interdependence of tumor necrosis factor, prostaglandin e2, and protein synthesis in...

Eur. J. Biochem. 191, 583-589 (1990) (cl FEBS 1990

Interdependence of tumor necrosis factor, prostaglandin EZ, and protein synthesis in lipopolysaccharide-exposed rat Kupffer cells Thomas PETERS, Ulrich KARCK and Karl DECKER Biochemisches Institut der Albert-Ludwigs-Universitat, Freiburg, Federal Republic of Germany

(Received March 13, 1990) - EJB 90 0265

Kupffer cells are the main producers of tumor necrosis factor-a (TNF; cachectin) and eicosanoids in the liver exposed to lipopolysaccharide (endotoxin; LPS). A very rapid but transient release of TNF is followed by a slow, steady synthesis of prostaglandin E2 (PGE2). TNF itself is able to provoke eicosanoid synthesis in Kupffer cells; the rate and pattern of prostaglandin production are similar to those observed after treatment with LPS. Anti-TNF antibodies completely neutralize TNF action on Kupffer cells, thus ruling out any participation of contaminating LPS. LPS stimulation of PGE2 production in Kupffer cells is reduced by the antiserum to 50%, indicating an involvement of TNF in the stimulatory action of LPS. On the other hand, PGE2, a potent inhibitor of LPS-elicited TNF release, is able to suppress LPS- but not TNF-stimulated eicosanoid synthesis in rat Kupffer cells.

In addition to this autocrine circuit, extrahepatic factors participate in the regulation of Kupffer cell activation : glucocorticoids not only inhibit TNF or prostaglandin production, they also reverse the LPS-specific changes in the prostaglandin pattern of Kupffer cells.

LPS, TNF or cycloheximide when given alone in the concentration range applied in this study do not affect the viability of rat Kupffer cells. However, the combinations of cycloheximide and either LPS or TNF cause rapid death of the cultured cells. The cytolytic potential of either combination cannot be alleviated by treatment with glucocorticoids.

Kupffer cells, the resident macrophages of the liver, are among the first immunocompetent cells to come into contact with gut-derived LPS. They are able to internalize and modify LPS [l] and they respond to LPS with a variety of signals.

It has been shown previously that exposure of Kupffer cells to LPS leads to the synthesis of potent inflammatory mediators including PGE2 [2], TNF [3], interleukin-1 [4] and interleukin-6 [5]. While TNF is an elicitor of prostaglandin synthesis in macrophages [6] including rat Kupffer cells [7], LPS-induced TNF synthesis is suppressed by PGE2 as well as by glucocorticoids [3].

TNF and PGEz influence independently and differently metabolic activities of diverse tissues including the liver and modulate the function of other mediators [8 - 121. PGEz was shown to suppress TNF synthesis in macrophages of different origin [3, 13, 141. TNF proved to be cytotoxic to some cell types, notably the fibroblast line L929 [15,16]; this effect was enhanced by inhibitors of protein synthesis [16]. Rat Kupffer cells are quite resistant to TNF as well as to LPS; however, it will be shown that this resistance is dependent on an intact

Correspondence to K . Decker, Biochemisches Institut, Albert- Ludwigs-Universitat, Hermann-Herderstrasse 7, D-7800 Freiburg, Federal Republic of Germany

Abbreviations. LPS, lipopolysaccharide (endotoxin); TNF, tumor necrosis factor-a; PG, prostaglandin; 6-oxo-PGFi,, 6-0x0-prosta- glandin F1,; TXB2, thromboxane B2; NCS, new-born calf serum.

Enzymes. Phospholipase A2 or phosphatide 2-acylhydrolase (EC 3.1 .I .4.); cyclooxygenase or arachidonate:oxygen oxidoreductase (cyclizing) (EC 1.14.99.-); lactate dehydrogenase or L-1actate:NAD' oxidoreductase (EC 1.1.1.27.).

machinery for protein synthesis. The viability and functional- ity of these macrophages which seems to be delicately regu- lated by their products is of great importance for the defence against certain inflammatory insults and liver metastases in malignancies [ 1 71.

