Báo cáo khoa học: Acylation of lysophosphatidylcholine plays a key role in the response of monocytes to lipopolysaccharide

doi:10.1046/j.1432-1033.2003.03649.x

Eur. J. Biochem. 270, 2782–2788 (2003) (cid:2) FEBS 2003

Acylation of lysophosphatidylcholine plays a key role in the response of monocytes to lipopolysaccharide

Bernhard Schmid1,2, Michael J. Finnen3, John L. Harwood1 and Simon K. Jackson2 1School of Biosciences, Cardiff University, UK; 2University of Wales College of Medicine, Cardiff, UK; and 3Yamanouchi Research Institute, Oxford, UK

50201 (3-hydroxyethyl 5,3¢-thiophenyl pyridine) strongly inhibited (up to 90%) TNF-a and IL-6 production in response to LPS in both unprimed MonoMac-6 cells and in cells primed with IFN-c. In similar experiments, these inhibitors also substantially decreased the response of both primed and unprimed peripheral blood mononuclear cells to LPS. Sequence-based amplification methods showed that SK&F 98625 inhibited TNF-a production by decreasing TNF-a mRNA levels in MonoMac-6 cells. Taken together, the data from these studies suggest that LPCAT is a key enzyme in both the pathways of activation (priming) and the inflammatory response to LPS in monocytes.

lysophosphatidylcholine acyltransferase;

Keywords: lyso- phosphosphatidate acyltransferase; inflammatory response; lipopolysaccharide.

Mononuclear phagocytes play a pivotal role in the pro- gression of septic shock by producing tumor necrosis factor- a (TNF-a) and other inflammatory mediators in response to lipopolysaccharide (LPS) from Gram-negative bacteria. Our previous studies have shown monocyte and macrophage activation correlate with changes in membrane phospholipid composition, mediated by acyltransferases. Interferon-c (IFN-c), which activates and primes these cells for enhanced inflammatory responses to LPS, was found to selectively activate lysophosphatidylcholine acyltransferase (LPCAT) (P < 0.05) but not lysophosphatidic acid acyltransferase (LPAAT) activity. When used to prime the human mono- cytic cell line MonoMac 6, the production of TNF-a and interleukin-6 (IL-6) was approximately five times greater in cells primed with IFN-c than unprimed cells. Two LPCAT inhibitors SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo2,3- dihydro-imidazole-1-yl)heptane phosphonate) and YM

LPS-induced mediators such as TNF-a has been the focus of much research aimed at developing specific therapies for septic shock.

Endotoxin, the lipopolysaccharide (LPS) component of the cell walls of Gram-negative bacteria, is an important microbial molecular pattern that initiates inflammatory and coagulation reactions as part of the host innate immune response to infection. However, excessive inflammatory responses to LPS can lead to septic shock.

increased

Many of the deleterious effects of LPS are mediated by inflammatory cytokines including tumor necrosis factor (TNF)-a, interleukin (IL)-1 and IL-6 produced by mono- nuclear phagocytes (e.g. monocytes and macrophages) [1]. TNF-a has been shown to be a key mediator in experimen- tal LPS-induced septic shock as neutralizing antibodies to TNF-a prevent mortality [2,3] and TNF-a receptor knock- out mice are less sensitive to the biological effects of LPS [4]. Understanding and modulating the production of

Studies in our laboratories have been concerned with elucidating mechanisms of responsiveness to low (nonlethal) doses of LPS which, we believe, underscore the excessive inflammatory responses leading to septic shock [5,6]. An important regulator of LPS-induced biological activity is interferon (IFN)-c [7,8] and the neutralization of IFN-c or the deletion of its receptors have been shown to be protective for the lethal outcomes of several forms of endotoxic shock [9,10]. An important contribution of IFN-c to the development of LPS-induced shock is by priming monocytes/macrophages inflammatory for responsiveness to LPS [11]. The molecular basis for such priming reactions remain obscure.

In previous studies investigating the priming of mono- cytes by IFN-c, we showed that this cytokine significantly modified the phospholipid composition of such cells and increased the unsaturated fatty acyl groups esterified at the sn-2 position of phosphatidylcholine (PtdCho) [5,6]. More- over, we found that IFN-c up-regulated monocyte lysoPC acyltransferase (LPCAT), a key enzyme in the regulation of PtdCho fatty acyl composition [6,12]. These results suggest that LPCAT may regulate the priming of monocytes by IFN-c and allow increased inflammatory cytokine produc- tion when these cells are stimulated by LPS. In studies of T-lymphocyte activation, Szamel and colleagues [13,14] have demonstrated that activation of the T-cell antigen

