(Received for publication, October 16, 1995; and in revised form, December 4, 1995)
From the
Monocyte chemotactic protein (MCP)-1, a member of the C-C (or
) branch of the chemokine superfamily, at chemotactic
concentrations, induced a rapid release of
[
H]arachidonic acid but not of
[
C]oleic acid from prelabeled human monocytes.
This effect was associated with an increase in the intensity of the
immunoreactive band corresponding to the phosphorylated form of
cytosolic phospholipase A
(cPLA
). To address
the role of cPLA
in the induction of monocyte chemotaxis,
cells were treated with a specific antisense oligonucleotide. Monocytes
cultured in the presence of 10 µM antisense
oligonucleotide for 48 h showed a marked decrease (57 ± 5%; n = 4) of cPLA
expression, as evaluated by
Western blot analysis and a nearly complete inhibition (81.8 ±
4.2%; n = 3) of [
H]arachidonic
acid release in MCP-1-stimulated cells. Monocyte chemotaxis in response
to MCP-1 also was inhibited in a concentration-dependent manner by
cPLA
antisense oligonucleotide (IC
=
1.9 ± 1.1 µM; n = 3), with complete
inhibition observed between 3 and 10 µM. No inhibition of
chemotactic response was observed in monocytes treated with a control
oligonucleotide. Monocyte migration in response to MCP-3, RANTES
(regulated on activation normal T cells expressed and secreted), and
MIP-1
/LD78 also was inhibited (>70%) in antisense
oligonucleotide-treated cells. On the contrary, the chemotactic
response elicited by formyl-methionyl-leucyl-phenylalanine and C5a, two
``classical'' chemotactic agonists, was minimally affected
(<20%) by antisense oligonucleotide treatment. These data show that
cPLA
plays a major role in
[
H]arachidonic acid release by MCP-1 in human
monocytes and provide direct evidence for the involvement of cPLA
in C-C chemokine-induced monocyte chemotaxis.
The recruitment of leukocytes from the blood compartment to the site of inflammation represents one of the characteristic elements of the inflammatory process(1) . Locally produced chemotactic agonists are believed to play a crucial role in the ``multistep paradigm'' of leukocyte accumulation in tissues(2, 3) .
In the past few years a new
superfamily of chemotactic cytokines, named chemokines, was described.
The hallmark of this family is a four conserved cysteine
motif(4, 5, 6, 7) . According to the
relative position of the first two cysteines it is possible to
distinguish two families: the C-X-C (or ) chemokines, active on
neutrophils and T lymphocytes (4, 5, 6, 7) , and the C-C (or
) chemokines that exert their action on multiple leukocyte
populations, including monocytes, basophils, eosinophils, T
lymphocytes, natural killer, and dendritic
cells(4, 5, 6, 7, 8, 9) .
Recently, a protein that may define a third family (the C or
chemokines) was described. This protein is characterized by the absence
of the first and third cysteines and is active on T lymphocytes (10) .
Chemokines, as well as classical chemotactic
agonists, such as formylated peptides (of which fMLP ()is
the prototype) and C5a, bind to and activate a family of
rhodopsin-like, GTP-binding protein-coupled seven-transmembrane domain
receptors (11, 12, 13) . Activated
chemotactic receptors induce remodeling of membrane phospholipids by
the action of phospholipases (C, D, and A
) and these events
ultimately lead to the induction of different biological responses:
chemotaxis, activation of the oxidative burst, and release of lysosomal
enzymes(12, 13, 14) . The role of individual
second messengers in the generation of different biological responses
is still unclear.
In previous studies aimed at better clarifying the
molecular bases for monocyte migration in response to monocyte
chemotactic protein (MCP)-1, a prototypic C-C
chemokine(8, 15, 16, 17) , we
reported that MCP-1 induces a rapid (<15 s) and transient (15
min) release of [
H]arachidonic acid from labeled
human monocytes(18, 19) . This effect was inhibited by
Bordetella pertussis toxin treatment, was dependent on the influx of
extracellular Ca
, and was increased in a synergistic
fashion by platelet-activating factor. Similar results were obtained
with other proteins of the C-C chemokine family (e.g. MCP-3,
RANTES, and MIP-1
/LD78)(18, 19, 20) . In
parallel, also the chemotactic response to MCP-1, MCP-3, RANTES, and
MIP-1
/LD78 were increased(18) . These results, together
with the finding that PLA
inhibitors block monocyte
chemotaxis(18) , suggest a role for arachidonic acid as a
second messenger in monocyte migration to chemokines.
