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Lysophospholipid acyltransferases in monocyte inflammatory responses and sepsis
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Immunobiology 209 (2004) 31–38 www.elsevier.de/imbio
REVIEW
Lysophospholipid acyltransferases in monocyte inflammatory responses and sepsis Simon K. Jackson*, Joan Parton Department of Medical Microbiology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, Wales, UK Received 1 April 2004; accepted 8 April 2004
Abstract Acyltransferases are important in the regulation of membrane phospholipid fatty acyl composition and together with phospholipase A2 enzymes control arachidonic acid incorporation and remodelling within phospholipids. In addition, monocyte and macrophage acyltransferase activity has been shown to respond to various inflammatory cytokines under conditions that can induce enhanced cellular responses. Work in our laboratory indicates that the enzyme lysophosphatidylcholine acyltransferase may mediate the priming reactions of monocytes to the cytokine interferon-g: Our recent studies suggest that this enzyme might also affect the responses of monocytes to the bacterial agent lipopolysaccharide that may be important in the development of sepsis. This article summarises the relationship between monocyte lysophosphatidylcholine acyltransferase, lipopolysaccharide and sepsis. r 2004 Elsevier GmbH. All rights reserved. Keywords: Sepsis, acyltransferase, monocytes
Lipopolysaccharide and sepsis Sepsis is a consequence of an overwhelming inflammatory response to infection which has proved exceptionally difficult to treat despite advances in antibiotic therapy and intensive care. Sepsis occurs in at least one and a half million people throughout the world each year. In the United States alone, sepsis develops in more than 500,000 patients each year, with a 30–70% mortality rate (Angus et al., 2001). Sepsis develops from the systemic inflammatory response to pathogens in the blood. Bacterial pathogens carry surface molecules Abbreviations: CoA, coenzyme A; IFN-g, interferon-gamma; LPCAT, lysoPCacyltransferase; LPS, lipopolysaccharide; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; TNF, tumour necrosis factor; TLR, toll like receptor *Corresponding author. Tel.: +44-29-2074-4725; fax: +44-29-20742161. E-mail address: [email protected] (S.K. Jackson). 0171-2985/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2004.04.006
termed pathogen associated molecular patterns (PAMP) which can induce a variety of inflammatory mediators. The resulting inflammatory response can produce cardiovascular derangements, hypotension, multiple organ failure and death (Cohen, 2002). The best characterised of these microbial PAMP is the bacterial molecule lipopolysaccharide (LPS), the endotoxin present in the outer membrane of Gram-negative bacteria (Rietschel et al., 1994; Van Amersfoot et al., 2003). Key target cells in the pathogenesis of LPS-induced sepsis are the monocytes and macrophages. These cells can respond to LPS in the circulation by the production of inflammatory mediators including cytokines and bioactive lipids, and the expression of cell-surface receptors and adhesion molecules (Bhatia and Moochhala, 2004; Glauser et al., 1991). It is the massive release of the inflammatory mediators that is a primary mechanism for the initiation of severe sepsis (Hesse et al., 1988; Tracey and Lowry, 1990). Two inflammatory cytokines,
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Tumour necrosis factor (TNF) and interferon-g (IFN-gÞ; have been shown to be particularly important in the development of septic shock. TNF is central to the pathogenesis as indicated by the relative resistance to LPS-induced toxicity in mice lacking the p55 TNFR, TNF protein or producing high levels of TNFR1 fusion protein (Pasparakis et al., 1996; Rothe et al., 1993). The control of TNF production has been a focus for the development of sepsis therapies. However, the results of clinical trials of specific anti-TNF therapies for sepsis have been disappointing (Abraham, 1999), reflecting the complexity of the syndrome and the involvement of several cytokines with overlapping functions. IFN-g is another important regulator of LPS-induced pathology (Doherty et al., 1992; Silva and Cohen, 1992). Administration of IFN-g or neutralizing antibody to IFN-g has been shown to modify the lethal outcomes of several forms of endotoxic shock and Gram-negative bacterial infections and IFN-gR-deficient mice are relatively resistant to LPS-induced shock (Car et al., 1994; Heinzel, 1990). The molecular events leading to inflammatory mediator production by cells in response to LPS are becoming well understood. LPS, liberated from invading bacteria either spontaneously during growth or as a consequence of immune-mediated lysis, exists in the circulation bound to numerous lipid binding proteins including albumin, transferrin, high-density lipoproteins and LPS-binding protein (LBP), an acute phase protein synthesised by the liver in response to infection. LBP complexes LPS and acts as a lipid transfer protein shuttling LPS to the surface of monocytes and macrophages (Cohen, 2002; Schumann et al., 1990). These cells express a receptor for LPS, CD14, a 55 kDa glycosylphosphoinositol (GPI)-anchored membrane protein, which has been shown to be a receptor for LPS (Pugin et al., 1994; Wright et al., 1990). CD14 associates with the signalling receptor TLR4, which in turn initiates the signal cascade resulting in NF-kB transcription factor activation and inflammatory gene transcription in response to LPS (Hoshino et al., 1999; Van Amersfoot et al., 2003). Regulation of this signalling pathway for LPS-mediated responses is a focus of research aimed at developing new therapies for sepsis and related inflammatory disease.
