mainly descriptive, and the underlying molecular mechanisms and the intracellular pathways activated by reverse signaling remain enigmatic. For instance, Watts et al. [14 ] reported that the constitutive phosphorylation of mTNF at serine 5 [15 ] is mediated by casein kinase I (CKI). In response to engagement of mTNF by sTNF receptor fragments, mTNF became dephosphorylated, and Ca2+ entry was increased concomitantly [14 ]. Domonkos et al. [16 ] observed that the proteolytic processing of mTNF caused the nuclear translocation of the remaining 10-kDa fragment comprising the transmembrane and cytoplasmic domain. They further claimed that this 10-kDa mTNF fragment induced the expression of interleukin-1ß (IL-1ß). Another report described that reverse signaling of CD40L in B cells induces production of immunoglobulins and subsequently, proliferation [5 ]. Wiley et al. [6 ] showed that IL-8 production is enhanced in response to the activation of CD30L in granulocytes. Suzuki and Fink [8 ] demonstrated that maximal proliferation of cytotoxic T lymphocytes requires reverse signaling through Fas-L. In addition, cross-linking CD137L induces activation of monocytes [9 ]. More recently, Harashima et al. [13 ] showed that reverse signaling via mTNF mediates an up-regulation of E-selectin by CD4+ T cells.

These observations testify to the importance of reverse signaling and warrant further investigations into the underlying signal-transduction mechanisms. This prompted us to examine the nature of LPS resistance induced by reverse signaling through mTNF. Our strategy was to use pharmacological inhibitors to examine pathways that play a role in LPS signaling and hence are candidates for interference by reverse signaling. Protein kinase C (PKC) plays a central role as a component of various signal-transduction pathways (reviewed in refs. [17 , 18 ]). It is also involved in LPS signaling [19 ], raising the question of whether reverse signaling by mTNF might affect PKC signaling. Main downstream effectors of PKC are mitogen-activated protein kinases (MAPKs)/extracellular signal-regulated kinases (ERKs; reviewed in ref. [20 ]). In this paper, we have investigated the role of PKC and MAPK/ERK in the reverse signaling by mTNF and present a working model of how reverse signaling renders MO/M{Phi} refractory against the inflammatory stimulus of LPS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents
Human umbilical vein endothelial cells were prepared according to the method of Jaffe et al. [21 ]. EC (passages 2–4) were cultured in EC growth medium, supplemented with low serum (5%; Promocell, Heidelberg, Germany). The human dermal microvascular EC line CDC/EU-HMEC (further referred to as EC) was kindly provided by the Centers for Disease Control and Prevention (Atlanta, GA) and has been established as described previously [22 ]. EC (passages 20–30) were cultured in MCDB131 medium, supplemented with 15% fetal calf serum (FCS), 1 µg/ml hydrocortisone (Sigma, Deisenhofen, Germany), 10 ng/ml epidermal growth factor (BD Biosciences, Heidelberg, Germany), and antibiotics (Gibco-BRL, Karlsruhe, Germany). The monocytic cell line Mono Mac 6 (MM6) was kindly provided by Prof. Dr. H. W. Loems Ziegler-Heitbrock (University of Leicester, UK) [23 ]. The MM6 was cultured in RPMI, supplemented with 15% FCS, L-glutamine, modified Eagle’s medium (MEM) nonessential amino acids, vitamins, antibiotics, MEM sodium pyruvate, and OPI supplement (Sigma). All cell-culture reagents were purchased from Gibco-BRL unless stated otherwise. The human anti-TNF monoclonal antibody (mAb) MAK195 was obtained from Knoll AG (Ludwigshafen, Germany) [24 ]; LPS (serotype Escherichia coli 055:B5) was from Sigma. All kinase inhibitors were from Calbiochem-Novabiochem GmbH (Bad Soden, Germany): PKC inhibitor (1–10 µM; bisindolylmaleimide I, GF109203X); MAPK kinase (MEK) inhibitor (20 µM; U0126). Kinetic analyses of the kinase inhibitors revealed that they were active in all preincubation periods tested up to at least 2 h (data not shown).

MO/M{Phi} constitutively express mTNF, depending on the particular cell type and intra-assay variations between 20%- and 40%-positive cells. Upon LPS stimulation, this number is usually doubled (data not shown).