MATERIALS AND METHODS

Culture medium RPMI 1640 (Instamed@, without phenol red but with L-glutamine) and newborn-calf serum (NCS) were from Biochrom (Berlin, FRG), pronase and collagenase from Boehringer Mannheim (Mannheim, FRG), and prosta- glandins from Sigma (Munchen, FRG). LPS (Salmonella minnesota R 595) was a kind gift from Dr. C. Galanos (Max- Planck-Institut f u r Immunhiologie, Freiburg). Recombinant mouse TNF was generously supplied by Dr. R.G. Adolf (Emst-Boehringer-Institut, Vienna), the monoclonal anti- bodies against PGE2 and thromboxane Bz by Dr. M. Reinke (Erlangen, FRG). The polyclonal antiserum against PGD2 was a gift of Prof. Dr. 0. Hayaishi (Osaka, Japan). The 3H- labelled eicosanoids were bought from Amersham (Frankfurt, FRG). All solvents used were of HPLC grade (Rathburn Chemicals, Scotland).

Kupffer cells were isolated from male Wistar rats (SPF animals, 250 - 350 g, Interfauna, Tuttlingen, FRG) by the method of Brouwer et al. [18] as modified by Eyhorn et al. [19]. Cells were maintained in primary culture with RPMI 1640 medium supplemented with 30% NCS for 72 h, medium was changed every 24 h. Experiments for prostaglandin syn- thesis were done in RPMI supplemented with 10% NCS.

584

Prostanoids were measured by radioimmunoassay as de- scribed earlier [19]. For HPLC analysis of radioactive prosta- glandins Kupffer cells were labeled with [3H]arachidonic acid (0.7 pCi/ml, specific activity 100 Ci/mmol) for 24 h, then the medium was changed and the experiment performed. Prosta- glandins were extracted from culture media using CI8 Sep- Pak cartridges, subsequently separated by reverse-phase HPLC and detected by liquid-scintillation counting. The elu- tion buffer for the prostanoids was a mixture of 29% acetonitrile and 71 O/O of an aqueous 0.05% trifluoroacetic acid solution, pH 2.3.

TNF was determined in the L929 cell lysis assay [15], applying serial dilutions of Kupffer cell supernatants to the TNF-sensitive L929 cells grown on 96-well microtiter plates. Cell survival was quantified by light microscopy as well as by measuring the uptake of crystal violet by the exposed cells using an Elisa Reader. The test was standardized with re- combinant mouse TNF.

Anti-(mouse TNF) antiserum was obtained by immuniz- ing rabbits twice with 0.1 mg recombinant mouse TNF dis- solved in ABM-komplett 2 (Sebak, FRG). The IgG fraction was purified by extraction with caprylic acid [20].

Viability of cell cultures in the cytotoxicity experiments was determined by light microscopy of unfixed cells as well as by measuring lactate dehydrogenase activity in the medium and cell homogenate according to Bergmeyer et al. [21]. LDH leakage in the media is expressed as thc percentage of total enzyme activity (i. e. intracellular plus extracellular).

RESULTS

Exposure of Kupffer cells to LPS led to the production of inflammatory mediators of different classes and by different kinetics (Fig. 1): an immediate release of TNF with limited duration (< 4 h after stimulation), and a rather slow (as compared to the response to zymosan or phorbol ester) but steady synthesis of PGE2 lasting more than 20 h.

The kinetic peculiarities raised the possibility of a sequen- tial stimulation. Therefore, PGE, synthesis by rat Kupffer cells after exogenous addition of recombinant mouse TNF was determined radioimmunologically and compared to that observed after LPS challenge (Fig. 2). TNF was able to elicit PGE2 synthesis with kinetics very similar to those after LPS exposure, although the total amount of PGE2 produced within 24 h was only 70 YO of that found in supernatants of LPS- exposed Kupffer cells.

The kinetics of synthesis and the HPLC profiles of the recovered prostanoids after LPS and TNF exposure, respec- tively, were very similar. However, the activity of recombinant mouse TNF necessary to obtain 80 YO of the PGE2 found after LPS challenge was quite high. It was about 40- 80 times the activity determined in the media of maximally stimulated rat Kupffer cells.

Comparison of the radioactive eicosanoids released from [3H]arachidonic-acid-labeled Kupffer cells (Fig. 3) revealed that both LPS and TNF elicited almost the same pattern: PGE2 (36%) and thromboxane (36%) were the most abun- dant prostanoids while smaller amounts of PGD2 and traces of 6-oxo-PGF1, were also present.