Sensitisation to LPS and priming It is well known from experimental models of endotoxin-induced sepsis that infectious agents can upregulate or sensitise responses to subsequent LPS challenge. Such sensitizing agents include bacterial infection with P. acnes (Berendt et al., 1980) and the BCG strain of mycobacteria (Peavy et al., 1979) which
can increase the responsiveness of leukocytes to LPS several thousand fold. Priming for enhanced responses to LPS is of considerable interest clinically because it is thought that underlying or sub-clinical infections may prime patients for exaggerated inflammatory responses to low concentrations of LPS. However, the molecular mechanisms of this priming are not well understood. IFN-g has been shown to be an important mediator of the sensitising actions of infectious agents, such as P. acnes, on macrophages for LPS (Billiau et al., 1987; Katschinski et al., 1992). Indeed, a major contribution of IFN-g to LPS-induced shock may be priming an enhanced activation state in monocytes/macrophages (Adams and Hamilton, 1984; Silva and Cohen, 1992). Upon subsequent exposure to LPS, the primed macrophages become hyper-activated and produce large amounts of TNF and IL-1 (Doherty et al., 1992; Heinzel, 1990; Heremans et al., 1990).
Mechanisms of INF-c mediated priming of macrophages The precise mechanisms underlying the priming of macrophages by INF-g for enhanced responses to LPS have remained elusive. Recent work has suggested that IFN-g might increase responsiveness to LPS by augmenting the signal transduction pathway including upregulating TLR4 expression (Bosisio et al., 2002) or promoting IL-1 receptor associated kinase expression and its association to MyD88 (Adib-Conquy and Cavaillon, 2002). Our laboratory has been concerned with elucidating the mechanisms of the priming responses of monocytes and macrophages to LPS. We have previously established that infections, such as BCG, which increase sensitivity to LPS in experimental models of sepsis, alter the membrane phospholipid profiles of macrophages and monocytes (Stark et al., 1990). Furthermore, these ‘priming’ infections were shown to induce the production of IFN-g which mediated the macrophage/monocyte responsiveness. We subsequently showed that IFN-g could produce similar alterations in macrophage phospholipid compositions that accompany the priming of these cells both in vivo and in vitro (Jackson et al., 1989, 1993). In particular, IFN-g stimulated the increased incorporation of unsaturated fatty acids into phosphatidylcholine (PC) which were then turned over into phosphatidylethanolamine (PE) (Darmani et al., 1993). The incorporation of unsaturated fatty acids into phospholipids is accomplished by the deacylation and reacylation of the phopsholipids (the Lands Cycle) mediated by the activity of the lipid modifying enzymes phospholipases and acyltransferases (Balsinde, 2002). Thus, we suggested that IFN-g might increase responsiveness to LPS
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in macrophages by up-regulating the activity of these enzymes. While the activity of phospholipases was not influenced by IFN-g; the activity of certain acyltransferases was significantly increased by this priming agent (Schmid et al., 2003).
Acyltransferases The acylation–deacylation of membrane phospholipids (the Lands Cycle) is a mechanism for incorporating unsaturated fatty acids (mainly arachidonic acid) into different phospholipids to provide a range of lipid mediators (Yamashita et al., 1997). The fatty acids are cleaved from phospholipids by the action of phospholipase A2 and re-incorporated by acyltransferases. Studies have revealed that arachidonic-acid is incorporated first into phospholipids containing a 1-acyl linkage by Coenzyme A (CoA)-dependent enzymes. The arachidonic acid is then transferred by CoAindependent transacylases from 1-acyl linked phospholipids to 1-alkyl and 1-alk-1-enyl lyso phospholipids to form 1-alkyl and 1-alk-1-enyl-2-arachidonyl phospholipids (Chilton et al., 1996), which are important in the synthesis of platelet activating factor (PAF) (Fig. 1). Both CoA-dependent acyltransferases and CoA-independent transacylases (CoAIT) in addition to their important role in providing substrates for lipid mediators of inflammation, have been found to be involved in lipid signalling pathways (Jackson, 1997; Prokazova et al., 1998) and leukocyte activation. They may also modulate the activities of other membrane-localised enzymes (Prokazova et al., 1998). However, little is known about the regulation of these enzymes during inflammation, although a study using human neutrophils described a PAF-induced increase in the arachidonoyl-CoA-specific lysophospholipid acyltransferase activity (Tou, 1987). Similarly, TNF was found to increase the CoAIT activity in human neutrophils (Winkler et al., 1994) and IL-1a was shown to increase the incorporation of arachidonate into phosphatidylinositol and phosphatidylserine in rat mesangial cells, implying an enhanced rate of arachidonate-selective lysophospholipid acyltransferase(s) (Winkler et al., 1995). Studies from our laboratory recently demonstrated that TNF can modify phospholipid compositions in monocytes via activation of CoA-independent transacylases (Neville et al., manuscript submission). Furthermore, we showed that IFN-g and concanavalin A, another priming agent, could selectively activate lysophosphatidyl choline acyltransferase (LPCAT) but not the lysophosphatidic acid acyltransferase (LPAAT) (Schmid et al., 2003) (Fig. 2). Thus, LPCAT activity is up-regulated under conditions of priming monocytes/ macrophages for increased responses to LPS.