Correspondence to S. K. Jackson, Department of Medical Microbio- logy, University of Wales College of Medicine, Cardiff CF14 4XN, UK. Fax: + 44 29 20742161, Tel.: + 44 29 20744725, E-mail: jacksonsk@cardiff.ac.uk Abbreviations: BSA, bovine serum albumin; DAG, diacylglycerol; IFN-c, interferon-c; IL-6, interleukin-6; LPS, lipopolysaccharide; LPAAT, lysophosphatidate acyltransferase; LPCAT, lysophospha- tidylcholine acyltransferase; NASBATM, nucleic acid sequence based amplification; PtdOH, phosphatidic acid; PKC, protein kinase C; PtdCho, phosphatidylcholine; TNF-a, tumor necrosis factor-a. (Received 11 September 2002, revised 17 March 2003, accepted 02 May 2003)

Monocyte acyltransferase in inflammation (Eur. J. Biochem. 270) 2783

(cid:2) FEBS 2003

receptor/CD3 complex leads to increased incorporation of polyunsaturated fatty acids into phosphatidylcholine, also mediated by a LPCAT. Thus, LPCAT may alter the cell membrane lipid environment so as to favor the assembly of a signaling complex which can then activate the cellular response [15].

isms and Cell Cultures (Braunschweig, Germany). Cells were grown in RPMI 1640 medium supplemented with 10% (v/v) low endotoxin fetal bovine serum, 2 mM L-glutamine, 100 UÆmL)1 penicillin, 100 UÆmL)1 streptomycin, 1 mM sodium pyruvate, 1% (v/v) nonessential amino acids (all from Life Technologies/Gibco, Paisley, UK) ((cid:2)complete(cid:3) RPMI medium) and 8 lgÆmL)1 bovine insulin (Sigma). Cells were grown at 37 (cid:3)C and 5% (v/v) CO2 in a humidified incubator.

To elucidate the role of LPCAT in the priming and activation of monocytes by IFN-c, we have utilized specific LPCAT inhibitors in an attempt to block this process. We present herein evidence to show that LPCAT (but not other acyltransferases) is a key regulator of both the priming of monocytes by IFN-c and for LPS-initiated TNF-a release.

Materials and methods

Materials

Unless stated otherwise, all reagents were from Sigma Chemical Co (Poole, UK) and were of the best available grades. Recombinant human IFN-c was from Peprotech (London, UK) and diluted in endotoxin-free water (Sigma).

To isolate human monocytes, blood (10 mL) was collec- ted from a healthy volunteer by venipuncture into a heparinized tube and 2 mL Hypaque-Ficoll (Sigma) was layered on top. The sample was centrifuged for 20 min at 400 g at room temperature and the mononuclear cell layer removed to a fresh tube. The pellet was washed twice with NaCl/Pi. RPMI 1640 medium without additives was added to cells to give a cell density of 5 · 106 mL)1. To each well of a 24-well plate, 400 lL of the cell suspension was added and incubated at 37 (cid:3)C for 2 h. The monolayer was washed thrice with warm RPMI 1640 medium. Fresh complete RPMI 1640 medium (with all additives detailed above) (1 mL) was added and the cells incubated at 37 (cid:3)C as indicated in the legends to the figures.

Acyl-transferase inhibitors

Cell viability was determined by dye exclusion. Equal volumes of cell suspension and a 0.4% (v/v) solution of trypan blue (Sigma) in 20 mM NaCl/Tris (pH 7.3) were mixed. In all experiments viability was greater than 95%.

2

1

The acyltransferase inhibitor SK&F 98625 [diethyl 7-(3,4, 5-triphenyl-2-oxo2,3-dihydro-imidazole-1-yl)heptane phos- phonate] [16] was purchased from Ferring Research Insti- tute, Southampton, UK. YM 50201 (3-hydroxyethyl 5,3¢-thiophenyl pyridine), was a kind gift from the Yama- nouchi Research Institute, Oxford, UK. Their structures are given in Fig. 1. YM 50201 is a non-competitive inhibitor of CoA-dependent LPCAT and has been shown to significantly inhibit the incorporation of [14C]linoleic acid into lysophos- . phatidylcholine (M. J. Finnen, unpublished results)

Cell culture

The acute monocytic leukemia cell line MonoMac-6 [17] was obtained from the German Collection of Microorgan-

Preparation of microsomes MonoMac-6 cells (5 · 106) were incubated in 25 mL complete RPMI 1640 medium under conditions indicated in the legend to Fig. 2 and harvested by centrifugation at 700 g for 2 min at 4 (cid:3)C. The pellet was washed twice in microsomal buffer (pH 7.4) containing sucrose 250 mM, Tris 10 mM, MgCl2 1 mM, EGTA 1 mM [18]. The cells were finally resuspended in 1 mL of microsomal buffer and cells were lysed in an ice-cooled Dounce homogenizer (2500 r.p.m., 2 min). Successful cell lysis was checked by light microscopy. Another 1 mL of microsomal buffer was added. To remove unbroken cells and large debris, the lysate was centrifuged at 700 g for 5 min and the resulting supernatant was spun at 20 000 g for 20 min and the final supernatant at 100 000 g for 1 h. The 100 000 g pellet thus produced was resuspended in 1 mL microsomal buffer (above) and stored at )80 (cid:3)C. Protein concentration was determined by the method of Bradford [19].

