(Received for publication, November 28, 1995; and in revised form, January 30, 1996)
From the
F-isoprostanes are free radical-catalyzed products
of arachidonic acid. One of these compounds, 8-epiprostaglandin
F
(8-epi-PGF
), is a mitogen and
vasoconstrictor. We have shown that 8-epi-PGF
, unlike
other F
-isoprostanes, is a minor product of the
prostaglandin endoperoxide synthase-1 (PG G/H S-1) expressed in human
platelets (Praticó, D., Lawson, J. A., and
FitzGerald, G. A.(1995) J. Biol. Chem. 270, 9800-9808).
Human monocytes express PG G/H S-1 constitutively and exhibit regulated
expression of PG G/H S-2. Induction of PG G/H S-2 by concanavalin A,
the phorbol ester, phorbol 12-myristate 13-acetate, and bacterial
lipopolysaccharide was confirmed with a specific antibody in monocytes
pretreated with aspirin to inhibit PG G/H S-1. Induction of PG G/H S-2
by all three stimuli coincided with increased formation of
prostaglandin E
(PGE
), thromboxane B
(TxB
), and 8-epi-PGF
, but not of
other F
-isoprostanes. Confirmation of PG G/H S-2 as the
source of 8-epi-PGF
formation was obtained by
down-regulating the enzyme with dexamethasone; preventing protein
synthesis with cycloheximide; and preventing synthesis of
PGE
, TxB
, and 8-epi-PGF
with
the specific PG G/H S-2 inhibitor, L 745,337.
Monocytes also exhibit
the facility to generate 8-epi-PGF in a free
radical-dependent manner. Thus, stimulation with opsonized zymosan or
coincubation with low density lipoprotein was unassociated with product
formation. However, coincubation of low density lipoprotein with
zymosan-stimulated human monocytes resulted in marked formation of
8-epi-PGF
, but not of PGE
or
TxB
. Production of 8-epi-PGF
coincided
with that of thiobarbituric acid-reactive substances and lipid
hydroperoxides, but was unaccompanied by PG G/H S-2 induction.
Pretreatment of monocytes with the antioxidant, butylated
hydroxytoluene or with superoxide dismutase, but not with L 745,337,
suppressed formation of 8-epi-PGF
, thiobarbituric
acid-reactive substances, and lipid hydroperoxides.
In conclusion,
human monocytes may form bioactive 8-epi-PGF either
via free radical- or enzyme-catalyzed pathways. 8-Epi-PGF
is a more abundant product of monocyte PG G/H S-2 than of
platelet PG G/H S-1. Formation by inducible PG G/H S-2 must be
considered as a source of this compound in vivo.
Monocytes are thought to play a central role in
atherogenesis(1) . They adhere to and transmigrate between
endothelial cells(2) . Monocyte-derived macrophages have high
affinity receptors for oxidized low density lipoprotein (LDL), ()and LDL ingestion results in their transformation into
foam cells(3, 4) . Discharge of the contents of foam
cells, themselves marked constituents of fatty streaks(5) , is
thought to contribute to formation of atherosclerotic
plaque(6) . Monocytes and macrophages exhibit the ability to
transform arachidonic acid to prostaglandins and related compounds via
the enzyme prostaglandin endoperoxide synthase (PG G/H S). There are
two forms of this enzyme(7, 8) . Monocytes express PG
G/H S-1 constitutively(9) , and PG G/H S-2 is up-regulated in
response to cytokines, growth factors, and bacterial lipopolysaccharide
(LPS) (9, 10, 11) . It is unknown what role,
if any, prostanoids may play in atherogenesis, although they have been
implicated in regulating cellular
proliferation(12, 13) , vascular tone and
permeability(14, 15) , and aspects of cholesterol
metabolism(16) . Additional to its susceptibility to
enzyme-catalyzed metabolism to biologically active compounds,
arachidonic acid may be subject to free radical attack, leading to
formation of prostaglandin isomers in situ in the cell
membrane phospholipid(17, 18) . These isoprostanes may
exert biological effects intra- or extracellularly. A prostaglandin
F
isomer, 8-epi-PGF
, induces
vasoconstriction and cellular proliferation, effects that are prevented
by thromboxane A
receptor
antagonists(19, 20) . Recently, we have shown that
this compound is also a minor product of PG G/H S-1 in human
platelets(21) .