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LPCAT and priming in macrophages and monocytes Cytokines are typical physiological priming agents and priming by several cytokines has been shown to upregulate PAF and eicosanoid generation in different cell types (Glaser et al., 1990). CoA-dependent acyltransferases and CoA-independent transacylases would also be expected to be key enzymes involved in the priming process for increased lipid mediator production. Interleukin-1a can increase the activity of arachidonic acidlysophospholipid acyltransferase and stimulates arachidonic acid incorporation into phospholipids in rat (Nakazato and Sedor, 1992) and stimulates lysoPA acyltransferase in human mesangial cells (Bursten et al., 1991). Increased acyltransferase activity would protect the mesangial cell membranes from the potentially damaging effects of PLA2-generated lysophospholipids. A CoA-independent transacylase was up-regulated in human neutrophils after treatment with TNF which was shown to prime these cells for enhanced arachidonic acid metabolism following activation with the agonist formyl peptide, f-MetLeuPhe (fMLP) (Gegner et al., 1995). Acyltransferases have been characterised in macrophages and evidence from our work suggests that priming cytokines such as IFN-g can increase the activity of both CoA-dependent and CoA-independent transacylases in a human monocyte cell line (Neville et al., 1997). We demonstrated that IFN-g could induce the altered phospholipid profiles seen in monocytes and macrophages both in vivo and in vitro which accompanied their increased inflammatory responses to LPS (Darmani et al., 1993; Jackson et al., 1993). In particular, we found that IFN-g could directly up-regulate the activity of lysophosphatidylcholine acyltransferase (LPCAT) which reacylates lysoPC with unsaturated fatty acids (Schmid et al., 2003). Such studies suggest that that LPCAT may regulate the priming of monocytes by IFNg and stimulate increased inflammatory cytokine production in response to LPS. In studies of T-lymphocyte activation, Szamel et al. (1993, 1998) have demonstrated that activation of the T-cell antigen receptor/CD3 complex leads to increased incorporation of polyunsaturated fatty acids into phosphatidylcholine, also mediated by an LPCAT. Thus, LPCAT may play a crucial role in the early phase of T-cell activation by elevated incorporation of polyunsaturated fatty acids into plasma membranes phospholipids. Results from our work (Schmid et al., 2003) suggest that LPCAT plays a similar role in the activation of monocytes by priming agents such as IFN-g: Interestingly, our results showed that neither of the priming agents IFN-g or concanavalin A, affected the activity of LPAAT, which has been reported to be up-regulated by the lipid A portion of LPS in mesangial cells (Bursten
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Fig. 1. The generation of phospholipids with different fatty acid compositions by the sequential activity of phospholipase A2 and Coenzyme A (CoA)-dependent and -independent acyl transferase enzymes.
et al., 1992). Therefore, priming of monocytes seems to specifically involve up-regulation of LPCAT. It is hypothesised that activation of LPCAT alters the cell membrane lipid environment so as to favour the assembly of a signalling complex which can then activate the cellular response (Jackson, 1997). Therefore, we suggest that increased activation of LPCAT facilitates the LPS-stimulated signalling pathways that initiate inflammatory gene transcription. Furthermore, inhibition of LPCAT would be expected to inhibit the LPS signalling and down-regulate inflammatory mediator production.
LPCAT activation and the inflammatory response We and others have suggested that acyltransferases in leukocytes might be important in the development of inflammatory disease including septic shock (Jackson, 1997; Neville et al., 1997, manuscript submitted; Schmid et al., 2003). Indeed one recent report indicates that inhibition of LPAAT suppresses neutrophil adherence and chemotaxis and decreases IL-8-induced injury in isolated rat lungs perfused with human neutrophils (Guidot et al., 1997). Protection from IL-8 mediated
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Fig. 2. The effect of priming agents concanavalin A and IFN-g on activity of LPCAT and LPAAT enzymes in human monocytes. po0:01; # po0:05: Adapted from Schmid (2002).
