Acylation of lysophosphatidylcholine plays a key role
in the response of monocytes to lipopolysaccharide
Bernhard Schmid
1,2
, Michael J. Finnen
3
, John L. Harwood
1
and Simon K. Jackson
2
1
School of Biosciences, Cardiff University, UK;
2
University of Wales College of Medicine, Cardiff, UK; and
3
Yamanouchi Research Institute, Oxford, UK
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

out mice are less sensitive to the biological effects of LPS
[4]. Understanding and modulating the production of
LPS-induced mediators such as TNF-a has been the focus
of much research aimed at developing specific therapies for
septic shock.
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 for increased inflammatory
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]

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).
Acyl-transferase inhibitors
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 [
14
C]linoleic acid into lysophos-
phatidylcholine (M. J. Finnen, unpublished results)
1
.
Cell culture
The acute monocytic leukemia cell line MonoMac-6 [17]
was obtained from the German Collection of Microorgan-
isms and Cell Cultures (Braunschweig, Germany). Cells
were grown in RPMI 1640 medium supplemented with 10%
(v/v) low endotoxin fetal bovine serum, 2 m
ML
-glutamine,
100 UÆmL
)1
penicillin, 100 UÆmL

(1 mL) was added and the cells incubated at 37 °Cas
indicated in the legends to the figures.
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 m
M
NaCl/Tris (pH 7.3) were
mixed. In all experiments viability was greater than 95%.
Preparation of microsomes
MonoMac-6 cells (5 · 10
6
) were incubated in 25 mL
complete RPMI 1640 medium under conditions indicated
in the legend to Fig. 2
2
and harvested by centrifugation at
700 g for 2 min at 4 °C. The pellet was washed twice in
microsomal buffer (pH 7.4) containing sucrose 250 m
M
,
Tris 10 m
M
,MgCl
2
1m
M
,EGTA1m
M
[18]. The cells
were finally resuspended in 1 mL of microsomal buffer and

4
; pH 7.4) [18]. Microsomes (approxi-
mately 1 lgprotein)wereaddedin25lL assay buffer
and the mixture incubated with shaking for 20 min at
37 °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.
Ó FEBS 2003 Monocyte acyltransferase in inflammation (Eur. J. Biochem. 270) 2783
chloroform/methanol (1 : 2, v/v). An additional 200 lL
chloroform, followed by 200 lL1
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 I
2
vapor
and their positions marked. Once the I
2
had evaporated,
individual lipid bands were scraped into scintillation vials
and radioactivity estimated in Optisafe ÔHisafeÕ scintillant
(Fisons, Loughborough, UK) using a LKB Wallac 1211

)1
) per well and incubated overnight. The plate
was washed thrice with 0.05% v/v Tween 20 in NaCl/P
i
and
blotted dry. To reduce nonspecific binding, 300 lL bovine
serum albumin (BSA; 1%, w/v) and sucrose (5%, w/v) in
NaCl/P
i
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 m
M
, BSA 0.1%, Tween 20 0.05%, v/v, Tris 20 m
M
,
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 lL200ngÆmL
)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

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
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.
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 l
M
) but not by the protein kinase
C inhibitor bisindolylmalamide (1 l

acyltransferase activity. In contrast, YM 50201 completely
2784 B. Schmid et al. (Eur. J. Biochem. 270) Ó FEBS 2003
inhibits LPCAT but does not affect LPAAT or CoA-
independent AT activities at this concentration (M. J.
Finnen, unpublished observations)
4
. 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 l
M
)orYM
50201 (1 l
M
) 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.
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

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
Table 1. Effect of interferon c on acyltransferase activity from Mono-
Mac-6 cells. MonoMac-6 cells (5 · 10
6
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
)for12h
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
)1
Ælgprotein
)1
)
LPCAT LPAAT
Control 6.33 ± 0.35 2.07 ± 0.19
IFN-c 7.73 ± 0.25* 1.96 ± 0.16
* P < 0.05.
Fig. 3. Effect of acyltransferase inhibitors on TNF-a (A) and IL-6 (B)

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.
Discussion
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 study has shown that IFN-c
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.
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,

2+
-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.
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).
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

amplified and detected as described in Materials and methods.
2786 B. Schmid et al. (Eur. J. Biochem. 270) Ó 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.
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.
Acknowledgments
We are grateful to the Yamanouchi Research Institute, Oxford, for
studentship support for BS.
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