Published online before print April 10, 2008
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* Centre for Research in Biomedicine, University of the West of England, Bristol, United Kingdom;
Department of Medical Microbiology, School of Medicine, and
School of Biosciences, Cardiff University, Cardiff, United Kingdom; and
School of Chemistry, University of Sheffield, Sheffield, United Kingdom
1Correspondence: Centre for Research in Biomedicine, University of the West of England, Bristol, BS16 1QY, UK. E-mail: simon.jackson{at}uwe.ac.uk
ABSTRACT
Sepsis, an overwhelming inflammatory response to infection, is a major cause of morbidity and mortality worldwide and has no specific therapy. Phospholipid metabolites, such as lysophospholipids, have been shown to regulate inflammatory responses in sepsis, although their mechanism of action is not well understood. The phospholipid-metabolizing enzymes, lysophospholipid acyltransferases, control membrane phospholipid composition, function, and the inflammatory responses of innate immune cells. Here, we show that lysophosphatidylcholine acyltransferase (LPCAT) regulates inflammatory responses to LPS and other microbial stimuli. Specific inhibition of LPCAT down-regulated inflammatory cytokine production in monocytes and epithelial cells by preventing translocation of TLR4 into membrane lipid raft domains. Our observations demonstrate a new regulatory mechanism that facilitates the innate immune responses to microbial molecular patterns and provide a basis for the anti-inflammatory activity observed in many phospholipid metabolites. This provides the possibility of the development of new classes of anti-inflammatory and antisepsis agents.
Key Words: lipopolysaccharide • lysophosphatidylcholine acyltransferase • sepsis
INTRODUCTION
Sepsis is a major cause of morbidity and mortality worldwide, with over 200,000 deaths annually in the United States alone [1
]. LPS is the endotoxin present in the outer membrane of Gram-negative bacteria and is an important trigger of sepsis [2
]. Macrophages and monocytes are prominent innate immune cells that recognize and respond to pM amounts of LPS with the production of inflammatory mediators including TNF-
and IL-6. Increased or prolonged exposure to LPS can lead to a rigorous, dysregulated expression of these inflammatory molecules, resulting in the cardiovascular derangements and organ damage characteristic of sepsis [3
]. Understanding the mechanisms that regulate the production of inflammatory mediators in response to LPS is an important goal in the development of new therapies for this condition.
Recently, it has been shown that a number of phospholipid metabolites, including oxidized phospholipids [4
] and lysophosphatidylcholine (LPC) [5
], have anti-inflammatory properties and have therapeutic potential in experimental models of sepsis. However, their mechanism of action has not been fully elucidated. Previously, it was shown that monocytes and macrophages activated in vivo or in vitro for enhanced inflammatory responses to LPS had altered molecular species of membrane phosphatidylcholine (PC) [6
7
]. This led us to identify a phospholipid-modifying enzyme, LPC acyltransferase (LPCAT), which can regulate inflammatory cytokine responses to LPS in monocytes and macrophages [8
9
]. Furthermore, we showed that various cytokines including IFN-
can up-regulate the activity of LPCAT in innate immune cells [10
]. This enzyme is of particular interest, as it may influence cellular inflammatory responses by two potential mechanisms. First, it uses LPC as a substrate, and increased LPCAT activity would be expected to consume this potential, antisepsis molecule and generate phospholipid products. Second, several reports show that alteration of lysophospholipid/phospholipid ratios can dramatically alter membrane properties and functions [11
12
]. Such altered properties can be expected to modulate the lateral movement of membrane proteins and may be crucial for the assembly of membrane receptor complexes. Moreover, changes in membrane phospholipid composition are known to affect cholesterol-rich microdomains known as lipid rafts [13
]. As the LPS receptor complex is known to be assembled in lipid rafts [14
], we sought to determine if LPCAT could regulate the translocation of the LPS receptor molecule, TLR4, into lipid raft domains. This report describes how inhibition of LPCAT in innate immune cells down-regulates the inflammatory response to LPS and other microbial stimuli by preventing translocation of TLR4 into membrane lipid raft microdomains, providing a novel, regulatory mechanism for inflammatory responses to infection.