LPS contamination of the recombinant mouse TNF prep- aration was ruled out by the finding that a partially purified antiserum against recombinant mouse TNF completely neu- tralized the stimulatory effect of recombinant mouse TNF (Table 1). This antiserum binds and neutralizes rat TNF in

200

100 - II) - - d

; 2

5

0

Y

U

0.5

0.1

I / J " 1

0 1 2 4 8 16 24

TIME AFTER LPS ADDITION [h]

Fig. 1. Kupffer cellsproduce T N F and PGEz upon LPS chullenge with very dijferent kinetics. Rat Kupffer cells were kept in primary culture for 72 h. Medium was changed and LPS (400 ng/ml) added. The amount of TNF (0 ) and PGEz (A) in the cell supernatant was determined at the times indicated. TNF was measured by serial di- lution in the LY2Y cell lysis assay, PGE2 by radioimmunoassay. Open symbols represent the control samples without LPS addition

T

2 4 8 16 24 time [h]

Fig. 2. LPS- and TNF-elicited PGE2 synthesis in rut Kupfer cells. Rat Kupffer cells kept in primary culture for 72 h were stimulated with LPS (400 ng/ml; A), recombinant mouse TNF (8000 Ujml; W ) or left untreated (0). Samples of the supernatants were taken at the times indicated and analysed for PGEz by radioimmunoassay. Results are given as means SD of the amount of PGEz accumubdted in the Supernatants; they represent four independent experiments

the mouse L929 cell lysis assay. According to this assay the antiserum was added in a tenfold excess over the amount necessary to neutralize the maximal production of rat Kupffer cells. The LPS effect, however, was reduced to 50 O/O only by this antiserum. Unrelated antigen-antibody complexes did not inhibit the response to LPS (data not shown). These findings suggest either that a substantial fraction of the LPS- triggered stimulation of PGE2 synthesis is mediated by TNF not exposed to the antibody, e. g. present inside the Kupffer cells, or that other, as yet unrecognized factors produced by LPS-stimulated cells, are able to trigger PGE, synthesis.

PGE2 proved to be very effective in suppressing TNF production. 1 nM PGE2 added 1 h before LPS to the cells reduced the TNF levels in the supernatants by 80 YO. The inhibitory effect was very rapid: addition of PGE, (1 pM) up

6-Oxo-PGFIo TXB, PGE, PGD,

t t ll

0.24

Elut ion t i m e (rnin)

Fig. 3. HPLC pattern of prostanoids released by rat Kupffer cells stimulated with recombinant mouse T N F ( A ) or L P S ( B ) . After 48 h of primary culture, Kupffer cells were labeled with [3H]arachidonic acid for 24 h. The media were then changed and cells stimulated with recombinant mouse TNF (8000 U/ml, lane A) or LPS (400 ng/ml, lane B). Unstimulated controls are shown in (C). Supernatants were collected 24 h after the addition of the stimulus and processed as described in Materials and Methods. Retention times of tritiated standards are indicated. Ordinate, radioactivity of the effluent in arbitrary units

Table 1. Neutralisation of the TNF- and LPS-elicited prostanoid syn- thesis by anti-[recombinant mouse TNF) antiserum Kupffer cells kept in primary culture for 72 h were stimulated either withTNF (8000 U/ml), LPS (400 ng/ml) or left untreated. For neutral- ization of TNF, partially purified (see Materials and Methods) anti- (recombinant mouse TNF) antiserum was used. The neutralization was monitored in the L929 cell-lysis-assay. PGE2 was measured by radioimmunoassay. Results represent mean values of four indepen- dent experiments f S. D. Ab, antiserum

Treatment PGEZ accumulated

pmol . cells. 24 h-’

None 9.8 f 4.5 TNF 36.7 f 14 TNF/anti-TNF Ab 9.5 * 3.9 LPS 75.3 f 17.9 LPSianti-TNF Ab 33.3 f I1

to 30 min after the LPS challenge, still resulted in a 70 % inhibition (Fig. 4). These results suggest a role of PGE2 as an immediate inhibitor in LPS-activated Kupffer cells. If prosta- glandin formation was suppressed with indomethacin, how- ever, Kupffer cells did not show an increased or prolonged TNF release (data not shown). This indicates that the level of PGE2 produced by non-stimulated cells is insufficient to prevent TNF release.