lung injury suggests a therapeutic role of acyltransferase inhibitors in patients at risk of developing acute respiratory distress syndrome (ARDS). Inhibitors of CoA-independent transacylase block the movement of arachidonic acid into 1-ether-linked phospholipids in human neutrophils further supporting the concept that blockade of CoA-independent transacylases is a therapeutic possibility in modulating inflammatory responses. Our studies have shown that LPCAT regulates monocyte and macrophage responses to LPS. Further evidence that LPCAT plays a central role in the inflammatory response of monocytes came from the use of specific LPCAT inhibitors. Several specific LPCAT inhibitors have been characterised and found to have good activity with an IC50 of 5–20 mM (Schmid, 2002; Schmid et al., 2003). These include SK&F 98625 [diethyl 7-(3,4,5-triphenyl-2-oxo2,3-dihydro-imidazole1-yl)heptane phosphonate] and YM 50201 (3-hydroxyethyl 5,30 -thiophenyl pyridine). These are non-competitive inhibitors of LPCAT that can inhibit the incorporation of 14C-linoleic acid into lysoPC (Schmid, 2002). Inhibition of LPCAT activity with specific inhibitors was found to almost completely block the production of TNF and IL-6 in LPS-stimulated, IFN-gprimed cells (Fig. 3) (Schmid et al., 2003). Moreover, LPCAT inhibition also down-regulated TNF production in LPS-stimulated cells that had not been primed with IFN-g and significantly reduced TNF mRNA production (Schmid et al., 2003). This suggests that the inhibition of LPCAT is affecting the LPS signalling pathway between receptor activation and gene transcription. It has been demonstrated recently that lysophosphatidylcholine (LPC) may have therapeutic potential in sepsis by moderating inflammatory cytokine
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expression (Yan et al., 2004). In particular, in an animal model of sepsis, it was shown that LPC induced a transient increase in T-helper type 1 cytokines (IFN-g; IL-2, IL-12) and a decrease in the inflammatory cytokines TNF and IL-1b. Although LPC was also found to enhance the clearance of microbes by neutrophils, it is tempting to speculate that LPCAT might increase LPS-stimulated inflammatory cytokine expression by removal of LPC (Fig. 1). Furthermore, inhibition of LPCAT activity would allow increases in LPC which could then down-regulate inflammatory cytokine production. This raises the fascinating possibility that macrophage and monocyte inflammatory responses might be controlled by the activity of LPCAT and the resultant membrane levels of LPC. To understand the mechanisms by which LPCAT might regulate monocyte responses to LPS further characterisation of the enzyme is required.
Further characterisation of monocyte LPCAT Despite the importance of LPCAT activity in the maintenance of the complex molecular species composition of biological membranes, the characterisation and isolation of the enzyme has been hampered by problems in solubilising the protein from membrane domains without inactivating it. An unstable enzyme activity was isolated from bovine brain and heart microsomes and from rat liver microsomes with detergent solubilisation (Mukherjee et al., 1992). Kerkhoff et al. (2000) have reported the extraction of membrane lysophospholipid acyltransferase which retained full enzyme activity. Recently, the enzyme has been isolated in newly formed PC vesicles by solubilisation of rat liver microsomes with two substrates LPC and acyl CoA and it has been found that the lipid environment plays an important role in the regulation of the enzyme’s affinity for its substrate (Fyrst et al., 1996). Purification of LPCAT using this method has enabled partial sequencing and identification of a putative DNA sequence for the enzyme (Schmid, 2002). The cDNA has sequence homology to other known acyltransferases. Current work in our laboratory is utilising the cDNA to produce cells over-expressing LPCAT activity in order to further study its role in monocyte activation.
Possible mechanisms of LPCAT regulating monocyte inflammatory responses Toll-like receptor 4 (TLR4) together with the accessory molecule MD-2 has been shown to be the final activating receptor for LPS on monocytes/macrophages (Zarember and Godowski, 2002). Our results
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Fig. 3. TNF-a (A) and IL-6 (B) production in isolated peripheral blood mononuclear cells after priming and inhibition of acyltransferase activity with two selected inhibitors (Pi4, Pi38). Results show means7SD from six independent experiments.
imply that LPCAT activity is necessary for effective signal transduction from TLR4/MD-2, and that this mechanism is enhanced by IFN-g: Recent studies suggest that IFN-g may either stimulate TLR4 (Bosisio et al., 2002) or promote interleukin-1 receptor-associated kinase (IRAK) as a mechanism of priming (AdibConquy and Cavaillon, 2002). Furthermore, it has been shown that in monocytes CD14 and TLR4 co-localise to cholesterol-rich membrane regions (‘lipid rafts’) to induce signal transduction in response to LPS (Jiang et al., 2000). 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 PC, are also components of lipid rafts (Rouquette-Jazdanian et al., 2002) and alteration of the saturation of PC within these regions would also alter the co-localisation of the signalling receptors for LPS. LPCAT, by controlling the physical state of the lipid microenvironment in the rafts, could modulate the signalling receptor response to LPS. Recently, the activity of lysophospholipid acyltransferases has been shown to alter membrane curvature and be important for membrane fission and vesicle formation (Schmidt et al.,
1999; Weigert et al., 1999). Thus, lysophospholipid acyltransferases might control monocyte and macrophage inflammatory responses by both controlling arachidonate availability for mediator formation and facilitating signalling complex formation and responses to inflammatory stimuli.