Production of MO/M{Phi} SN
For the activation of mTNF reverse signaling, MO/M{Phi} were treated with human anti-TNF mAb MAK195 (anti-TNF, 20 µg/ml) for 30 min in the presence or absence of inhibitors. Cells were then vigorously washed and subsequently treated with LPS or left untreated. LPS treatment (100 ng/ml) was performed in the presence or absence of pharmacological inhibitors for 4 h. Cells were then washed thoroughly to remove the LPS and incubated another 4 h in normal growth medium before SN were collected.

Apoptosis assays
Death factor X was assayed by its ability to induce apoptosis of EC [4 ]. To detect apoptosis of EC mediated by death factor X, 1 x 105 EC were seeded into 35 mm petri dishes (Falcon, Gräfelfing-Locham, Germany) and cocultured for 48 h with SN of differently stimulated MO/M{Phi}, which were left untreated or incubated in the presence of LPS (100 ng/ml) for 4 h, washed thoroughly to remove the LPS, and subsequently, incubated another 4 h before SN were collected. To perform the apoptosis assays, EC were fixed with methanol/acetone (1:1) for 2 min. Then, EC were washed in phosphate-buffered saline (PBS) and stained with 4,6-diamidino-2-phenylindole (DAPI; 0.5 µg/ml; Sigma), dissolved in 25% glycerin/PBS, mounted on glass slides, and subjected to microscopic analysis. Nuclear condensation as revealed by DAPI staining in the absence of trypan blue uptake is considered characteristic of apoptosis as opposed to necrosis [25 , 26 ]. The quantitative analysis included counting the number of apoptotic cells relative to all identifiable cells from 16 digital micrographs, with an average of 30 cells per field.

Western blot analysis
After stimulation of MO/M{Phi}, whole cell lysates were used for Western blot analysis by following the standard method. Cell pellets were washed in 1 ml wash buffer (40 mM Tris-HCl, 0.3 mM NaCl, pH 8, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.5 mM orthovanadate, 0.2 mM natriumfluoride; all reagents were from Boehringer Mannheim, Mannheim, Germany, or Sigma). After washing, the cell pellets were lysed in lysis buffer (same as wash buffer+1% Nonidet P-40) for 30 min at 4°C and were then centrifuged at 13,000 gfor 15 min. An aliquot of SN was used to determine the protein concentration by the Biuret method. Equal amounts of protein were mixed 1:1 with 2x sample buffer [20% glycerol, 4% sodium dodecyl sulfate (SDS), 10% ß-mercaptoethanol, 0.02% bromphenol blue, and 1.25 mM Tris, pH 6.8; all chemicals were from Sigma], loaded onto a 10% SDS-polyacrylamide gel electrophoresis gel, and separated at 130 V for 2 h. Proteins were transferred to nitrocellulose (Amersham Pharmacia Biotech, Freiburg, Germany). To avoid unspecific protein binding, the membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h, washed, and then incubated with the primary antibody in 5% bovine serum albumin/TBST overnight at 4°C. The blots were washed three times and then incubated with the second antibody in 5% milk/TBST for 1 h. Immunoreactive bands were developed using an enhanced chemiluminescent kit (Amersham Pharmacia Biotech). The antibodies used were from New England Biolabs (Frankfurt A.M., Germany) or Sigma. Computer-guided densitometry was done with Image MasterTM software (Amersham Pharmacia Biotech).

Enzyme-linked immunosorbent assays (ELISAs)
sTNF in the SN of MO/M{Phi} was determined by the ELISA sandwich technique according to the manufacturer’s instructions (R&D Systems, Wiesbaden-Nordenstadt, Germany).