As PGE2 was able to suppress the LPS-elicited TNF pro- duction by Kupffer cells, and TNF was involved in the LPS stimulation of prostanoids, PGE2 was tested for its ability to suppress prostanoid formation after an LPS challenge. It was

n

0 0.5 1

concentration of PGE, hM] tirneof additionrh]

loo l o O 1 I PGE,lpM

Fig. 4. PGEz inhibits LPS-elicited T N F release very rapidly and at nunomolar concentrations. Kupffer cells were kept in primary culture for 72 h, then media were changed and the cells treated with various concentrations of PGE2 or solvent (ethanol, final concentration 0.1 YO ; trace C). LPS (400 ng/ml) was added 1 h later. For determination of time dependence, PGE2 was kept at a constant (1pM) concentration and added 1 h prior (- 1 h), simultaneously with (0 h), 30 min (0.5 h) and 1 h (1 h) after LPS to the Kupffer cell cultures. 4 h after the addition of LPS the media were removed and tested for cytotoxicity in the L929 assay. The bars indicate the mean TNF activity (as a percentage of the control value) of three independent experiments

6-OXO-PGFla TXB, PGE, PGD,

t t t t

Elut ion t i m e (rnin)

Fig. 5. PGE2 inhibits the LPS-triggered prostanoid synthesis in rat Kupfler Cells. Kupffer cells kept in primary culture for 72 h were labeled with [3H]arachidonic acid as described in Materials and Methods. They were exposed to LPS (400 ng/ml) and unlabeled pros- taglandins for 24 h in the following way: (A), LPS only; (B), LPSjl pMPGD2;(C), LPS/lOnM PGE2;(D),LPS/IOOnMPGE2;(E), LPS/ 1 pM PGE2 ; (F), untreated control. The radioactive prostaglandins synthesized from the intracellular arachidonate pool were analyzed by radio-HPLC. Retention times of tritiated standards are indicated. Radioactivity is shown in arbitrary units. Experiments were carried out in triplicate with similar results

found that PGE2 inhibited this process in a dose-dependent manner (Fig. 5) . Maximal inhibition was seen at a concen- tration of I pM, but 10 nM PGE2 already showed some inhibition. The effect was quite specific for this prostaglandin since PGD2 was ineffective.

586

In contrast, the addition of PGE2 to TNF-stimulated Kupffer cells did not lead to an inhibition of prostanoid for- mation ; rather, a further stimulation of prostanoid synthesis was observed (Table 2). It should be mentioned that PGE2 did not suppress the prostanoid synthesis in zymosan-triggered Kupffer cells.

Glucocorticoids have previously been shown to inhibit eicosanoid formation in Kupffer cells [22] ; this effect was attributed to the suppression of phospholipase A2 [23] and took several hours of incubation for full manifestation. This pathway can be activated by a variety of stimuli, hence its inhibition is not specifically a LPS-related phenomenon. How- ever, LPS was also found to influence eicosanoid synthesis distal to prostaglandin H synthase. Quiescent Kupffer cells in primary culture, when incubated in a medium supplemented with arachidonic acid or exposed to some non-inflammatory activators of the eicosanoid pathway such as zymosan or phorbol ester, produce only small amounts of PGE2 and thromboxane relative to PGD2 [19]. After treatment with LPS, the absolute amount of PGE2 increases [24]. This change of the prostanoid pattern was also observed in studies on prostanoid synthesis by cell-free extracts of LPS-treated rat Kupffer cells [25]. The shift towards an enhanced capacity of PGE2 synthesis can still be seen 24 h after LPS exposure [24]. Dexamethasone, a potent and rapidly acting inhibitor of the TNF release caused by LPS, also reversed the changes in the PGE2/PGD2 ratio provoked by LPS (Table 3) .

To circumvent the inhibitory action of the glucocorticoid on eicosanoid synthesis from endogenous substrates (diacyl-

Table 2. PGEz stimulates the TNF-elicited prostanoid release in rat Kupjfer cells Kupffer cells, kept in primary culture for 72 hand labeled as described in Materials and Methods, were stimulated with TNF (8000 U . ml- '), and a combination of unlabeled PGEz (1 pM) and TNF. Radioactive PGE2 and thromboxane synthesized from the intracellular arachi- donate pool were analyzed by radio-HPLC. Radioactivity (arbitrary units) in the regions of interest was integrated. n, number of indcpen- dent experiments

Treatment 3H incorporated into n

PGEz TXBZ

None 21 f 18 34f 19 5 TN F 203 f 63 207-t 40 3 TNF/PGEZ 382 f 33 354 f 109 3

phosphoglycerol-bound arachidonic acid) and still observe the PGE2/PGD2 shift, free arachidonic acid was supplied to the medium of the Kupffer cells thereby providing treated and control cells with the same amount of precursor.