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Immunobiology 209 (2004) 31–38 www.elsevier.de/imbio
REVIEW
Lysophospholipid acyltransferases in monocyte inflammatory responses and sepsis Simon K. Jackson*, Joan Parton Department of Medical Microbiology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, Wales, UK Received 1 April 2004; accepted 8 April 2004
Abstract Acyltransferases are important in the regulation of membrane phospholipid fatty acyl composition and together with phospholipase A2 enzymes control arachidonic acid incorporation and remodelling within phospholipids. In addition, monocyte and macrophage acyltransferase activity has been shown to respond to various inflammatory cytokines under conditions that can induce enhanced cellular responses. Work in our laboratory indicates that the enzyme lysophosphatidylcholine acyltransferase may mediate the priming reactions of monocytes to the cytokine interferon-g: Our recent studies suggest that this enzyme might also affect the responses of monocytes to the bacterial agent lipopolysaccharide that may be important in the development of sepsis. This article summarises the relationship between monocyte lysophosphatidylcholine acyltransferase, lipopolysaccharide and sepsis. r 2004 Elsevier GmbH. All rights reserved. Keywords: Sepsis, acyltransferase, monocytes
Lipopolysaccharide and sepsis Sepsis is a consequence of an overwhelming inflammatory response to infection which has proved exceptionally difficult to treat despite advances in antibiotic therapy and intensive care. Sepsis occurs in at least one and a half million people throughout the world each year. In the United States alone, sepsis develops in more than 500,000 patients each year, with a 30–70% mortality rate (Angus et al., 2001). Sepsis develops from the systemic inflammatory response to pathogens in the blood. Bacterial pathogens carry surface molecules Abbreviations: CoA, coenzyme A; IFN-g, interferon-gamma; LPCAT, lysoPCacyltransferase; LPS, lipopolysaccharide; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; TNF, tumour necrosis factor; TLR, toll like receptor *Corresponding author. Tel.: +44-29-2074-4725; fax: +44-29-20742161. E-mail address: [email protected] (S.K. Jackson). 0171-2985/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2004.04.006
termed pathogen associated molecular patterns (PAMP) which can induce a variety of inflammatory mediators. The resulting inflammatory response can produce cardiovascular derangements, hypotension, multiple organ failure and death (Cohen, 2002). The best characterised of these microbial PAMP is the bacterial molecule lipopolysaccharide (LPS), the endotoxin present in the outer membrane of Gram-negative bacteria (Rietschel et al., 1994; Van Amersfoot et al., 2003). Key target cells in the pathogenesis of LPS-induced sepsis are the monocytes and macrophages. These cells can respond to LPS in the circulation by the production of inflammatory mediators including cytokines and bioactive lipids, and the expression of cell-surface receptors and adhesion molecules (Bhatia and Moochhala, 2004; Glauser et al., 1991). It is the massive release of the inflammatory mediators that is a primary mechanism for the initiation of severe sepsis (Hesse et al., 1988; Tracey and Lowry, 1990). Two inflammatory cytokines,
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Tumour necrosis factor (TNF) and interferon-g (IFN-gÞ; have been shown to be particularly important in the development of septic shock. TNF is central to the pathogenesis as indicated by the relative resistance to LPS-induced toxicity in mice lacking the p55 TNFR, TNF protein or producing high levels of TNFR1 fusion protein (Pasparakis et al., 1996; Rothe et al., 1993). The control of TNF production has been a focus for the development of sepsis therapies. However, the results of clinical trials of specific anti-TNF therapies for sepsis have been disappointing (Abraham, 1999), reflecting the complexity of the syndrome and the involvement of several cytokines with overlapping functions. IFN-g is another important regulator of LPS-induced pathology (Doherty et al., 1992; Silva and Cohen, 1992). Administration of IFN-g or neutralizing antibody to IFN-g has been shown to modify the lethal outcomes of several forms of endotoxic shock and Gram-negative bacterial infections and IFN-gR-deficient mice are relatively resistant to LPS-induced shock (Car et al., 1994; Heinzel, 1990). The molecular events leading to inflammatory mediator production by cells in response to LPS are becoming well understood. LPS, liberated from invading bacteria either spontaneously during growth or as a consequence of immune-mediated lysis, exists in the circulation bound to numerous lipid binding proteins including albumin, transferrin, high-density lipoproteins and LPS-binding protein (LBP), an acute phase protein synthesised by the liver in response to infection. LBP complexes LPS and acts as a lipid transfer protein shuttling LPS to the surface of monocytes and macrophages (Cohen, 2002; Schumann et al., 1990). These cells express a receptor for LPS, CD14, a 55 kDa glycosylphosphoinositol (GPI)-anchored membrane protein, which has been shown to be a receptor for LPS (Pugin et al., 1994; Wright et al., 1990). CD14 associates with the signalling receptor TLR4, which in turn initiates the signal cascade resulting in NF-kB transcription factor activation and inflammatory gene transcription in response to LPS (Hoshino et al., 1999; Van Amersfoot et al., 2003). Regulation of this signalling pathway for LPS-mediated responses is a focus of research aimed at developing new therapies for sepsis and related inflammatory disease.