MATERIALS AND METHODS
Reagents
All reagents were obtained from Sigma (Poole, UK), unless stated otherwise. The human MonoMac 6 cell line was obtained from German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany), and the A549 lung epithelial cell line was from American Type Culture Collection (Manassas, VA, USA). Palmitoyl-3-cysteine-serine-lysine-4 [Pam3Cys-Ser-(Lys)4] hydrochloride (Pam3Cys) and TLC plates were supplied by Merck Chemicals Ltd. (Nottingham UK). Depyrogenated glass vials, pyrogene-free water, and the Limulus ameboycte lysate (LAL) assay kit were obtained from Lonza Ltd. (Wokingham, UK). ELISA DuoSet kits were obtained from R&D Systems (Oxford, UK). Precast gels were from Invitrogen (Paisley, UK), antibodies for Western blotting and anti-G2A-blocking antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies for flow cytometry were from e-Bioscience (San Diego, CA, USA). ECL Plus detection kit and [1-14C] acyl-CoA were from Amersham Biosciences (Buckinghamshire, UK).
Cell lines
MM6 cells were maintained in RPMI-1640 medium, supplemented with 200 mM L-glutamine, 1% penicillin/streptomycin, 10% (v/v) FCS, 1 mM sodium pyruvate, and 1% (v/v) nonessential amino acids. A549 cells were grown using RPMI-1640 medium supplemented only with 5% FCS, 200 mM L-glutamine, and 1% penicillin/streptomycin. Both cell lines were seeded at a density recommended by the respective supplier and grown at 37°C in humidified air and 5% CO2. All media components were screened for the presence of endotoxin using a LAL assay sensitive to 5 pg endotoxin/ml (Lonza, Wokingham, UK).
LPS and Pam3Cys
A stock solution of LPS was prepared by dissolving 2 mg LPS in 1 mL pyrogen-free water. A stock solution of Pam3Cys was prepared by dissolving 5 mg Pam3Cys in pyrogen-free water in depyrogenated glass vials. These solutions were mixed by vortexing and kept at –80°C until use. The commercial LPS preparation was found to be free from protein and nucleic acid contamination by Coomassie blue binding and 260/280 nm absorbance, respectively, and from proteinase and DNase/RNase treatment and cellular responses as described recently [15
]. The Pam3Cys solution was found to be free of detectable levels of LPS (<10 pg/ml) by the LAL assay.
Cell stimulation
MonoMac 6 cells (5x105) were stimulated with LPS (100 ng/mL) or Pam3Cys (500 ng/mL) for 6 h, and the supernatants were collected and stored at –80°C until assayed. To assess the effect of LPC on the induction of LPS-induced cytokine release, the cells were preincubated with LPC (100 µM) for one-half hour in the presence or absence of anti-G2A (5 µg/mL)-neutralizing antibody to the LPC receptor before being stimulated with LPS for a further 6 h.
A549 epithelial cells (105) were seeded in 12-well plates and grown to confluence while changing the media daily. The cells were stimulated with LPS (100 ng/mL) in the presence of 1% human serum, with or with out the LPCAT inhibitor for 24 h.
Cytokine assay
The cytokines TNF-, IL-6, and IL-8 in the cell culture supernatants were assayed by ELISA using DuoSet kits according to the manufacturer’s instructions.
Isolation of lipid rafts
The lipid raft domains were isolated from cells as described by Triantafilou et al. [14
] with slight modifications. Briefly, cells were lysed in 300 µl MEB buffer (150 mM NaCl, 20 mM MES, pH 6.5) containing 200 mM Na3VO4, 1% Triton X-100, and protease inhibitor cocktails for 1 h on ice. Lysates were homogenized by sonicating for three 10 s bursts and mixed with an equal volume of 90% sucrose in MEB and placed at the bottom of polyallomer centrifuge tubes. Samples were overlaid with 1 ml 30% sucrose and 500 µl 5% sucrose in MEB buffer and centrifuged at 100,000 g for 16 h using a TLS-55 rotor and OptimaTM TL ultracentrifuge. Fractions (250 µl) were gently removed from the top of the gradient and 4 µl 5% sodium deoxycholate was added to each fraction to solubilize rafts.
Lipid raft-containing fractions were identified by dot-blotting for GM-1 ganglioside with HRP-conjugated cholera toxin. Proteins in lipid raft fractions were concentrated by methanol precipitation as described previously [16 ].
TLR4 detection in membrane fractions by Western blotting
Cell membrane fractions with equal amounts of protein were separated on 10% SDS-PAGE and blotted onto a nitrocellulose membrane using a blot module. TLR4 in each fraction was determined by immunoblotting with anti-TLR4 antibody and detected using an ECL Plus detection kit.
Expression of TLR4
Surface expression of TLR4 was assessed in A549 cells by flow cytometry using anti-TLR4-PE-conjugated antibody and isotype control on a FACSVantageTM flow cytometer (BD Biosciences, San Jose, CA, USA). The data were acquired using CELLQuest (BD Biosciences) and analyzed using WinMDI 2.8 software (Joe Trotter, PharMingen, San Diego, CA, USA).