Inhibition of protein synthesis by cycloheximide (300 ng/ ml) was well tolerated by primary cultures of rat Kupffer cells for at least 4 h, as shown by light microscopy (Fig. 6B) and measurements of the leakage of lactate dehydrogenase into the medium. Addition of TNF (8000 Ujml) or LPS (400 ng/ ml) (Fig. 6C, Table 4) did not influence cell viability. Cell functions like phagocytosis, superoxide and eicosanoid pro- duction were not impaired (data not shown). However, the simultaneous addition of TNF and cycloheximide, or of LPS and cycloheximide, led to severe morphological changes within 4 h: cells became rounded, detached and were lysed (Fig. 6 D). Leakage of lactate dehydrogenase into the medium was increased to about 50% of the total activity. The cytotoxicity of TNF was not due to LPS contamination, as inactivation of the TNF solution by heating to 95°C (15 min), as well as by addition of anti-TNF antiserum, completely abolished cell lysis (Table 4).

Dexamethasone has been shown to be a potent inhibitor of LPS-induced eicosanoid synthesis and TNF release. It also

Table 4. Lytic uctivity of TNF and L P S on rat Kupffer cells treated with cycloheximide Kupffcr cells were kept in primary culture for 72 h. Then medium was changed to RPMI/l% bovine serum albumin and the substances (cycloheximide, 300 ng/ml; TNF, 8000 U/ml; LPS, 400 ng/ml) added as indicated above. After 4 h, media were removed, cells were scraped into ice-cold phosphate-buffered saline and sonicated. Lactate de- hydrogenase was determined in the media as well as in the cells. Cell leakage is lactate dehydrogenase activity in the medium as a percentage of the total activity in medium and cells. Results represent five independent experiments f S. D. Ab, antiserum

Treatment ~- ~

Cell leakage

None Cycloheximide TN F TNF/cycloheximide LPS LPS/cycloheximide TNF/anti-TNF Ab/cycloheximide TNF (95"C)/cycloheximide

~~

%

6.0f 3.9 8.9 f 7.1 7.5 f 5.2

50.9 f 18.9 5.8 f 2.0

51.2 f 15.6 3.5 * 0.5 5.5 f 2.1

Table 3. Dexumethasone reverses LPS-induced changes in the PGE2/PGD2 ratio Kupffer cells kept in primary culture for 48 h were treated either with LPS (400 ngiml), dexamethasone (Dex, 1 pM) or a combination of both for another 24 h. For measurement of prostaglandin release, the medium was changed and 30 pM unlabeled arachidonic acid added. PGDz and PGE2 were determined by radioimmunoassay. In parallel experiments 30 pM (2 pCi) 3H-labeled arachidonic acid was used. These samples were analyzed by radio-HPLC. Results are given as means of three independent cxperiments S. D.

Pretreatment Prostaglandin released Ratio Label incorporated into Ralio PGDZ/PGEZ PGD2/PGEZ

PGE2 PGDz PGEZ PGDz

pmol . cell. h -

None 12.0 -t 7.0 87.5 f 5.7 7.3 136 f 14 893f 11 6.6 LPS 45.5 f 0.3 84.5 f 2.6 1.9 294 f 41 885f 89 3 .0 LPS/Dex 15.9 f 6.6 94.7 f 6.3 5.9 147f 9 947 f 160 6.4 Dex 12.0 f 0.2 81.6 f 10.0 6.8 137f 5 865f 85 6.3

58 7

Fig. 6. Morphology of Kupffer cell lysis after treatment with LPS and cycloheximide. Kupffer cells kept 72 h in primary culture left without treatment (A), 4 h after addition of LPS (400 ng/ml) (B), 4 h after addition of cycloheximide (300 nglml) (C), after 4 h treatment with LPS (400 ng/ml) and cycloheximide (300 ng/ml) (D). Light microscopy of unfixed cells was performed at a magnification x 200

Table 5 . Dexamethasone does not prevent LPS- or TNF-induced lysis of vat Kupffer cells sensitized by cycloheximide After 48 h in primary culture, Kupffer cells were either treated with dexamethasone (1 pM) or left untreated. After another 24 h, the medium was changed to RPMI/I% bovine serum albumin and the substances (cycloheximide, 300 ng/ml; TNF, 8000 U/ml; LPS, 400 ng/ml) were added as indicated ; dexamcthasone treatment was con- tinued. Cell leakage was determined 4 h later as described in Table 4. Results are means of three independent experiments f S. D.