Measurement of coenzyme A-dependent acyltransferase activity

(0.5 nmol;

Enzyme activity was determined by measuring the incor- poration of radioactivity from acyl-CoA into diacyl- phosphoglycerides. Aliquots 0.7 kBq) of [1–14C]oleoyl-CoA (Amersham) and 1 nmol 1-palmitoyl lysophosphatidylcholine or 1-oleoyl lysophosphatidic acid for each assay were dried under vacuum and resuspended in 75 lL assay buffer (150 mM NaCl, 1 mM EGTA and 10 mM Na2HPO4; pH 7.4) [18]. Microsomes (approxi- mately 1 lg protein) were added in 25 lL assay buffer and the mixture incubated with shaking for 20 min at 37 (cid:3)C. The reaction was stopped by the addition of 100 lL

Fig. 1. Structures of the acyltransferase inhibitors used. (A) SK&F 98625 [14] and (B) YM50201.

2784 B. Schmid et al. (Eur. J. Biochem. 270)

(cid:2) FEBS 2003

Multiskan plate reader using the absorbance difference, A450)A540.

Detection of human TNF-a mRNA by NASBATM TNF-a mRNA levels were measured by NASBATM (nucleic acid sequence-based amplification) as described previously [21]. Briefly, a silica-based extraction system was used to prepare nucleic acids from MonoMac-6 cells. TNF-a mRNA was amplified in the NASBATM process and analyzed by polyacrylamide gel electrophoresis. Dried gels were scanned on a Hewlett Packard photosmart scanner.

Results

Stimulation of monocyte acyltransferase activity by IFN-c

chloroform/methanol (1 : 2, v/v). An additional 200 lL chloroform, followed by 200 lL 1 M potassium chloride were then added. After vortexing, the mixture was centri- fuged at 400 g for 2 min, the aqueous phase discarded and the organic phase dried under vacuum at room temperature. The latter was redissolved in 25 lL chloroform and applied to a TLC plate (Silica gel 60, Merck, Darmstadt, Germany). The samples were separated using a solvent of chloroform/ methanol/water (65 : 35 : 7, v/v/v). Authentic lipid stand- ards (Avanti Polar Lipids, Alabaster, AL, USA) were separated alongside the samples for identification. After drying the plates, lipids were lightly stained with I2 vapor and their positions marked. Once the I2 had evaporated, individual lipid bands were scraped into scintillation vials and radioactivity estimated in Optisafe (cid:2)Hisafe(cid:3) scintillant (Fisons, Loughborough, UK) using a LKB Wallac 1211 betacounter. Automatic quench correction was made by the external standard channels ratio method. The data was analyzed by Student’s unpaired t-test.

3

Our previous work [5,6] and that of others [22] has shown that IFN-c can profoundly alter the acyl chain compo- sition of monocyte membrane phospholipids. Such alter- ations could be attributed to Lands-type remodeling [23] and were coincident with increased LPCAT activity [12]. To confirm that the modifications of phospholipids were due to altered acyltransferase activity, the present study measured the effect of IFN-c on monocyte LPCAT and LPAAT.

Measurement of TNF-a MonoMac-6 cells (5 · 105 cells per well) were incubated in a 24-well tissue culture plate as detailed in the legend to Fig. 2. Phorbol 12-myristate 13-acetate (PMA) (0.5 ngÆmL)1) was added to the incubation medium to aid the secretion of TNF-a from the MonoMac-6 cells. At this concentration PMA alone does not lead to a change in TNF-a levels [20]. Cells were harvested by centrifugation (1 min, 10 000 g, room temperature) and nucleic acids extracted for nucleic acid sequence based amplification (NASBATM) (see below). The supernatants from the incubations were stored at )80 (cid:3)C.

Acyltransferase activities were measured in microsomes prepared from MonoMac-6 cells incubated in the presence or absence of 250 UÆmL)1 IFN-c. It has been shown previously that this concentration of IFN-c modulates the response of monocytes to LPS [24]. The cytokine was found to significantly increase LPCAT activity (P < 0.05), while having no effect on LPAAT activity (Table 1). However, LPS, added before or after IFN-c, did not affect the LPCAT activity (data not shown). The stimulation of LPCAT by IFN-c could be inhibited by the tyrosine kinase inhibitor tyrphostin (2.5 lM) but not by the protein kinase C inhibitor bisindolylmalamide (1 lM) (data not shown). This suggests that the signaling pathway from the IFN-c receptor to the acyltransferase involves tyrosine kinases but not protein kinase C. Such a finding is not unexpected as the Janus family of cytoplasmic tyrosine kinases is known to relay signals from IFN-c receptors to the nucleus, thereby initiating gene activation [25].