Cells of the
monocytic lineage posses at least three different types of
PLA(21, 22) : a low molecular mass
(
14 kDa) secreted form that requires for its catalytic activity
millimolar concentrations of Ca
and does not show a
selectivity for the fatty acid esterified at the sn-2
position(23, 24) ; a 85-kDa cytosolic PLA
(cPLA
) that shows a certain degree of specificity for
arachidonic acid and that translocates to the membrane fraction by a
Ca
(nanomolar)-dependent mechanism upon receptor
stimulation(25, 26, 27) ; and a
Ca
-independent ATP-regulated cytosolic PLA
that does not show a preference for the fatty acid at the sn-2
position (28) . Because of the lack of specific inhibitors, the
relative contribution of these enzymes in arachidonic acid metabolism
in human monocytes is still uncertain.
In this paper we report that
MCP-1-stimulated monocytes selectively released
[H]arachidonic acid; no detectable release of
[
C]oleic acid was observed. This effect
paralleled the phosphorylation of cPLA
evaluated by Western
blot analysis. In addition, by the use of a specific antisense
oligonucleotide, we show that cPLA
plays a crucial role in
the chemotactic response of human monocytes to C-C chemokines.
Figure 1:
Effect of MCP-1
on the release of [H]arachidonic acid
([
H] 20:4) and [
C]oleic
acid ([
C] 18:1) from labeled human monocytes.
Human monocytes were separated and labeled as detailed under
``Experimental Procedures.'' Cells (10
/ml) were
stimulated with 100 ng/ml MCP-1 for the times indicated. The reaction
was stopped by the addition of 2 ml of chloroform/methanol/formic acid
(1:2:0.2, v/v/v) followed by lipid extraction. Accumulation of labeled
free fatty acids was evaluated in the extracted organic phase by TLC on
silica gel G plates using a solvent system of hexane/ethyl ether/formic
acid (15:10:1, v/v/v). The results are expressed as the percentage of
radioactivity in the fatty acid (FA) band at the net of their
respective control values. Each point represents the average value of
three different experiments ± S.E. For
[
C]oleic acid, the S.E. is contained within the
symbol size.**, p < 0.01 by paired Student's t test.
Figure 2:
Effect of MCP-1 stimulation on the
electrophoretic mobility of cPLA in human monocytes.
Monocytes (2
10
/ml) were prewarmed at 37 °C for
5 min, pretreated with 100 nM platelet-activating factor (PAF) or ethanol (1 µl/ml) for 1 min, and then stimulated
with 100 ng/ml MCP-1 for an additional 3 min or 10
M fMLP for 1 min. Whole cell lysates were prepared in
the presence of 1% Nonidet P-40 and analyzed by immunoblotting with
specific cPLA
rabbit antisera as described under
``Experimental Procedures.''
Figure 3:
Time course of MCP-1 stimulation on
cPLA electrophoretic mobility. Monocytes were prewarmed at
37 °C for 5 min and stimulated with 100 ng/ml MCP-1 for the times
indicated. Then cells were sonicated and fractionated as detailed under
``Experimental Procedures.'' Autoradiographies were analyzed
by densitometric analysis using an IBAS 2 Kontron image processing
system. The results are expressed as ratios (mean ± S.D.; n = 4) of arbitrary optical unit of slower over the faster
migrating immunoreactive band. *, p < 0.05;**, p < 0.01 by paired Student's t test.
Figure 4:
Effect of cPLA antisense
oligonucleotide on human monocyte chemotaxis. Human monocytes, obtained
as detailed under ``Experimental Procedures,'' were cultured
in the absence or in the presence of different concentrations of
antisense or control oligonucleotides for 48 h. Cells were then washed,
resuspended (1.5
10
/ml) in RPMI 1640 medium in the
presence of 1% FCS, and tested for their ability to migrate across a
polycarbonate filter in response to an optimal concentration of MCP-1
(50 ng/ml) or fMLP (10
M). One experiment
performed in triplicate, representative of three similar experiments,
is shown. The results are expressed as the number of migrated monocytes
in five high power oil immersion microscopic
fields.