This study demonstrates that PG G/H S-2 may
also form 8-epi-PGF. Interestingly, it is a more
abundant product of this enzyme in monocytes than of the PG G/H S-1
isoform in platelets. Monocytes also retain the ability to form this
compound in a free radical-catalyzed manner. Indeed, coincubation of
monocytes with LDL results in a time-dependent formation of
8-epi-PGF
, coincident with LDL oxidation.
The
cells were plated in 24-well multiplates and maintained at 37 °C in
a temperature-controlled, humidified 95% air, 5% CO incubator. The nonadherent cells were removed by washing the
plates twice with Dulbecco's phosphate-buffered saline after 2 h
of incubation, and the adherent cells were maintained in RPMI 1640
medium enriched with 5% inactivated fetal calf serum, L-glutamine, and antibiotics. The resultant harvested adherent
cells routinely contained >92% monocytes as determined by morphology
and staining for nonspecific esterase(25) . The contribution of
PG G/H S-1 activity to eicosanoid production in response to the
different stimuli was suppressed by pretreating the blood with aspirin
(10 µg/ml) in the sampling syringe and at time 0. This was
confirmed by product analysis. Isolated monocytes were incubated for 1,
4, 8, and 24 h in the absence and presence of LPS (10 µg/ml),
concanavalin A (10 µg/ml), or PMA (100 nM). Fresh medium
containing 10 µM arachidonic acid was added at each time
point after removing the incubation medium; the supernatants were
harvested after 30 min.
Concentrations of PGE,
TxB
, and 8-epi-PGF
were determined by gas
chromatography/mass spectrometry (GC/MS) as described
previously(21, 26) . The contribution of induced PG
G/H S-2 to formation of these products was elucidated by use of a
protein synthesis inhibitor, cycloheximide(27) ; a specific
inhibitor of this isoform of the enzyme, L 745,337(28) ; or
dexamethasone, which has previously been shown to down-regulate PG G/H
S-2(29) .
Figure 1: Time course of eicosanoid formation in human monocytes stimulated with LPS (10 µg/ml). The supernatant was assayed for prostanoid production by GC/MS at the indicated times. Values are reported as mean ± S.D. from four experiments.
Figure 2:
Western blot analysis of PG G/H S-2
protein expression in monocytes treated with LPS (10 µg/ml).
Isolated human monocytes (1 10
/ml) were incubated
with saline for 24 h (control (c)); with LPS alone for 1, 4,
8, and 24 h; with LPS + dexamethasone (D) for 24 h; and
with LPS + cycloheximide (C) for 24 h. The figure is
representative of three experiments.
Figure 3:
Selected ion monitoring of
8-epi-PGF. The upper panel shows a peak at m/z 699 corresponding to authentic
O
-labeled internal standard. The center
panel shows a peak (m/z 695) observed during
LPS-stimulated human monocytes, corresponding to the retention time of
authentic 8-epi-PGF
. No other isoprostanes were
present. The lower panel shows the suppression of that peak (m/z 695) when the sample was treated with
cycloheximide.
Similar results were obtained with
concanavalin A (10 µg/ml) and PMA (100 nM). Both compounds
caused a time-dependent increase on 8-epi-PGF,
PGE
, and TxB
. The respective ratios of product
formed at 24 h of incubation were 1:5.3:160 for concanavalin A (Fig. 4) and 1:5:130 for PMA (Fig. 5). Again, both
concanavalin A (Fig. 6A) and PMA (Fig. 6B) caused induction of PG G/H S-2 protein, which
was suppressed, together with product formation, by cycloheximide and
dexamethasone ( Table 2and Table 3). L 745,337 suppressed
formation of concanavalin A (IC
= 40 ± 6
nM), and PMA (IC
= 35 ± 8
nM) stimulated formation of 8-epi-PGF
, along
with that of the other eicosanoids. The induced PG G/H S-2 protein was
again unaffected by L 745,337.