Effect of priming on TNF-a production

IFN-c is well-known to up-regulate monocyte responses to LPS [11,24] although the mechanism for this priming effect is not well understood. We previously showed that mono- cytes activated by IFN-c showed phospholipid modifi- cations consistent with increased LPCAT activation. Therefore, in this study we wished to assess the effect of blocking LPCAT activity on priming of monocytes by IFN-c. To do this we utilized two acyltransferase inhibitors. SK&F 98625, originally described as a CoA-independent acyltransferase inhibitor [16], was included as it inhibits both LPCAT and LPAAT in MonoMac 6 cells with IC50 values of 10 lM and 30 lM, respectively (data not shown). At 20 lM, the concentration used in our experiments, it completely inhibits both LPCAT and CoA-independent acyltransferase activity. In contrast, YM 50201 completely

Production of TNF-a protein by MonoMac-6 cells and peripheral blood mononuclear cells was determined by an ELISA method. All incubations were performed at room temperature. An ELISA plate (Nunc Maxisorb) was loaded with 100 lL antihuman TNF-a mouse monoclonal capture antibody (R&D, Minneapolis, MN, USA) in NaCl/Pi (4 lgÆmL)1) per well and incubated overnight. The plate was washed thrice with 0.05% v/v Tween 20 in NaCl/Pi and blotted dry. To reduce nonspecific binding, 300 lL bovine serum albumin (BSA; 1%, w/v) and sucrose (5%, w/v) in NaCl/Pi were added to each well and the plates incubated for at least 1 h and washed as above. Human recombinant TNF-a (R&D) was diluted in dilution buffer (NaCl 150 mM, BSA 0.1%, Tween 20 0.05%, v/v, Tris 20 mM, pH 7.3). Samples (supernatants from the incubations) were diluted in the same buffer where appropriate. Standards ranging from 15.6 to 1000 pgÆmL)1 and samples (100 lL) were added to the plate and incubated for 1 h. The plate was washed as above and 100 lL 200 ngÆmL)1 biotinylated detection antibody (R&D, Abingdon, UK) in dilution buffer added. After incubating for 2 h, the plate was washed, 100 lL streptavidin–horseradish peroxidase conju- gate (0.6 lgÆmL)1, Zymed, San Francisco, CA, USA) added and the plate incubated for 20 min. The plate was washed and 100 lL of tetramethylbenzidine substrate containing 0.01% H2O2 (Kirkegaard and Perry, Gaithersburgh, MD, USA) per well. The plate was incubated in the dark for 20 min and the reaction stopped by the addition of 50 lL of 0.5 M sulfuric acid. The plate was read on a Labsystems

Monocyte acyltransferase in inflammation (Eur. J. Biochem. 270) 2785

(cid:2) FEBS 2003

Table 1. Effect of interferon c on acyltransferase activity from Mono- Mac-6 cells. MonoMac-6 cells (5 · 106 in 25 mL RPMI 1640 medium with the additives detailed in the Materials and methods section) were incubated in triplicate with or without IFN-c (250 UÆmL)1) for 12 h and microsomes prepared as described in Materials and methods. LPCAT and LPAAT activities were measured in vitro in duplicate. Results are presented as mean ± SD from six independent experi- ments. Differences were analyzed by Student’s t-test.

Acyltransferase activity (nmolÆmin)1Ælgprotein)1)

LPCAT LPAAT

6.33 ± 0.35 7.73 ± 0.25* 2.07 ± 0.19 1.96 ± 0.16 Control IFN-c

4

inhibits LPCAT but does not affect LPAAT or CoA- independent AT activities at this concentration (M. J. Finnen, unpublished observations) . Neither inhibitor was toxic to the cells at the concentrations used in this study (data not shown).

MonoMac-6 cells were preincubated with or without the acyltransferase inhibitors SK&F 98625 (20 lM) or YM 50201 (1 lM) for 30 min and then with IFN-c (250 UÆmL)1) for 12 h. After this period LPS was added as indicated in Fig. 2 and the cells incubated further for 3 h after which the cell supernatant was taken for cytokine assay.

* P < 0.05.

Incubation of the cells with IFN-c alone did not change TNF-a production (Fig. 2). Stimulation with LPS alone induced significant TNF-a production compared with unstimulated cells and this was increased approximately a further fourfold by priming with IFN-c (Fig. 2). Preincu- bation with either SK&F 98625 or YM 50201 almost completely inhibited TNF-a production in cells challenged with LPS either with or without priming by IFN-c.