Statistical analysis
The significance of differences between experimental values was assessed by means of the Student’s t-test. Analysis of the differences between EC apoptosis-inducing versus noninducing SN of MO/M{Phi} and ELISA SN gave values of P < 0.001 in all cases unless stated otherwise.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Multiple roles of PKC: activation of the MAPK/ERK pathway by LPS and by reverse signaling through mTNF
As PKC often activates the MAPK/ERK cascade [20 ], we asked whether this also applies to LPS signaling and whether reverse signaling through mTNF might influence this. To engage mTNF and stimulate reverse signaling, we used (Fab')2 fragments of the anti-TNF antibody MAK195 (20 µg/ml). They are referred to as anti-TNF throughout the manuscript. MO/M{Phi} were treated with anti-TNF and LPS or both stimuli sequentially in the presence or absence of the PKC inhibitor GF109203X. Whole-cell lysates were assayed for the activation of MEK and ERK using phosphorylation-specific antibodies that specifically detect the activated forms of these kinases (pMEK and pERK, respectively). In LPS (Fig. 1 , lane 5) and anti-TNF-treated MO/M{Phi} (Fig. 1 , lane 3), the levels of pMEK and pERK were elevated. This was completely abrogated by GF109203X (Fig. 1 , lanes 4 and 6). The fact that GF109203X also inhibited the constitutive pMEK and pERK expression indicates that PKC is also involved in the maintenance of the basal level MAPK/ERK activation. The preincubation of MO/M{Phi} with anti-TNF before LPS activation (Fig. 1 , lane 7) significantly lowered pMEK and pERK levels. This suggests that reverse signaling by mTNF could render the MEK–ERK pathway unresponsive to LPS stimulation in MO/M{Phi}. The presence of GF109203X during the preincubation with anti-TNF slightly relieved the suppression of LPS-induced MEK and ERK phosphorylation by reverse signaling (Fig. 1 , compare lanes 6 and 8). This effect was moderate, but this was to be expected. The ability of GF109203X to interfere with the suppression of MEK and ERK phosphorylation induced through reverse signaling is likely to be in great part counterbalanced by the ability of GF109203X to suppress the basal level of MEK and ERK phosphorylation (Fig. 1 , lane 2).



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  		Figure 1. Effect of the PKC inhibitor GF109203X on the MEK–ERK pathway in MO/M{Phi}. MO/M{Phi} cells were treated as indicated, and cell lysates were analyzed for the activation of the MEK–ERK pathway by immunoblotting for the activated forms of these kinases as described in Materials and Methods. Even protein loads were demonstrated by ß-actin and whole MEK and ERK protein stains. Shown is a representative out of four independent experiments.

 
Reverse signaling by mTNF renders MO/M{Phi} refractory to LPS-induced MEK and ERK activation
A puzzling observation was that anti-TNF and LPS activate the MEK–ERK pathway and that this pathway is required for LPS to induce cytokines and death factor X. Yet, anti-TNF could block the LPS effects. One possibility that would reconcile these findings is that the stimulation with anti-TNF before LPS activation might induce an exhaustion of the MAPK pathway so that the cells can no longer respond to LPS. The MEK–ERK pathway features multiple feedback controls that could be responsible for a rapid desensitization of the pathway after stimulation [27 ]. To test this hypothesis, MO/M{Phi} were treated with anti-TNF for various time periods, washed, and then challenged with LPS (Fig. 2 ). MEK and ERK activation was monitored by Western blot analysis with phosphorylation-specific antibodies (Fig. 2) . Anti-TNF induced a transient phosphorylation of MEK and ERK, which had returned to basal levels 45 min after stimulation. As expected, LPS caused a robust activation of MEK and ERK. However, the preincubation of MO/M{Phi} with anti-TNF for 15 or 30 min before LPS treatment progressively blocked the LPS-induced MEK and ERK activation. This is especially apparent at the level of ERK phosphorylation, which is suppressed even below basal levels after 15 min pretreatment with anti-TNF, and is almost completely abolished after 30 min pretreatment. These experiments strongly suggest that the mTNF-triggered desensitization toward LPS might be a result of a desensitization of the ERK/MAPK pathway that renders it refractory to further stimulation.



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  		Figure 2. Reverse signaling through mTNF activates MEK and ERK and subsequently renders MEK and ERK refractory against activation in response to LPS. MO/M{Phi} cells were treated as indicated, and cell lysates were analyzed for the activation of the MEK–ERK pathway by immunoblotting for the activated forms of these kinases as described in Materials and Methods. Even protein loads were demonstrated by ß-actin and total MEK and ERK protein stains. Shown is a representative out of five independent experiments.