Figure 4: Time course of eicosanoid formation in human monocytes stimulated with concanavalin A (10 µg/ml). The supernatant was assayed for prostanoid production by GC/MS at the indicated times. Values are reported as mean ± S.D. from five experiments.
Figure 5: Time course of eicosanoid formation in human monocytes stimulated with PMA (100 nM). The supernatant was assayed for prostanoid production by GC/MS at the indicated times. Values are reported as mean ± S.D. from four experiments.
Figure 6: Western blot analyses of PG G/H S-2 protein expression in monocytes treated with concanavalin A (10 µg/ml) or PMA (100 nM). A, isolated human monocytes were incubated with saline for 24 h (control (c)); with concanavalin A alone for 1, 4, 8, and 24 h; with concanavalin A + dexamethasone (D) for 24 h; and with concanavalin A + cycloheximide (C) for 24 h. This figure is representative of four experiments. B, monocytes were incubated with saline for 24 h (control (c)); with PMA alone for 1, 4, 8, and 24 h; with PMA + dexamethasone (D) for 24 h; and with PMA + cycloheximide (C) for 24 h. The figure is representative of three different experiments.
Activation of human monocytes with opsonized zymosan in the presence
of LDL caused a marked time-dependent increase in
8-epi-PGF, but not in PGE
or
TxB
. The increase in 8-epi-PGF
was
associated with an increase in TBARS and hydroperoxide levels (Table 4). This phenomenon was prevented by the oxygen free
radical scavengers superoxide dismutase (300 units/ml) and BHT (20
µM), but not by L 745,337 (100 nM) (Fig. 7) or by the nonselective inhibitor of PG G/H S, aspirin
(data not shown). The quantities of 8-epi-PGF
formed
under these conditions, in the presence of an excess of lipid (LDL)
substrate, are not comparable with those formed in isolated stimulated
human monocytes in the earlier experiments. In contrast to the data
presented in Fig. 3, an array of peaks corresponding to
F
-isoprostanes was evident in the supernatants of
zymosan-stimulated human monocytes incubated with LDL (Fig. 8, center panel). One of these corresponds to the retention time
of the 8-epi-PGF
standard (m/z 699) (Fig. 8, upper panel). Pretreatment with antioxidants
suppressed the peaks, reflecting free radical-catalyzed isoprostane
formation under these experimental conditions (Fig. 8, lower
panel). The retention time of authentic PGF
is
well away from that of the F
-isoprostanes. Finally,
coincubation of zymosan-activated human monocytes with LDL did not
result in induction of PG G/H S-2 protein (data not shown).
Figure 7:
Formation of 8-epi-PGF during cell-mediated LDL oxidation. LDL (400 µg of
protein/ml) was incubated with human monocytes stimulated with
opsonized zymosan for 3 and 24 h. LDL was also incubated with
zymosan-treated monocytes for 24 h in the presence of superoxide
dismutase (SOD; 300 units/ml), BHT (20 µM), or L
745,337 (100 nM). Values are reported as mean ± S.D.
from four experiments.
Figure 8:
Selected ion monitoring of
8-epi-PGF. The upper panel shows a peak at m/z 699 corresponding to authentic
O
-labeled internal standard. The center panel shows a peak (m/z 695) corresponding to the
retention time of authentic 8-epi-PGF
. The array of
peaks seen in the chromatogram most likely corresponds to
F
-isoprostanes. The lower panel shows the
reduction of the peaks (m/z 695) when the sample was incubated
with butylated hydroxytoluene supporting the free radical-catalyzed
origin of them.
8-Epi-PGF is an abundant isoprostane formed
in response to free radical attack on arachidonic acid(17) .
Urinary excretion of 8-epi-PGF
is increased in
syndromes putatively associated with increased oxidant stress in
vivo, including paracetamol and paraquat poisoning(35) ,
cigarette smoking (36) , and vascular reperfusion(37) .