To confirm that the results seen with the MonoMac-6 cells were representative of human monocytes, which play a prominent role in LPS recognition and subsequent TNF-a production in vivo [26], peripheral blood monocytes were

isolated and incubated in the same way as the experiments with MonoMac 6 cells (Fig. 3). In addition to TNF-a, the inflammatory cytokine IL-6 was also measured to check that the results were not just specific for TNF-a. IFN-c again was an effective priming agent for LPS-induced responses and the acyltransferase inhibitors SK&F 98625 and YM 50201 inhibited both TNF-a and IL-6 production significantly (Fig. 3A,B). These results confirm that Mono- Mac-6 cells can be used as a model for peripheral blood monocytes and that the acyltransferase LPCAT plays a significant role in the production of TNF-a and IL-6 in LPS-stimulated monocytes.

Fig. 3. Effect of acyltransferase inhibitors on TNF-a (A) and IL-6 (B) production in isolated peripheral blood mononuclear cells challenged with IFN-c and/or LPS. Peripheral blood monocytes (5 · 104 mL)1) were incubated as indicated using concentrations and conditions as detailed in the legend to Fig. 2. Results show means ± SD from six inde- pendent experiments each carried out in duplicate.

TNF-a is synthesized as a 26-kDa precursor and is then cleaved to yield the mature 17 kDa protein and it has been suggested that biological activity is regulated by the balance between the two forms [27]. Inhibition of precursor processing offers protection against a lethal dose of endotoxin [28]. To assess whether an acyltransferase inhi- bitor affects transcription or the processing and release of the TNF-a protein, MonoMac-6 cells were preincubated with SK&F 98625 (20 lM) for 30 min as indicated in Fig. 4. Cells were then incubated with IFN-c and subsequently with LPS. TNF-a protein levels in the supernatants were measured by ELISA and nucleic acids were extracted from the cell pellets. Two NASBA reactions were set up: One for the low copy housekeeping gene U1A and one for TNF-a. The level of U1A mRNA was similar in all samples as shown in Fig. 4A. This indicated equal extraction efficiency and that SK&F 98625 was not a general transcription inhibitor. There was a background level of TNF-a mRNA in unstimulated cells (Fig. 4B, lane 1) but a strong increase could be seen after IFN-c/LPS treatment (lane 2). Addition

Fig. 2. Effect of priming and acyltransferase inhibitors on TNF-a pro- duction in MonoMac-6 cells. MonoMac-6 cells (5 · 105 cellsÆmL)1 in a 24-well plate) were preincubated for 30 min with SK&F 98625 (20 lM) or YM 50201 (1 lM) and then further incubated with or without IFN-c (250 UÆmL)1) for 12 h as indicated. After this period, LPS (100 ngÆmL)1) was added and cells were incubated for a further 3 h. Results are means ± SD from four independent experiments each carried out in duplicate.

2786 B. Schmid et al. (Eur. J. Biochem. 270)

(cid:2) FEBS 2003

phospholipids [32]. Our results suggest that LPCAT may play a similar role in the activation of LPS-induced monocytes.

Our study has shown that IFN-c significantly enhances LPCAT activity in monocytes under conditions that prime these cells for responses to LPS. In contrast, LPAAT activity, reported to be up-regulated by lipid A in mesangial cells [33], is not altered by IFN-c priming of human monocytes. Moreover, LPCAT also appears to be critical to inhibition of this enzyme monocyte responses to LPS; almost completely inhibited LPS-induced TNF-a protein and mRNA production suggesting a role for LPCAT in the LPS-induced signaling pathways in monocytes. In addition, LPCAT was found to be crucial for IL-6 production in a similar manner, indicating that it may generally influence monocyte inflammatory cytokine responses to LPS.

Our results suggest that acyltransferases play a key role in the production of inflammatory cytokines from LPS- stimulated monocytes. our findings support those of Yamada et al., who showed that SK&F 98625 significantly reduced TNF production from peritoneal macrophages stimulated with the endomembrane Ca2+-ATP-ase inhi- bitor thapsigargin [34]. They assumed this effect to be due to inhibition of CoA-independent acyltransferase activity as SK&F 98625 was originally described as a CoA-indepen- dent acyltransferase inhibitor [15]. However, in our system, TNF-a production was significantly inhibited by YM 50201, a selective inhibitor of LPCAT, suggesting that LPS-stimulated TNF-a production may also be regulated via this CoA-dependent acyltransferase.

of SK&F 98625 reduced TNF-a mRNA levels to control levels (lane 3). In supernatants corresponding to samples from lanes 1–3, TNF-a protein levels were also measured by ELISA and were 50, 2099 and 103 pgÆmL)1, respectively. These values reflect the relative intensity of the detected mRNA in the three samples (Fig. 4). This result suggests that LPCAT inhibitors can inhibit TNF-a production in MonoMac-6 cells at the level of mRNA.