Figure 5:
MCP-1 and fMLP dose-responses for monocyte
chemotaxis. Human monocytes, obtained as detailed under
``Experimental Procedures,'' were cultured in the absence or
in the presence of 10 µM antisense or control
oligonucleotides for 48 h. Cells were then washed, resuspended (1.5
10
/ml) in RPMI 1640 medium in the presence of 1%
FCS, and tested for their ability to migrate across a polycarbonate
filter in response to different concentrations of MCP-1 or fMLP. One
experiment performed in triplicate, representative of three similar
experiments, is shown. The results are expressed as the number of
migrated monocytes in five high power oil immersion microscopic
fields.
Figure 6:
Effect of cPLA antisense
oligonucleotide on monocyte chemotaxis to C-C chemokines and C5a. Human
monocytes, obtained as detailed under ``Experimental
Procedures,'' were cultured in the absence or in the presence of
10 µM antisense or control oligonucleotides for 48 h.
Cells were then washed, resuspended (1.5
10
/ml) in
RPMI 1640 medium in the presence of 1% FCS, and tested for their
ability to migrate across a polycarbonate filter in response to an
optimal concentration of chemokines, C5a or fMLP. The results are
expressed as the percentage of inhibition of chemotactic response of
cells treated with the antisense oligonucleotide at the net of basal
migration (49 ± 10; n = 24). Chemotactic
response of cells treated with control oligonucleotide at the net of
basal migration (49 ± 9) to each agonist was assumed as 100% (93
± 17, n = 18, 50 ng/ml MCP-1; 93 ± 23, n = 5, 50 ng/ml MCP-3; 82 ± 25, n = 3, 50 ng/ml MIP-1
/LD78; 70 ± 8, n = 3, 100 ng/ml RANTES; 122 ± 24, n =
7, 50 ng/ml C5a; 135 ± 24, n = 21,
10
M fMLP). The results are the average
numbers (±S.E.) of multiple experiments (see above) performed
with different monocyte cultures each one in triplicate. The results
obtained with chemokines were statistically different (p <
0.01, by paired Student's t test) from the control group
(see ``Results'').
The release of arachidonic acid and the production of
eicosanoids is an early event in the activation of phagocytic cells by
several inflammatory agonists including chemotactic
factors(14, 44, 45) . PLA activation represents the most direct and the main mechanism of
arachidonic release from the sn-2 position of membrane phospholipids.
Thus, activation of PLA
is the rate-limiting step in
arachidonic acid mobilization(21, 22, 46) .
In the present study we report that chemotactic concentrations of
MCP-1, a prototypic C-C chemokine, induced
[H]arachidonic acid release and phosphorylation
of cPLA
in a time-dependent manner (Fig. 1Fig. 2Fig. 3). Similar results (not shown)
were obtained with MCP-3, another member of the C-C chemokine family
that shows 72% homology to (8) and shares binding sites (19, 47) with MCP-1, in human monocytes. The kinetics
of cPLA
phosphorylation after MCP-1 and MCP-3 stimulation
were fast and correlated with arachidonic acid release from labeled
monocytes. Both release and phosphorylation were already detectable 1
min after stimulation, peaked between 3 and 10 min, and returned to
baseline within the following 10 min ( (18) and (19) and Fig. 1and 2). cPLA
is a 85-kDa
protein that preferentially hydrolyzes phospholipids containing
arachidonic acid at the 2 position and that was recently purified and
cloned from the cytosol of myelomonocytic cell
lines(25, 26, 27, 48, 49, 50) .
Ca
is not required for cPLA
catalytic
activity(51, 52) , but nanomolar concentrations of
Ca
are needed for interfacial association with the
lipid bilayer(25, 26) . In ionophore-permeabilized
human monocytes it was shown that maximal arachidonic acid release by
MCP-1 was observed in the presence of 300-700 nM free
Ca
concentration(18) . These concentrations
are compatible with MCP-1-activated intracellular Ca
levels in monocytes (30, 53) and with the
calcium concentrations required for cPLA
membrane
association(25, 26) . In the experimental conditions
used, MCP-1 did not release oleic acid from labeled monocytes,
suggesting that the activated phospholipase(s) is specific for
arachidonic acid-labeled phospholipid pools (Fig. 1). Finally,
cPLA
antisense oligonucleotide-treated monocytes released
only a minute fraction (<20% of control oligonucleotide-treated
cells) of [
H]arachidonic acid when challenged
with MCP-1 (Table 1). Taken together, these data indicate that
cPLA
plays a major role in the mobilization of arachidonic
acid in MCP-1-stimulated monocytes.