Pretreatment Treatment Cell leakage

% - None 5 k 3 - cycloheximide 8 + I - TNF 8 + 4 - TNF/cycloheximide 4 3 k Y - LPS 8 & 4 - LPS/cycloheximide 40f 17 Dexamethasone LPS/cycloheximide 49 * 22 Dexamethasone TNFicycloheximide 41& 5

Table 6. Desensitization of Kupffer cells towards the lytic action of T N F and LPS in combination with cyclohexirnide by treatment with TNF or LPS After 48 h in primary culture, Kupffer cells were either treated with TNF (8000 Ujml), LPS (400 ng/ml) or left untreated. After another 24 h medium was changed to RPMIjl % bovine serum albumin and the substances (cycloheximide, 300 ng/ml; TNF, 8000 U/ml; LPS, 400 ng/ml) added. Leakage was determined 4 h later. Results are means of three independent experiments f S. D.

_ _ _ _ ~

Pretrealmen t Treatment Cell leakage

-

cycle heximide TNF TNF/cycloheximide LPS LPS/cycloheximide TNF/cyclo heximide LPS/cycloheximide TNF/cycloheximide LPS jcycloheximide

Yo

Y + 7 Y + _ 5 9 f 5

36+_ 16 l o + _ 6 37 +_ 13

8 f 4 4 + _ 4

I l k 1 1 3 k 5

reduces TNF-induced cell lysis in certain tumor cell lines [26, 271. Therefore, dexamethasone was tested for 24 h and during the lysis experiment to find if it could prevent cell death. As shown in Table 5, dexamethasone treatment had no beneficial effect on Kupffer cell survival.

Induction of tolerance to in vivo toxicity of LPS as well as of TNF has been described [28]. For this reason the effect of

incubation of Kupffer cells with LPS and TNF prior to ex- posure to cycloheximide and LPS or TNF was examined. TNF and LPS induced cross-tolerance towards each other (Table 6).

588

LPS PTNF

Fig. I . Tentative scheme of the inverse effect of PGE2 on its own synthesis elicited by LPS and TNF

DISCUSSION

Several factors may be responsible for the incomplete sup- pression of the LPS-elicited PGE2 synthesis by anti-(re- combinant mouse TNF) antisera. Considerable species speci- ficity of TNF has been reported [29] and, therefore, neutraliza- tion of rat TNF by anti-(mouse TNF) antiserum in the mouse L929 cell lysis assay may not correspond to its neutralization capacity in a rat Kupffer cell assay. Membrane-bound forms of TNF have been described [30, 311. These are active in killing tumor cell targets and may exist as membrane-spanning molecules [32] or as a cytokine bound to its own receptor [33]. It has not been determined whether these forms of TNF contribute to an autocrine effect on the producer cell and whether an antibody prevents this autocrine action as it does in the case of the soluble form. Finally, a second TNF-inde- pendent mechanism may exist by which LPS exerts its stimu- latory activity on Kupffer cells.

PGE2 inhibits LPS-stimulated prostanoid formation very specifically and at nanomolar concentrations, but it does not inhibit the prostanoid synthesis evoked by TNF. PGE2 added to TNF -stimulated Kupffer cells even has an enhancing effect that raises eicosanoid levels in the medium into the range of LPS-stimulated cells. PGEz per se has no stimulatory activity. Assuming a single pathway of eicosanoid synthesis from arachidonic acid in rat Kupffer cells, it may be concluded that the signal transducing pathway triggered by TNF has an immediate, PGE2-insensitive access to the process of prosta- glandin formation, whereas the conversion of the LPS-elicited signal includes a PGE2-inhibitable step (Fig. 7). Thus, the existence of a TNF-independent pathway cannot be excluded, although TNF plays a major role in mediating LPS action.