Effect of mycostatin treatment
Cells were preincubated with mycostatin (0, 5, 25, 50, or 75 ug/mL) for 30 min. The cells were then washed three times with PBS and incubated for 2 h in fresh medium before they were stained to detect surface expression of TLR4 using flow cytometry.
Measurement of LPCAT activity
Enzyme activity was determined by measuring radioactive PC formed by incorporation of acyl groups from acyl-CoA into LPC as described previously [10
]. Briefly, an aliquot (0.5 nmol; 720 Bq) of [1-14C] acyl-CoA and 1 nmol 1-palmitoyl LPC was dried under vacuum and resuspended in 75 ml, 80 mM Tris-HCl assay buffer. Test samples (1 mg protein) were resuspended in 25 ml assay buffer, added to the mixture, and incubated for 20 min at 37°C. The reaction was stopped by the addition of 100 ml chloroform: methanol (1:2 v/v). Phase separation was facilitated by the addition of 200 ml chloroform and after 15 min, the addition of 200 ml 1 M potassium chloride. After centrifugation at 400 g for 2 min, the upper, aqueous phase was discarded, and the organic phase was dried under vacuum at room temperature.
The lipids were resolubilized in 25 ml chloroform and applied to a TLC plate (Silica gel 60) and cochromatographed with standards in chloroform/methanol/water (65:35:7, by vol). The plates were dried, and lipids were revealed with I2 vapor. After allowing the I2 to evaporate, phospholipid bands were scraped off into scintillation vials. Scintillant (6 ml) was added, and samples were counted in a Betacounter. Alternatively, electronic autoradiography was performed using a Canberra-Packard Instant Imager (Meriden, CT, USA).
LPCAT inhibitor
5-Hydroxyethyl 5,3' thiophenyl pyridine (HETP), a noncompetitive, specific inhibitor of CoA-dependent LPCAT [8
], was synthesized by a method adapted from Yamada et al. [17
]. In the cell systems used, HETP was found to have an IC50 of 10 µM for the inhibition of LPCAT activity. To investigate the role of LPCAT in cellular responses, a concentration of 20 µM was used routinely, as this was found to significantly inhibit LPCAT activity and did not affect cell viability.
Data analysis
Statistical comparisons among groups were determined by one-way ANOVA using Minitab software Version 14 (Minitab Inc., UK).
RESULTS
Several recent reports suggest that phospholipid metabolites have anti-inflammatory and potentially antisepsis properties, although their mechanism of action is not well understood [4
5
]. Previous work from our laboratories showed that inhibition of LPCAT can significantly reduce TNF- production at the transcriptional and post-transcriptional levels [8
]. Inhibition of LPCAT activity is expected to result in the lack of acylation of the substrate LPC, which has previously been reported to inhibit various inflammatory responses in innate immune cells [5
]. To investigate if the reduction in TNF- production seen when LPCAT was inhibited is a result of LPC, we sought to block the monocyte receptor for LPC, G2A [18
]. Treatment of the human monocyte cell line, MM6, with LPC resulted in a significant down-regulation in TNF- production (Fig. 1 A
). When anti-G2A antibody was included in the incubation, the inhibitory effect of the LPC was largely abolished (Fig. 1A)
, confirming that LPC acts through G2A in the MM6 monocytes. However, when anti-G2A antibody was added to the cells treated with the LPCAT inhibitor HETP and LPS, the levels of TNF- remained depressed (Fig. 1B)
, suggesting that the modulatory effect of LPCAT inhibition on TNF- was not mediated via LPC acting on G2A or alternatively, that endogenous and exogenous pools of LPC act differently.
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Figure 1. LPCAT inhibition down-regulates TNF- production in MM6 cells independently of the LPC receptor G2A. (A) Treatment of MM6 cells with 10 ng/ml LPS in the presence of 100 µM LPC caused a reduction of TNF- production that was inhibited by the anti-G2A antibody. (B) MM6 cells treated with 10 ng/ml LPS and 20 µM of the LPCAT inhibitor HETP caused down-regulation of TNF- that was not restored by anti-G2A. Inset shows the inhibition of LPCAT activity by HETP as the amount of PC formed from radiolabeled substrate; *, P < 0.05, versus LPS.
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or to monocytes. MM6 cells responded to incubation with Pam3Cys, a TLR2-stimulating molecule, with a vigorous production of TNF- (Fig. 2
). Preincubation of the cells with HETP significantly inhibited the production of TNF- (Fig. 2)
. Furthermore, incubation of the alveolar epithelial cell line, A549, with LPS induced the production of IL-8, which was significantly inhibited by preincubation with HETP (Fig. 3
).