 
Differential duration of MEK activation during reverse signaling versus LPS signaling
The above results suggest the hypothesis that the LPS resistance mediated by mTNF reverse signaling is a result of the exhaustion of the MAPK pathway. This poses the question of what difference between the mTNF and the LPS signaling is responsible for discriminating between the anti-inflammatory (mTNF) and the inflammatory action (LPS) of the MAPK/ERK pathway. Possibly, the duration of the MAPK/ERK activation signal could be critical. Therefore, MO/M{Phi} were stimulated with LPS or anti-TNF and assayed for MEK activation (Fig. 3 ). The level of pMEK approached the baseline level 45 min after anti-TNF stimulation, whereas the LPS-triggered MEK activation persisted. Quantification of the bands by laser densitometry confirmed these results. These data suggest that a main difference between anti-TNF and LPS stimulation is the duration of MAPK/ERK activation. They further suggest that a long-term activation may favor an LPS-mediated inflammatory response, whereas the antagonistic reverse signaling pathway features a transient MAPK/ERK activation.



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  		Figure 3. Differential duration of MEK activation by mTNF reverse signaling versus LPS signaling. MO/M{Phi} were treated with anti-TNF or LPS for the indicated times. MEK activation was analyzed by Western blotting with phospho-specific antibodies. Blots were quantified by laser densitometry. The quantification is displayed as a bar graph, which shows the relative phosphorylation of MEK activation in response to anti-TNF or LPS. Data shown are an example of three independent experiments.

 
The LPS response of MO/M{Phi} is PKC-dependent and can be suppressed by reverse signaling through mTNF
As outlined before, we reasoned that PKC is a good candidate for being involved in reverse signaling by mTNF. To examine this hypothesis, we used the selective PKC inhibitor GF109203X. MO/M{Phi} were treated with LPS (100 ng/mL) in the presence or absence of the PKC inhibitor GF109203X (10 µM). Subsequently, SN were assayed for the production of death factor X and sTNF, as described in Materials and Methods.

As depicted in Figure 4A , left bar graph, LPS induced the production of death factor X in a PKC-dependent manner. After costimulation of MO/M{Phi} with LPS and GF109203X, the death factor X was no longer produced, as evident by the lack of EC apoptosis. GF109203X also prevented the LPS-induced production of sTNF (Fig. 4A , right bar graph). The fact that the LPS signal-transduction pathway for the induction of cytokines was PKC-dependent is in line with previously published data [28 ].



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  		Figure 4. The LPS response (A) and the suppression by reverse signaling of mTNF (B) are dependent on PKC. (A) The PKC inhibitor GF109203X prevents the production of death factor X in response to LPS (left bar graph), as assayed by the potential of MO/M{Phi} SN to induce EC apoptosis. GF109203X also prevents the production of sTNF as a read-out for LPS-induced cytokine production (right bar graph). *, P= 0.059, of LPS versus LPS + GF109203X. (B) Pretreatment of MO/M{Phi} with anti-TNF prevents the production of death factor X in response to LPS. The prevention is inhibited by GF109203X (upper bar graph). The effect of anti-TNF pretreatment on the production of sTNF is independent of PKC. **, P= 0.003, of anti-TNF > LPS versus anti-TNF + GF109203X > LPS. For experimental details, see Materials and Methods. These are representatives out of three independent experiments.

 
Consistent with previous data [4 ], the preincubation of MO/M{Phi} with anti-TNF resulted in the suppression of the LPS-induced production of death factor X (Fig. 4B , upper graph) and sTNF (Fig. 4B , lower graph). The PKC inhibitor GF109203X prevented the suppression of death factor X (Fig. 4B , upper graph) but not of sTNF (Fig. 4B , lower graph). Similar results were obtained for IL-1 (data not shown). These results suggest the existence of at least two different signaling pathways emanating from mTNF as a receptor, one that involves a PKC-dependent (death factor X suppression) pathway and another that works via a PKC-independent (cytokine suppression) route. It can be ruled out from previous studies [4 ] that anti-TNF interfered with a function of mTNF as an autocrine ligand, as the blockade of the TNF receptors with neutralizing antibodies did not affect reverse signaling at all.

Reverse signaling by mTNF uses the MAPK/ERK pathway for the suppression of death factor X but not for the suppression of cytokines
The above findings have several corollaries. One is that the inhibition of the MEK–ERK pathway during treatment with anti-TNF should prevent reverse signaling and hence also prevent the desensitization of the MEK–ERK pathway. Further, on a functional level, the inhibition of the MEK–ERK pathway should prevent reverse signaling from suppressing the production of death factor X. However, it should not affect the inhibition of cytokine induction by reverse signaling. The experiments shown in Figure 1 place the MEK–ERK pathway in the PKC-dependent arm of reverse signaling, which is involved in the suppression of death factor X production but not in the suppression of cytokine production.