Sensitisation to LPS and priming It is well known from experimental models of endotoxin-induced sepsis that infectious agents can upregulate or sensitise responses to subsequent LPS challenge. Such sensitizing agents include bacterial infection with P. acnes (Berendt et al., 1980) and the BCG strain of mycobacteria (Peavy et al., 1979) which
can increase the responsiveness of leukocytes to LPS several thousand fold. Priming for enhanced responses to LPS is of considerable interest clinically because it is thought that underlying or sub-clinical infections may prime patients for exaggerated inflammatory responses to low concentrations of LPS. However, the molecular mechanisms of this priming are not well understood. IFN-g has been shown to be an important mediator of the sensitising actions of infectious agents, such as P. acnes, on macrophages for LPS (Billiau et al., 1987; Katschinski et al., 1992). Indeed, a major contribution of IFN-g to LPS-induced shock may be priming an enhanced activation state in monocytes/macrophages (Adams and Hamilton, 1984; Silva and Cohen, 1992). Upon subsequent exposure to LPS, the primed macrophages become hyper-activated and produce large amounts of TNF and IL-1 (Doherty et al., 1992; Heinzel, 1990; Heremans et al., 1990).
Mechanisms of INF-c mediated priming of macrophages The precise mechanisms underlying the priming of macrophages by INF-g for enhanced responses to LPS have remained elusive. Recent work has suggested that IFN-g might increase responsiveness to LPS by augmenting the signal transduction pathway including upregulating TLR4 expression (Bosisio et al., 2002) or promoting IL-1 receptor associated kinase expression and its association to MyD88 (Adib-Conquy and Cavaillon, 2002). Our laboratory has been concerned with elucidating the mechanisms of the priming responses of monocytes and macrophages to LPS. We have previously established that infections, such as BCG, which increase sensitivity to LPS in experimental models of sepsis, alter the membrane phospholipid profiles of macrophages and monocytes (Stark et al., 1990). Furthermore, these ‘priming’ infections were shown to induce the production of IFN-g which mediated the macrophage/monocyte responsiveness. We subsequently showed that IFN-g could produce similar alterations in macrophage phospholipid compositions that accompany the priming of these cells both in vivo and in vitro (Jackson et al., 1989, 1993). In particular, IFN-g stimulated the increased incorporation of unsaturated fatty acids into phosphatidylcholine (PC) which were then turned over into phosphatidylethanolamine (PE) (Darmani et al., 1993). The incorporation of unsaturated fatty acids into phospholipids is accomplished by the deacylation and reacylation of the phopsholipids (the Lands Cycle) mediated by the activity of the lipid modifying enzymes phospholipases and acyltransferases (Balsinde, 2002). Thus, we suggested that IFN-g might increase responsiveness to LPS
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in macrophages by up-regulating the activity of these enzymes. While the activity of phospholipases was not influenced by IFN-g; the activity of certain acyltransferases was significantly increased by this priming agent (Schmid et al., 2003).
Acyltransferases The acylation–deacylation of membrane phospholipids (the Lands Cycle) is a mechanism for incorporating unsaturated fatty acids (mainly arachidonic acid) into different phospholipids to provide a range of lipid mediators (Yamashita et al., 1997). The fatty acids are cleaved from phospholipids by the action of phospholipase A2 and re-incorporated by acyltransferases. Studies have revealed that arachidonic-acid is incorporated first into phospholipids containing a 1-acyl linkage by Coenzyme A (CoA)-dependent enzymes. The arachidonic acid is then transferred by CoAindependent transacylases from 1-acyl linked phospholipids to 1-alkyl and 1-alk-1-enyl lyso phospholipids to form 1-alkyl and 1-alk-1-enyl-2-arachidonyl phospholipids (Chilton et al., 1996), which are important in the synthesis of platelet activating factor (PAF) (Fig. 1). Both CoA-dependent acyltransferases and CoA-independent transacylases (CoAIT) in addition to their important role in providing substrates for lipid mediators of inflammation, have been found to be involved in lipid signalling pathways (Jackson, 1997; Prokazova et al., 1998) and leukocyte activation. They may also modulate the activities of other membrane-localised enzymes (Prokazova et al., 1998). However, little is known about the regulation of these enzymes during inflammation, although a study using human neutrophils described a PAF-induced increase in the arachidonoyl-CoA-specific lysophospholipid acyltransferase activity (Tou, 1987). Similarly, TNF was found to increase the CoAIT activity in human neutrophils (Winkler et al., 1994) and IL-1a was shown to increase the incorporation of arachidonate into phosphatidylinositol and phosphatidylserine in rat mesangial cells, implying an enhanced rate of arachidonate-selective lysophospholipid acyltransferase(s) (Winkler et al., 1995). Studies from our laboratory recently demonstrated that TNF can modify phospholipid compositions in monocytes via activation of CoA-independent transacylases (Neville et al., manuscript submission). Furthermore, we showed that IFN-g and concanavalin A, another priming agent, could selectively activate lysophosphatidyl choline acyltransferase (LPCAT) but not the lysophosphatidic acid acyltransferase (LPAAT) (Schmid et al., 2003) (Fig. 2). Thus, LPCAT activity is up-regulated under conditions of priming monocytes/ macrophages for increased responses to LPS.