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Figure 2. LPCAT inhibition down-regulates inflammatory responses to TLR2 ligands. Inhibition of LPCAT with HETP was effective in inhibiting TNF- production in response to Pam3Cys in MM6 cells. Results are the mean ± SD from three independent experiments; *, P < 0.05, versus Pam3Cys.
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Figure 3. HETP treatment resulted in down-regulation of IL-8 production in A549 lung epithelial cells. Results are the mean ± SD from three independent experiments; *, P < 0.05, versus LPS.
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Figure 4. Inhibition of LPCAT does not affect membrane expression of TLR4 in MM6 cells, which were incubated with no antibody, anti-TLR4 antibody, or isotype control antibody and analyzed by flow cytometry. (A) Untreated MM6 cells. (B) MM6 cells pretreated with HETP to inhibit LPCAT. (C) Overlay of results from A and B. Representative results from three independent experiments are shown. FL2, Fluorescence 2.
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was inhibited (Fig. 5 A
). However, the maximal dose of mycostatin (75 µg/ml) did not decrease expression of TLR4 (Fig. 5B
and 5C)
, suggesting that in our cell system, disruption of lipid rafts directly affected TLR4 signaling. To explore how any membrane-altering properties of LPCAT inhibition might influence TNF- production in response to LPS, we examined the translocation of the LPS receptor, TLR4, into membrane lipid microdomains (lipid rafts; Fig. 6 A
). Lipid raft fractions were identified in membrane fractions from MM6 cells by the raft marker ganglioside GM-1, which was visualized by staining with the ligand cholera toxin β-subunit [14
]. Immunostaining of the TLR4 receptor in unstimulated MM6 cells revealed that TLR4 is located essentially only in nonraft membrane domains. After stimulation with LPS, a significant fraction of the TLR4 was seen to be located in the lipid raft fractions of the cell membrane. When the cells were treated with the LPCAT inhibitor and stimulated with LPS, TLR4 was prevented from translocating into the raft fractions (Fig. 6A)
. When the raft and nonfractions from several experiments were pooled and blotted for the presence of TLR4, there was a significant lack of TLR4 in raft fractions from cells in which LPCAT activity was inhibited (Fig. 6B)
.
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Figure 5. Lipid raft disruption with mycostatin treatment inhibits TNF- production but does not affect TLR4 expression. Incubation of cells with 0–75 µg/ml mycostatin dose-dependently inhibited TNF- release, as determined by ELISA (*, P<0.01, vs. no mycostatin; A). Cells were incubated with (B) or without (C) 75 µg/ml mycostatin, and the expression of TLR4 was determined by flow cytometry. Representative results from three independent experiments are shown.
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Figure 6. Inhibition of LPCAT prevents translocation of the LPS receptor TLR4 into lipid raft membrane domains. (A) Fractions of MM6 cell membranes were separated by sucrose density gradient ultracentrifugation and lipid raft-containing fractions identified by the presence of the GM1 ganglioside by dot-blotting with HRP-conjugated cholera toxin β-subunit. Western blotting of the membrane fractions with anti-TLR4 antibody showed that LPS activation of MM6 cells stimulated the translocation of TLR4 into lipid raft domains, and this was prevented by treatment with the LPCAT inhibitor HETP. (B) Pooled raft and nonraft fractions clearly show the effect of HETP treatment on TLR4 translocation. Representative blots of membrane fractions are shown with a densitometry plot, indicating the changes in each membrane fraction from four independent experiments.
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DISCUSSION
The results from this study demonstrate that inhibition of the enzyme LPCAT can inhibit cellular responses to LPS by controlling the translocation of TLR4 into membrane lipid raft domains. This provides evidence that LPCAT, in addition to controlling the reacylation of membrane LPC, can regulate cellular responses to LPS via regulation of the translocation of TLR4 into membrane raft domains. Moreover, our results with Pam3Cys suggest that this enzyme can also control inflammatory responses to other bacterial ligands signaling via TLR2 [24 ], via regulation of assembly of their receptor complexes in membrane raft domains.
Inhibition of LPCAT with a specific inhibitor, HETP, was shown previously to inhibit TNF- production by monocytes at the mRNA and protein level [8
], suggesting LPCAT is important for the regulation of TNF- production. However, the mechanisms by which the enzyme might control such responses remained unknown.