The results from Figure 1 also suggested that the suppressive effects of PKC inhibition on the induction of death factor X and cytokines by LPS may be mediated via its inhibitory effects on MEK and ERK phosphorylation. To test this hypothesis, we measured the effects of the selective MEK inhibitor U0126 on LPS induction of death factor X and cytokines (Fig. 5A ). Cells were treated as described for Figure 4A except that U0126 was used instead of GF109203X. The MEK inhibitor yielded the same results as the PKC inhibitor, blocking the ability of LPS to induce death factor X and cytokines, which is in line with previously published data (reviewed in ref. [29 ]). These results indicate that these functional effects are mediated via PKC stimulation of the MEK–ERK pathway.



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  		Figure 5. The suppression of death factor X production but not the suppression of TNF secretion by reverse signaling through mTNF is dependent on the MEK–ERK pathway. (A) The MEK inhibitor U0126 prevents the accumulation of LPS-induced death factor X (left bar graph), as assayed by the potential of MO/M{Phi} SN to induce EC apoptosis. U0126 also prevents the production of sTNF as a read-out for LPS-induced cytokine production (right bar graph). These are representatives of four independent experiments. (B) MO/M{Phi} were treated as indicated in the figure, and the production of death factor X was assayed by the potential of SN to induce apoptosis in EC. ELISA analyzed the production of sTNF by MO/M{Phi}, as described in Materials and Methods. Data shown are representatives of four independent experiments. *, P= 0.003, of anti-TNF > LPS versus anti-TNF + U0126 > LPS.

 
To test whether U0126 would also block reverse signaling of mTNF, MO/M{Phi} were treated as for the experiments shown in Figure 4B in the presence or absence of U0126. Subsequently, SN of these cells were assayed for the production of death factor X and sTNF. U0126, indeed, also significantly impaired the ability of anti-TNF to suppress the production of death factor X. However, it failed to block the ability of anti-TNF to suppress the production of cytokines in response to LPS stimulation. Similar results were obtained for IL-1 (data not shown). It is interesting that these results reproduce the same pattern as the PKC inhibitor (Fig. 4) . They show that MEK activation was needed for the suppression of death factor X by reverse signaling but that MEK activation was not necessary for mTNF-mediated suppression of LPS-triggered sTNF. These results also suggest that the MAPK/ERK pathway is a downstream effector of PKC involved in the reverse signaling of mTNF.

Taken together with the functional data presented in Figure 4 , these data suggest that PKC has at least two discernible roles in this scenario. First, PKC is involved in the LPS-induced production of death factor X and cytokines (Fig. 4A) . This effect of PKC is mediated through the activation of the MEK–ERK signaling pathway (Fig. 1) . Second, PKC participates in the reverse signaling pathway that emanates from mTNF and suppresses death factor X. In contrast, PKC is not involved in the suppression of cytokine production by reverse signaling (Fig. 4B) . PKC inhibition appears to counteract the ability of anti-TNF to suppress the activation of the MEK–ERK pathway by LPS (Fig. 1) , suggesting that the PKC-dependent arm of reverse signaling is mediated through the MEK–ERK pathway.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we provide evidence that the LPS resistance caused by reverse signaling through mTNF [4 ] is a result of the functional exhaustion of the ERK/MAPK cascade. This pathway is shared between LPS and mTNF reverse signaling. Its prior activation by reverse signaling through mTNF prevents its subsequent stimulation by LPS.

Downstream of mTNF as a receptor, the signal subsequently bifurcates into a PKC-dependent pathway (suppression of LPS-induced death factor X) and a PKC-independent pathway (suppression of LPS-induced, soluble cytokines). The PKC-dependent route activates the MAPK cascade in a similar manner, such as LPS does, but apparently, without the final consequence of the transcriptional activation that leads to the production of death factor X. Conversely, the LPS-stimulated production of death factor X is antagonized by reverse signaling. Curiously, this antagonism seems to be exerted through the same pathway that mediates the induction of death factor X by LPS, i.e., the ERK/MAPK pathway. The crucial difference appears to lie in the activation kinetics. There is now accumulating evidence that the duration and relative strength of MAPK/ERK signaling can determine specific, biological outcomes [30 ].