Monocytes treated with a
specific antisense oligonucleotide were used to address the role of
cPLA in the induction of monocyte chemotaxis by C-C
chemokines. In 19 of 21 experiments performed with different monocyte
cultures, a nearly complete inhibition of cell migration was observed
(>85%) in response to an optimal concentration of MCP-1 ( Fig. 4and Fig. 5). Inhibition by the antisense
oligonucleotide was concentration-dependent and -specific, because it
was not observed in cells treated with a control oligonucleotide ( Fig. 4and Fig. 5) or with a c-myb-specific
antisense oligonucleotide (data not shown). Inhibition of chemotactic
response was not caused by toxicity of the treatment because: (i) cell
viability was always higher than 90% by trypan blue dye exclusion, and
treated monocytes were similar to untreated cells in terms of
morphology (data not shown) and spontaneous migration (Fig. 4);
(ii) cells exposed to control oligonucleotides showed a normal
migration to MCP-1 (Fig. 4); and (iii) cPLA
antisense oligonucleotide-treated cells migrated normally to fMLP (Fig. 4, 5 and 6). Finally, inhibition was not caused by
homologous receptor desensitization (8) because comparable
levels of MCP-1 were present in the supernatants of untreated and
control or antisense oligonucleotide-treated cells.
A more extensive
analysis showed that monocyte chemotaxis to all the C-C chemokines
tested was strongly (>70%) inhibited by the antisense
oligonucleotide treatment, whereas monocyte migration to fMLP or to C5a
was only minimally (<20%) affected (Fig. 6). Thus, according
to their requirement for cPLA, it is possible to divide the
chemotactic agonists tested in two groups, a first one, highly
sensitive to the action of the antisense oligonucleotide that includes
all the C-C chemokines investigated, and a second one that was poorly
sensitive to this treatment and that comprises classical chemotactic
factors. At the moment, the reason for this difference is unknown. It
is possible that fMLP and C5a but not chemokine receptors might have
access to the surviving cPLA
molecules. fMLP and C5a
receptors could also induce the required levels of free arachidonic
acid through the activation of other types of PLA
that are
not efficiently coupled to chemokine receptors. Alternatively, fMLP and
C5a receptors could bypass cPLA
inhibition through the
stronger activation of signaling pathways alternative to arachidonic
acid mobilization. A similar hypothesis can be formulated to explain
the ability of sopraoptimal concentrations of MCP-1 to overcome
oligonucleotide inhibition. The optimal chemotactic concentration (50
ng/ml MCP-1) is similar to or less than the K
value of MCP-1 receptors(47, 54) . A higher
degree of receptor occupancy could activate residual cPLA
or trigger alternative signaling pathways.
A direct role for
arachidonic acid and its metabolites in cell movement was recently
suggested in different cell types. Both 5-lypoxygenase and
cyclooxygenase products were found to regulate epidermal growth
factor-induced actin remodeling in A431 cells (55) and
neutrophil migration in vivo(56) .
cPLA-mediated arachidonic acid release was found to be
required for basic fibroblast growth factor-stimulated migration of
endothelial cells(57) . A direct role for arachidonic acid in
monocyte and macrophage adherence, expression of adhesion molecules,
and chemotaxis was suggested (58, 59, 60) .
Recently, three chemotactic factors for phagocytic cells: macrophage
colony-stimulating factor(40, 41) , transforming
growth factor-
(61) , and fMLP (62) were shown to
activate cPLA
in human monocytes, elicited guinea pig
macrophages, and human neutrophils, respectively.
In a previous
study we found a strict correlation between C-C chemokine-induced
arachidonic acid release and monocyte migration(18) . In the
present study, we show that cPLA appears to be the main
effector enzyme for chemokine-elicited arachidonic acid release in
human monocytes. In addition, by the use of specific antisense
oligonucleotides, we provide evidence that arachidonic acid by itself
or through its metabolites is strictly implicated in the induction of
monocyte migration to C-C chemokines.