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LPCAT and priming in macrophages and monocytes Cytokines are typical physiological priming agents and priming by several cytokines has been shown to upregulate PAF and eicosanoid generation in different cell types (Glaser et al., 1990). CoA-dependent acyltransferases and CoA-independent transacylases would also be expected to be key enzymes involved in the priming process for increased lipid mediator production. Interleukin-1a can increase the activity of arachidonic acidlysophospholipid acyltransferase and stimulates arachidonic acid incorporation into phospholipids in rat (Nakazato and Sedor, 1992) and stimulates lysoPA acyltransferase in human mesangial cells (Bursten et al., 1991). Increased acyltransferase activity would protect the mesangial cell membranes from the potentially damaging effects of PLA2-generated lysophospholipids. A CoA-independent transacylase was up-regulated in human neutrophils after treatment with TNF which was shown to prime these cells for enhanced arachidonic acid metabolism following activation with the agonist formyl peptide, f-MetLeuPhe (fMLP) (Gegner et al., 1995). Acyltransferases have been characterised in macrophages and evidence from our work suggests that priming cytokines such as IFN-g can increase the activity of both CoA-dependent and CoA-independent transacylases in a human monocyte cell line (Neville et al., 1997). We demonstrated that IFN-g could induce the altered phospholipid profiles seen in monocytes and macrophages both in vivo and in vitro which accompanied their increased inflammatory responses to LPS (Darmani et al., 1993; Jackson et al., 1993). In particular, we found that IFN-g could directly up-regulate the activity of lysophosphatidylcholine acyltransferase (LPCAT) which reacylates lysoPC with unsaturated fatty acids (Schmid et al., 2003). Such studies suggest that that LPCAT may regulate the priming of monocytes by IFNg and stimulate increased inflammatory cytokine production in response to LPS. In studies of T-lymphocyte activation, Szamel et al. (1993, 1998) have demonstrated that activation of the T-cell antigen receptor/CD3 complex leads to increased incorporation of polyunsaturated fatty acids into phosphatidylcholine, also mediated by an LPCAT. Thus, LPCAT may play a crucial role in the early phase of T-cell activation by elevated incorporation of polyunsaturated fatty acids into plasma membranes phospholipids. Results from our work (Schmid et al., 2003) suggest that LPCAT plays a similar role in the activation of monocytes by priming agents such as IFN-g: Interestingly, our results showed that neither of the priming agents IFN-g or concanavalin A, affected the activity of LPAAT, which has been reported to be up-regulated by the lipid A portion of LPS in mesangial cells (Bursten
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Fig. 1. The generation of phospholipids with different fatty acid compositions by the sequential activity of phospholipase A2 and Coenzyme A (CoA)-dependent and -independent acyl transferase enzymes.
et al., 1992). Therefore, priming of monocytes seems to specifically involve up-regulation of LPCAT. It is hypothesised that activation of LPCAT alters the cell membrane lipid environment so as to favour the assembly of a signalling complex which can then activate the cellular response (Jackson, 1997). Therefore, we suggest that increased activation of LPCAT facilitates the LPS-stimulated signalling pathways that initiate inflammatory gene transcription. Furthermore, inhibition of LPCAT would be expected to inhibit the LPS signalling and down-regulate inflammatory mediator production.
LPCAT activation and the inflammatory response We and others have suggested that acyltransferases in leukocytes might be important in the development of inflammatory disease including septic shock (Jackson, 1997; Neville et al., 1997, manuscript submitted; Schmid et al., 2003). Indeed one recent report indicates that inhibition of LPAAT suppresses neutrophil adherence and chemotaxis and decreases IL-8-induced injury in isolated rat lungs perfused with human neutrophils (Guidot et al., 1997). Protection from IL-8 mediated
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Fig. 2. The effect of priming agents concanavalin A and IFN-g on activity of LPCAT and LPAAT enzymes in human monocytes. po0:01; # po0:05: Adapted from Schmid (2002).