Our hypothesis that premature ERK signaling can lead to an anergic state is also supported by the literature. Tominaga et al. [31 ] published that the compound GLA-58, analogous to the LPS-derived lipid A, activates ERK and thus renders cells refractory against proinflammatory stimulation by LPS. It has also been reported that stimulation of mTNF leads to an increased calcium influx [14 ]. With regard to other models of endotoxin tolerance [32 ], this Ca2+ entry before the LPS stimulus might also contribute to the refractory state, similar or in addition to the ERK pathway. There is also evidence for a role for Toll-like receptors (TLR) in providing LPS resistance in other experimental systems, with a specificity for the respective bacterial strain [33 ]. However, in our hands, preliminary data show that TLR-4 expression and function are not significantly affected by reverse signaling through mTNF in comparison with LPS signaling alone (data not shown).

It is unclear how the short cytoplasmic tail of mTNF [34 , 35 ] leads to the activation of the PKC–MEK–ERK pathway; as this part of the molecule does not contain any discernible motifs indicative of catalytic activity, this link is most likely made via associated proteins. Furthermore, post-translational modifications seem to play a role. The constitutive phosphorylation of serine 5 [15 ] in the cytoplasmatic mTNF tail by CKI is likely to be involved in signal transduction. Preliminary data from our laboratory suggest that blocking CKI with specific inhibitors restores the capacity of MO/M{Phi} to respond to LPS (data not shown).

The ERK pathway is often triggered by the interaction of Raf kinases with activated Ras, i.e., Ras-guanosine 5'-triphosphate. Ras activation involves the tyrosine phosphorylation-dependent recruitment of exchange factors, such as SOS, into the vicinity of Ras at the plasma membrane [36 ]. Therefore, we are currently investigating whether Ras and Raf are also involved in reverse signaling by mTNF.

The role of PKC could be a catalytical function through its ability to activate the MEK–ERK pathway (reviewed in ref. [37 ]). Alternatively, it could operate as a chaperone, supporting the translocation of downstream kinases to the cell membrane, a pivotal step in their activation [37 ]. PKC also could regulate Raf-1 inhibitors, such as Raf kinase inhibitor protein (RKIP), which associates with Raf-1 and prevents Raf-1 from interacting and phosphorylating MEK, thereby blocking Raf-mediated activation of the MEK–ERK pathway [27 ]. During mitogenic stimulation, RKIP dissociates from Raf-1, enabling it to couple to MEK [38 ]. Very recently, PKC {delta} has been reported to phosphorylate RKIP, causing it to dissociate from Raf-1 [38 ]. We are currently investigating whether LPS and mTNF might activate different PKC isozymes and thus differentially influence RKIP/Raf-1 interactions.

Technically, the source of the TNF-reactive agents used for inducing reverse signaling of mTNF might be critical. In contrast to our observations, a humanized anti-TNF mAb (infliximab) induces apoptosis in monocytes and activates, rather than deactivates, the cells by engaging the p38 MAPK pathway [39 , 40 ]. This could have implications for clinical anti-TNF strategies, where a total monocytic anergy or loss of function, respectively, might be undesirable, e.g., in inflammatory bowel diseases, where there is still a need for fighting infections.

Other members of the TNF/NGF superfamily, where reverse signaling has been observed, could also share engagement of the ERK/MAPK cascade [5 , 6 , 8 , 9 ], not least because the cytoplasmic portion is well conserved between these family members. One also could postulate additional signal-transduction mechanisms, as many of these reverse signals are of an activating type [41 42 43 44 45 ].

Clonal exhaustion or anergy is a general principle in the adaptive-immune system, leading to the deletion of self-reactive T and B lymphocytes. Here, we provide evidence that it might also occur in innate immunity, e.g., for the self-limitation of toxic and/or proinflammatory effects of transmembrane cytokines.


   ACKNOWLEDGEMENTS
 
Grants DFG Ei68/1-1 and DFG Ei68/3-1 from the Deutsche Forschungsgemeinschaft (DFG), a Yamagiwa-Yoshida Memorial Cancer Study Grant of the UICC, and Cancer Research UK supported this work.

Received July 23, 2003; accepted October 15, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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