We have previously shown that it is a minor product of the PG G/H S-1
expressed in human platelets(21) . However, this route of
formation appears to contribute trivially to overall
8-epi-PGF
biosynthesis, even in a setting such as
chronic cigarette smoking(36) , in which platelet activation (38) is thought to coincide with increased oxidant stress.
Human monocytes contain PG G/H S-2 additional to the isoform
expressed in platelets. This form of the enzyme is highly regulated by
cytokines, LPS, hormones, and mitogenic
factors(9, 10, 11) . It is thought to be the
source of prostanoid production in inflammatory states (39) and, perhaps, in cancer(40) . The primary
sequences of both human enzymes exhibit 60%
similarity(8, 41) ; however, mutational analysis
suggests that the active site of PG G/H S-2 is the more accommodating.
For example, although aspirin is a relatively nondiscriminate PG G/H S
inhibitor(42, 43) , inhibition of PG G/H S-2, but not
of PG G/H S-1, by aspirin is associated with increased production of
(15R)-hydroxyeicosatetraenoic acid, coincident with inhibition
of prostanoid formation(44) . The present studies demonstrate
that 8-epi-PGF may be formed in a PG G/H S-2-dependent
manner. Using three independent stimuli, formation coincided in time
with the kinetics of the de novo synthesis of PG G/H S-2.
Furthermore, reduction in activity of the induced enzyme, by either
cycloheximide or steroids, prevented 8-epi-PGF
formation coincident with that of the conventional PG G/H S-2
products of these cells, PGE
and thromboxane A
.
A selective PG G/H S-2 inhibitor suppressed generation of all three
products without preventing synthesis of the enzyme. PG G/H S-1 was not
induced during stimulation of 8-epi-PGF
generation in
these experiments; thus, the contribution of the two enzymes to its
production is clearly segregated.
Unlike the case with platelet PG
G/H S-1, 8-epi-PGF is formed in greater abundance
relative to the conventional products of PG G/H S-2 in monocytes. Thus,
whereas maximal 8-epi-PGF
formation in platelets is
roughly that of TxB
, maximal formation by PG G/H S-2 in
monocytes is and of the corresponding production of PGE
and TxB
, respectively, the most abundant conventional
products of PG G/H S-2 in these cells. This observation raises the
possibility that the mitogenic properties of 8-epi-PGF
(20) may contribute to the role of PG G/H S-2 activation
in syndromes of vascular proliferation(45, 46) .
Selective inhibitors of this enzyme might be used in clarifying the
utility of urinary 8-epi-PGF
as an index of free
radical generation in syndromes putatively associated with oxidant
stress, in which PG G/H S-2 induction is possible. Although
enzyme-catalyzed formation seems to be a feature of
8-epi-PGF
, but not of other
F
-isoprostanes, similar caution might be applied to
estimates of ``F
-isoprostanes''(47) , of
which 8-epi-PGF
is an abundant
constituent(48) .
Monocytes are the first example of cells
in which both enzyme- and free radical-catalyzed formation of
8-epi-PGF have been demonstrated. Activation of
monocytes with zymosan, in contrast to LPS, concanavalin A, or PMA,
fails to induce PG G/H S-2 expression or 8-epi-PGF
generation. However, when coincubation with LDL affords the
availability of an abundant lipid substrate, zymosan-activated
monocytes catalyze the formation of a substantial amount of
8-epi-PGF
. This occurs coincident with production of
TBARS and hydroperoxides, both indices of lipid oxidation, but not with
either induction of PG G/H S-2 or generation of either PGE
or TxB
. Under these circumstances, selective PG G/H
S-2 inhibitors are ineffective in preventing 8-epi-PGF
formation. Rather, antioxidants, such as superoxide dismutase or
BHT, inhibit its production. Incubation of human monocytes with LDL in
the absence of zymosan activation fails to stimulate
8-epi-PGF
production.
Previous work has
demonstrated that copper-induced oxidation of LDL (49) is
associated with increased 8-epi-PGF generation.
Whether it or indeed other arachidonic acid products contribute to the
role of monocytes and their cellular derivatives in the process of
atherogenesis remains to be determined.