Fig. 4. Analysis of housekeeping gene U1A and TNF-a mRNA levels in primed and SK&F 98625 treated cells. MonoMac-6 cells (5 · 105 cellsÆ mL)1) were incubated for a total of 15 h. Lane 1, no additives; lane 2, incubation with IFN-c (250 UÆmL)1) for 12 h and subsequently with LPS (100 ngÆmL)1) for a further 3 h; lane 3, preincubated with SK&F 98625 (20 lM) for 30 min and then as lane 2; lane 4: negative control (probe alone); lane 5, positive control (TNF-a mRNA). The low copy housekeeping gene U1A (A) and TNF-a (B) mRNA were extracted, amplified and detected as described in Materials and methods.

Discussion

shown that

Interferon-c plays a critical role in priming monocytes and macrophages for LPS-induced cytokine production [11,22] that may have profound consequences for the development of LPS-induced septic shock [7,8]. However, the mechanism by which IFN-c activates mononuclear phagocytes for enhanced inflammatory responses to LPS has remained poorly defined. This IFN-c study has up-regulates LPCAT activity in monocytic cells and suggest that this enzyme plays a critical role in both the priming of these cells by IFN-c and their inflammatory cytokine response to LPS.

Several possibilities exist to explain the mechanisms through which LPCAT activity may regulate the mono- cyte priming of and responses to LPS. First, activation of plasma membrane LPCAT would likely result in elevated incorporation of polyunsaturated fatty acids into PtdCho. Exchange of fatty acid chains would lead to more unsaturated PtdCho species which might provide a substrate for PtdCho-specific phospholipase-C (PLC), resulting in elevation of diacylglycerol (DAG) species carrying polyunsaturated fatty acids [13]. It has recently been shown that PKC was activated only by 1,2-DAG carrying polyunsaturated fatty acids in activated T-cells [14]. In support of this, we have recently found that such DAG-stimulated PKC isoforms are required for the production of TNF-a in LPS-stimulated monocytes (unpublished observation).

Host sensitivity to LPS may be a crucial determinant in the development of lethal septic shock [7,8] and IFN-c has been shown to prime for such lethality in experimental models of septic shock [29]. Our previous studies [5,6] and those of others [22], have suggested that IFN-c may mediate such exaggerated responses, at least in part, through altered phospholipid metabolism. Indeed, the current investigation confirms that IFN-c can increase the activity of an enzyme, LPCAT, that participates in the rapid turnover of PtdCho. Lysophospholipid acyltransferases maintain membrane lipid composition and the asymmetrical distribution of unsaturated fatty acids within phospholipids [30] and control free arachidonic acid levels [31]. In addition, LPCAT has been suggested to play a crucial role in the early phase of T-cell activation by the elevated incorpor- ation of polyunsaturated fatty acids into plasma membrane

Second, LPS is known to activate monocytes via binding to the glycosylphosphatidylinositol (GPI)-linked surface receptor CD14 [35]. CD14 is localized in membrane microdomains enriched in sphingolipids and cholesterol known as lipid rafts [36]. CD14, as a GPI-linked receptor, lacks a transmembrane domain and the membrane protein TLR4 in association with MD2 mediate signal transduction to LPS [37]. It has been shown that in monocytes CD14 and TLR4 colocalize in rafts to induce signal transduction in response to LPS [38]. It would be expected that the composition of the monocyte membrane could influence the fluidity and hence movement of lipids and proteins within and about the lipid raft regions. Furthermore, recent studies have shown that glycerophospholipids such as PtdCho, are also components of the rafts [39] and alteration of the saturation of PtdCho within these regions would also

Monocyte acyltransferase in inflammation (Eur. J. Biochem. 270) 2787

(cid:2) FEBS 2003

alter the colocalization of the signaling receptors for LPS. In addition, IFN-c has recently been shown to augment TLR4 mRNA and surface expression in human monocytes which was associated with enhanced nuclear factor-kappa B acti- vation and cytokine production [40]. LPCAT, by controlling the physical state of the lipid microenvironment in the rafts, could modulate the signaling receptor response to LPS.

phocytes by selectively preventing a transmembrane signal trans- duction pathway leading to sustained activation of a protein kinase C isoenzyme, protein kinase C-beta. Eur. J. Immunol. 23, 3072–3081.

14. Szamel, M., Kaever, V., Leufgen, H. & Resch, K. (1998) The role of lysophosphatide acyltransferases and protein kinase C isoforms in the regulation of lymphocyte responses. Biochem. Soc. Trans. 26, 370–374.