Little is known about molecular mechanisms involved in LPS action. Although an LPS-receptor has not yet been iden- tified, several intracellular signal-transduction pathways are discussed [34]. LPS exposure of rat Kupffer cells leads to a slow, steady increase in intracellular cAMP levels [2]. TNF synthesis is regulated at the transcriptional as well as the posttranscriptional level [35]. Stimulation of TNF release by LPS was reported to be associated with a rise in the intracellu- lar cGMP level [14]. TNF and inhibitory doses of PGE2 raise intraccllular cAMP levels in peritoneal macrophages [14]. Eicosanoid synthesis seems to be regulated by the intracellular Ca2+ concentration controlling the activity of phospholipase

LPS activation of Kupffer cells is not only controlled by autocrine mechanisms, but also by extrahepatic hormones. Glucocorticoids released from the adrenal glands in severe

~361.

states of shock, and frequently used as anti-inflammatory and immunosuppressive drugs, inhibit LPS activation of rat Kupffer cells in three ways: (a) They very effectively inhibit TNF release; (b) they inhibit LPS-induced PGE2 release; (c) they completely reverse the LPS-induced changes in the pro- stanoid pattern of Kupffer cells.

The LPS-induced PGE2/PGD2 shift implicates that phospholipase A2 is not the only regulated enzyme of the pathway. Regulation of prostaglandin synthases also seems likely as a significant increase in the conversion of free arachidonic acid to PGE2 takes place in cell-free extracts of LPS-treated Kupffer cells [25]. Selective degradation of PGD2 to 9-deoxy-d9-PGD2 and d "-PGJ2 which is observed after incubation times [37,38] of several hours does not ac- count for the PGE2/PGDz shift seen Ih after the addition of arachidonic acid (Table 3), as the radioimmunoassay for PGDz cross-reacts with these metabolites and HPLC analysis reveals hardly any degradation products. The effect of glucocorticoids on this phenomenon requires further studies.

TNF or LPS cause rapid cell death of rat Kupffer cells in primary culture if combined with cycloheximide. TNF-related cytotoxicity enhanced by inhibitors of transcription or trans- lation has been observed in vivo in tumor models [39] and in vitro in some cell lines [26]. It is now shown that inhibition of protein synthesis also renders TNF cytotoxic to cells in pri- mary culture which have not been transformed, show no growth in culture and are well-differentiated macrophages [19]. This cell type is considered to be important in the defense against infections and migrating tumor cells. In these situ- ations one finds elevated levels of TNF in serum. The source of the cytokine may be, to some extent, the Kupffer cell itself. Destruction of Kupffer cells by a TNF-mediated mechanism may, therefore, be disadvantageous. A situation of depressed protein synthesis and elevated LPS or TNF levels is typically found in cytostatic treated patients suffering from bacteremia or in tumor therapy using a recently recommended [40] combi- nation of TNF and cytostatic drugs.

LPS, highly toxic in vivo, shows little toxicity in cell culture. LPS/cycloheximide-induced cytotoxicity does not seem to be due to endogenous TNF production by the Kupffer cell itself, as TNF levels after dexamethasone treatment did not exceed the levels of the untreated or cycloheximide-treated controls.

Involvement of phospholipase A2 activation in the cytolytic process of TNF [26,27] seems rather unlikely, as dexamethasone, used at a concentration that blocks eicosanoid formation almost completely, does not inhibit TNF/cycloheximide- or LPS/cycloheximide-induced cytoly- sis. Kupffer cell lysis most likely corresponds to the rapid, dexamethasone-insensitive cytolysis of TA1 pre-adipocytes [41] rather than to the slow dexamethasone-inhibitable cell destruction observed in other cell lines [26, 271.

The lytic action of TNF/cycloheximide and LPS/ cycloheximide can be almost completely abolished by a 24 h treatment of the Kupffer cells with TNF or LPS. This desensitization could be either due to the down-regulation of receptors or the activation of a protective, protein synthesis- dependent mechanism which should be the same for LPS and TNF. Studies on TNF-binding sites of other cell types have shown a down-regulation by TNF itself [42]. Therefore down- regulation of TNF receptors on Kupffer cells by TNF as well as by LPS, which leads to endogenous TNF production, is likely [43]. The LPS receptor has not yet been characterized, its down-regulation by LPS as well as by TNF may be a new element in the reciprocal action of LPS and TNF. The fact that unimpaired protein synthesis protects Kupffer cells

589

against TNF and LPS toxicity, and that several intracellular proteins are induced by TNF 144, 451, favours a protective mechanism.

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