lung injury suggests a therapeutic role of acyltransferase inhibitors in patients at risk of developing acute respiratory distress syndrome (ARDS). Inhibitors of CoA-independent transacylase block the movement of arachidonic acid into 1-ether-linked phospholipids in human neutrophils further supporting the concept that blockade of CoA-independent transacylases is a therapeutic possibility in modulating inflammatory responses. Our studies have shown that LPCAT regulates monocyte and macrophage responses to LPS. Further evidence that LPCAT plays a central role in the inflammatory response of monocytes came from the use of specific LPCAT inhibitors. Several specific LPCAT inhibitors have been characterised and found to have good activity with an IC50 of 5–20 mM (Schmid, 2002; Schmid et al., 2003). These include SK&F 98625 [diethyl 7-(3,4,5-triphenyl-2-oxo2,3-dihydro-imidazole1-yl)heptane phosphonate] and YM 50201 (3-hydroxyethyl 5,30 -thiophenyl pyridine). These are non-competitive inhibitors of LPCAT that can inhibit the incorporation of 14C-linoleic acid into lysoPC (Schmid, 2002). Inhibition of LPCAT activity with specific inhibitors was found to almost completely block the production of TNF and IL-6 in LPS-stimulated, IFN-gprimed cells (Fig. 3) (Schmid et al., 2003). Moreover, LPCAT inhibition also down-regulated TNF production in LPS-stimulated cells that had not been primed with IFN-g and significantly reduced TNF mRNA production (Schmid et al., 2003). This suggests that the inhibition of LPCAT is affecting the LPS signalling pathway between receptor activation and gene transcription. It has been demonstrated recently that lysophosphatidylcholine (LPC) may have therapeutic potential in sepsis by moderating inflammatory cytokine
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expression (Yan et al., 2004). In particular, in an animal model of sepsis, it was shown that LPC induced a transient increase in T-helper type 1 cytokines (IFN-g; IL-2, IL-12) and a decrease in the inflammatory cytokines TNF and IL-1b. Although LPC was also found to enhance the clearance of microbes by neutrophils, it is tempting to speculate that LPCAT might increase LPS-stimulated inflammatory cytokine expression by removal of LPC (Fig. 1). Furthermore, inhibition of LPCAT activity would allow increases in LPC which could then down-regulate inflammatory cytokine production. This raises the fascinating possibility that macrophage and monocyte inflammatory responses might be controlled by the activity of LPCAT and the resultant membrane levels of LPC. To understand the mechanisms by which LPCAT might regulate monocyte responses to LPS further characterisation of the enzyme is required.
Further characterisation of monocyte LPCAT Despite the importance of LPCAT activity in the maintenance of the complex molecular species composition of biological membranes, the characterisation and isolation of the enzyme has been hampered by problems in solubilising the protein from membrane domains without inactivating it. An unstable enzyme activity was isolated from bovine brain and heart microsomes and from rat liver microsomes with detergent solubilisation (Mukherjee et al., 1992). Kerkhoff et al. (2000) have reported the extraction of membrane lysophospholipid acyltransferase which retained full enzyme activity. Recently, the enzyme has been isolated in newly formed PC vesicles by solubilisation of rat liver microsomes with two substrates LPC and acyl CoA and it has been found that the lipid environment plays an important role in the regulation of the enzyme’s affinity for its substrate (Fyrst et al., 1996). Purification of LPCAT using this method has enabled partial sequencing and identification of a putative DNA sequence for the enzyme (Schmid, 2002). The cDNA has sequence homology to other known acyltransferases. Current work in our laboratory is utilising the cDNA to produce cells over-expressing LPCAT activity in order to further study its role in monocyte activation.
Possible mechanisms of LPCAT regulating monocyte inflammatory responses Toll-like receptor 4 (TLR4) together with the accessory molecule MD-2 has been shown to be the final activating receptor for LPS on monocytes/macrophages (Zarember and Godowski, 2002). Our results
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Fig. 3. TNF-a (A) and IL-6 (B) production in isolated peripheral blood mononuclear cells after priming and inhibition of acyltransferase activity with two selected inhibitors (Pi4, Pi38). Results show means7SD from six independent experiments.
imply that LPCAT activity is necessary for effective signal transduction from TLR4/MD-2, and that this mechanism is enhanced by IFN-g: Recent studies suggest that IFN-g may either stimulate TLR4 (Bosisio et al., 2002) or promote interleukin-1 receptor-associated kinase (IRAK) as a mechanism of priming (AdibConquy and Cavaillon, 2002). Furthermore, it has been shown that in monocytes CD14 and TLR4 co-localise to cholesterol-rich membrane regions (‘lipid rafts’) to induce signal transduction in response to LPS (Jiang et al., 2000). 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 PC, are also components of lipid rafts (Rouquette-Jazdanian et al., 2002) and alteration of the saturation of PC within these regions would also alter the co-localisation of the signalling receptors for LPS. LPCAT, by controlling the physical state of the lipid microenvironment in the rafts, could modulate the signalling receptor response to LPS. Recently, the activity of lysophospholipid acyltransferases has been shown to alter membrane curvature and be important for membrane fission and vesicle formation (Schmidt et al.,
1999; Weigert et al., 1999). Thus, lysophospholipid acyltransferases might control monocyte and macrophage inflammatory responses by both controlling arachidonate availability for mediator formation and facilitating signalling complex formation and responses to inflammatory stimuli.
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