In conclusion, we have shown that inhibition of mono- cyte LPCAT activity significantly inhibits LPS-induced TNF-a and IL-6 production, strongly suggesting that LPCAT plays an important role in mediating the signaling pathways for LPS-activation of these cells.

15. Jackson, S.K. (1997) The role of lipid metabolites in the signalling and activation of macrophages by lipopolysaccharide. Prog. Lipid Res. 36, 227–244.

Acknowledgments

16. Chilton, F.H., Fonteh, A.N., Sung, C.M., Hickey, D.M., Torphy, T.J., Mayer, R.J., Marshall, L.A., Heravi, J.D. & Winkler, J.D. (1995) Inhibitors of CoA-independent transacylase block the movement of arachidonate into 1-ether-linked phospholipids of human neutrophils. Biochemistry 34, 5403–5410.

We are grateful to the Yamanouchi Research Institute, Oxford, for studentship support for BS.

17. Ziegler-Heitbrock, H.W., Thiel, E., Fotterer, A., Herzog, V., Wirtz, A. & Riethmu¨ ller, G. (1988) Establishment of a human cell line (MonoMac-6) with characteristics of mature monocytes. Int. J. Cancer 41, 456–461.

References

1. Glauser, M.P., Zanetti, G., Baumgartner, J.D. & Cohen, J. (1991) 18. Winkler, J.D., Sung, C.M., Bennett, C.F. & Chilton, F.H. (1991) Characterization of CoA-independent transacylase activity in U937 cells. Biochim. Biophys. Acta 1081, 339–346. 5 Septic shock: pathogenesis. Lancet 338, 732–736 .

2. Silva, A.T., Bayston, K.F. & Cohen, J. (1990) Prophylactic and therapeutic effects of a monoclonal antibody to tumor necrosis factor-alpha in experimental gram-negative shock. J. Infect. Dis. 162, 421–427.

19. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 20. Pradines-Figueres, A. & Raetz, C.R. (1992) Processing and secretion of tumor necrosis factor alpha in endotoxin-treated MonoMac-6 cells are dependent on phorbol myristate acetate. J. Biol. Chem. 267, 23261–23268.

3. Ashkenazi, A., Marsters, S.A., Capon, D.J., Chamow, S.M., Figari, I.S., Pennica, D., Goeddel, D.V., Palladino, M.A. & Smith, D.H. (1991) Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin. Proc. Natl Acad. Sci. USA 88, 10535–10539. 21. Darke, B.M., Jackson, S.K., Hanna, S.M. & Fox, J.D. (1998) Detection of human TNF-alpha mRNA by NASBA. J. Immunol. Methods 212, 19–28.

22. Furlong, S.T., Mednis, A. & Remold, H.G. (1992) Interferon- gamma stimulates lipid metabolism in human monocytes. Cell. Immunol. 143, 108–117. 23. Lands, W.E.M. (1960) Metabolism of glycerolipids. J. Biol. Chem. 4. Erickson, S.L., de Sauvage, F.J., Kikly, K., Carver-Moore, K., Pitts-Meek, S., Gillett, N., Sheehan, K.C., Schreiber, R.D., Goeddel, D.V. & Moore, M.W. (1994) Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature 372, 560–563. 235, 2233–2237.

5. Jackson, S.K., Darmani, H., Stark, J.M. & Harwood, J.L. (1992) Interferon-c increases macrophage phospholipid polyunsatura- tion: a possible mechanism of endotoxin sensitivity. Int. J. Exp. Pathol. 73, 783–791. 24. Burchett, S.K., Weaver, W.M., Westall, J.A., Larsen, A., Kronheim, S. & Wilson, C.B. (1988) Regulation of tumor necrosis factor/cachectin and IL-1 secretion in human mononuclear phagocytes. J. Immunol. 140, 3473–3481. 25. Haque, S.J. & Williams, B.R. (1998) Signal transduction in the interferon system. Semin. Oncol. 25, 14–22. 6. Darmani, H., Harwood, J.L. & Jackson, S.K. (1993) Interferon-c stimulated uptake and turnover of linoleate and arachidonate in macrophages: a possible pathway for hypersensitivity to endo- toxin. Cell. Immunol. 152, 59–71. 7. Heinzel, F.P. (1990) The role of IFN-gamma in the pathology of experimental endotoxemia. J. Immunol. 145, 2920–2924.

26. Wang, H. & Tracey, J.K. (1999) Basic Principles and Clinical Correlates. In Inflammation (Gallin, J.I. & Snyderman, R., eds), pp. 471–486. Lippincott Williams & Wilkins, Philadelphia. 27. Watanabe, N., Nakada, K. & Kobayashi, Y. (1998) Processing and release of tumor necrosis factor alpha. Eur. J. Biochem. 253, 576–582. 8. Doherty, G.M., Lange, J.R., Langstein, H.N., Alexander, H.R., Buresh, C.M. & Norton, J.A. (1992) Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor- alpha. J. Immunol. 149, 1666–1670.