It has been shown recently that membrane microdomains known as "lipid rafts" are used as platforms for the assembly of a functional receptor complex for LPS. The lateral mobility of TLR4 into the membrane lipid raft domains has been shown to be crucial for LPS signaling [14 25 ]. Molecules that disrupt lipid raft integrity such as mycostatin are known to inhibit signal transduction in response to LPS in a monocytic cell line [14 ].
Here, we demonstrate that inhibition of LPCAT inhibits TNF- production by preventing the translocation of TLR4 into membrane lipid raft domains in MM6 cells. This result suggests that LPCAT may regulate the assembly of a functional LPS receptor complex in the membrane by controlling membrane PC composition. Indeed, up-regulation of LPCAT activity, by cytokines such as IFN-, can enhance cellular responses to LPS by stimulating the assembly of the LPS receptor (S. K. Jackson and W. Abate, manuscript in preparation).
Furthermore, our results show that the LPCAT modulation of the inflammatory response is not restricted to the MM6 cell line. This report shows that IL-8 induction from A549 lung epithelial cells is also regulated by inhibition of this enzyme, and previous work showed that IL-6 and TNF- responses from human peripheral blood monocytes were linked to LPCAT activity [8
9
].
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. Recent studies have shown that glycerophospholipids such as PC are also components of lipid rafts [26 ], and alteration of the saturation of PC within these regions would also alter the colocalization of the signaling receptors for LPS. LPCAT, by controlling the physical state of the lipid microenvironment in the rafts, could modulate the signaling receptor response to LPS. Thus, lysophospholipid acyltransferases might control monocyte and macrophage inflammatory responses by controlling arachidonate availability for mediator formation and facilitating signaling complex formation and responses to inflammatory stimuli.
LPCAT is responsible for the reacylation of LPC with unsaturated fatty acids to form new PC molecular species [27
28
]. The unrestrained synthesis of PC has recently been shown to be important for cell survival [29
]. In addition, evidence suggests that PC biosynthesis may be regulated in response to the lipid requirements of vesicular trafficking [30
31
]. Inhibition of LPCAT would decrease the reacylation of LPC into PC and might be expected to increase concentrations of LPC, which has been shown to have many immunomodulatory functions and play an important role in inflammation [32
]. Exogenous LPC has been shown to be internalized before acylation into PC [33
], and in our experiments, we blocked the reported monocyte receptor for LPC (G2A) [18
] and found that inhibition of LPCAT still caused a down-regulation of inflammatory cytokine production. This suggests that the immunomodulating activity of LPCAT inhibition is independent of the direct actions of LPC acting via this receptor and that other mechanisms must be operative. We show here that these other mechanisms include alteration of receptor protein recruitment into membrane lipid raft domains. By controlling the LPC/PC ratios and the composition of PC, LPCAT would be expected to modulate membrane fluidity and function. Indeed, our previous work showed that agents such as IFN- can increase LPCAT activity and thereby, alter membrane phospholipid composition and modulate membrane fluidity [6
7
8
9
]. Such changes in membrane composition and fluidity would be expected to modulate protein translocation to lipid raft domains.
In support of this hypothesis [6 34 ], a number of laboratories have reported an effect of inflammatory cytokines on CoA-dependent and independent lysophosphoglyceride acyltransferase activity [35 36 37 38 ], consistent with a role for these enzymes in inflammatory disease.
The balance between LPC and its acylation into PC catalyzed by LPCAT may represent an important control point for the functioning and survival of cells of the innate immune system. Dysregulation of this balance may result in inappropriate cellular responses to infectious stimuli that may allow the progression of systemic inflammation and organ injury [39 ].
In conclusion, our observations demonstrate a new regulatory mechanism that facilitates the innate immune responses to microbial molecular patterns and provides a basis for the anti-inflammatory activity observed in many phospholipid metabolites. This provides the possibility of the development of new classes of anti-inflammatory and antisepsis agents.
Received September 3, 2007; revised January 23, 2008; accepted February 5, 2008.
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  G. John, A. O. Yildirim, B. K. Rubin, D. C. Gruenert, and M. O. Henke TLR-4-Mediated Innate Immunity Is Reduced in Cystic Fibrosis Airway Cells Am. J. Respir. Cell Mol. Biol., April 1, 2010; 42(4): 424 - 431. [Abstract] [Full Text] [PDF]
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 W. Abate, A. A. Alghaithy, J. Parton, K. P. Jones, and S. K. Jackson Surfactant lipids regulate LPS-induced interleukin-8 production in A549 lung epithelial cells by inhibiting translocation of TLR4 into lipid raft domains J. Lipid Res., February 1, 2010; 51(2): 334 - 344. [Abstract] [Full Text] [PDF]
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