28. Mohler, K.M., Sleath, P.R., Fitzner, J.N., Cerretti, D.P., Alderson, M., Kerwar, S.S., Torrance, D.S., Otten-Evans, C., Greenstreet, T. & Weerawarna, K. (1994) Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing. Nature 370, 218–220. 9. Car, B.D., Eng, V.M., Schnyder, B., Ozmen, L., Huang, S., Gallay, P., Heumann, D., Aguet, M. & Ryffel, B. (1994) Inter- feron-c receptor deficient mice are resistant to endotoxic shock. J. Exp. Med. 179, 1437–1344. 10. Silva, A.T. & Cohen, J. (1992) Role of interferon-c in experimental Gram-negative sepsis. J. Inf. Dis. 166, 331–335.

29. Heremans, H., Van Damme, J., Dillen, C., Dijkmans, R. & Biliau, A. (1990) Interferon-c, a mediator of lethal lipopolysaccharide- induced Shwartzman-like shock reactions in mice. J. Exp. Med. 171, 1853–1869.

11. Gifford, G.E. & Lohman-Matthes, M.L. (1989) Gamma-inter- feron priming of mouse and human macrophages for induction of TNF production by bacterial lipopolysaccharide. J. Natl Cancer Inst. 78, 121–124. 30. MacDonald, J.I.S. & Sprecher, H. (1991) Phospholipid fatty acid remodeling in mammalian cells. Biochim. Biophys. Acta 1084, 105–121.

31. Chilton, F.H., Fontech, A.N., Surette, M.E., Triggiani, M. & (1996) Control of arachidonate levels within 12. Neville, N.T., Jackson, S.K. & Harwood, J.L. (1997) The effects of inflammatory cytokines on acyl coenzyme-A-dependent acyl- transferase. Biochem. Soc. Trans. 25, 496S. Winkler, J.D. inflammatory cells. Biochim. Biophys. Acta 1299, 1–15. 13. Szamel, M., Bartels, F. & Resch, K. (1993) Cyclosporin A inhibits T cell receptor-induced interleukin-2 synthesis of human T lym-

2788 B. Schmid et al. (Eur. J. Biochem. 270)

(cid:2) FEBS 2003

37. Zarember, K.A. & Godowski, P.J. (2002) Tissue expression of human toll-like receptors and differential regulation of toll-like receptor mRNAs in leukocytes in response to microbes, their products and cytokines. J. Immunol. 168, 554–561. 32. Kerkhoff, C., Gehring, L., Habben, K., Resch, K. & Kaever, V. (1997) The mitogen-induced lysophospholipid: acyl-CoA acyl- transferase (LAT) expression in human T-lymphocytes is dimin- ished by hydrocortisone. Biophys. Biochem. Res. Commun. 237, 632–638. 33. Bursten, S.L. & Harris, W.E.

38. Jiang, Q., Akashi, S., Miyake, K. & Petty, H.R. (2000) Lipopo- lysaccharide induces physical proximity between CD14 and toll- like receptor 4 (TLR-4) prior to nuclear translocation of NF-kappaB. J. Immunol. 165, 3541–3544. (1991) Rapid activation of phosphatidate phosphohydrolase in mesangial cells by lipid A. Biochemistry 30, 6195–6203.

35. Ulevitch, R.J. & Tobias, P.S. 39. Rouquette-Jazdanian, A.K., Pelassy, C., Breittmayer, J.-P., Cousin, J.-L. & Aussel, C. (2002) Metabolic labeling of membrane microdomains/rafts in Jurkat cells indicates the presence of glyc- erophospholipids implicated in signal transduction by the CD3 T-cell receptor. Biochem. J. 363, 645–655.

34. Yamada, M., Ichinowatari, G., Tanimoto, A., Yaginuma, H. & Ohuchi, K. (1998) Inhibition of tumor necrosis factor-a produc- tion by SK&F 98625, a CoA-independent transacylase inhibitor, in cultured rat peritoneal macrophages. Life Sci. 62, PL297–302. (1995) Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu. Rev. Immunol. 13, 437–443. 36. Simons, K. & Ikonen, E. (1997) Functional rafts in cell mem- 40. Bosisio, D., Polentarutti, N., Sironi, M. et al. (2002) Stimulation of toll-like receptor 4 expression in human mononuclear phagocytes by interferon-c: a molecular basis for priming and synergism with bacterial lipopolysaccharide. Blood 99, 3427–3431. branes. Nature 387, 569–572.