(Received for publication, March 13, 1995; and in revised form, June 26, 1995)
From the
The present study was aimed at defining the chemotactic activity of phosphatidic acid, which is rapidly produced by phagocytes in response to chemotactic agonists. Exogenously added phosphatidic acid induced human monocyte directional migration across polycarbonate filters with an efficacy (number of cell migrated) comparable to that of ``classical'' chemotactic factors. In lipid specificity studies, activity of phosphatidic acid decreased with increasing acyl chain length but was restored by introducing unsaturation in the acyl chain with the most active form being the natural occurring 18:0,20:4-phosphatidic acid. Lysophosphatidic acid was also active in inducing monocyte migration. No other phospholipid and lysophospholipid tested was effective in this response. Monocyte migration was regulated by a gradient of phosphatidic acid and lysophosphatidic acid bound to the polycarbonate filter, in the absence of detectable soluble chemoattractant. Migration was also observed if phospholipids were bound to fibronectin-coated polycarbonate filters. Thus, phosphatidic acid and lysophosphatidic acid, similarly to other physiological chemoattractants (e.g. C5a and interleukin-8), induce cell migration by an haptotactic mechanism. Phosphatidic acid caused a rapid increase of filamentous actin and, at higher concentrations, induced a rise of intracellular calcium concentration. Monocyte migration to phosphatidic acid and lysophosphatidic acid, but not to diacylglycerol, was inhibited in a concentration-dependent manner by Bordetella pertussis toxin, while cholera toxin was ineffective. In the chemotactic assay, phosphatidic acid and lysophosphatidic acid induced a complete homologous desensitization and only partially cross-desensitized one with each other, or with diacylglycerol and monocyte chemotactic protein-1. Suramine inhibited monocyte chemotaxis with a different efficiency: phosphatidic acid > lysophosphatidic acid diacylglycerol. On the contrary, monocyte chemotactic protein-1-induced chemotaxis was not affected by the drug. Collectively, these data show that phosphatidic acid induces haptotactic migration of monocytes that is at least in part receptor-mediated. These results support a role for phosphatidic acid and lysophosphatidic acid in the regulation of leukocyte accumulation into tissues.
The recruitment of leukocytes from the blood compartment into
tissues is a highly regulated process which involves receptor and
counter-receptor interactions and secretion of chemotactic factors (for
reviews, see (1, 2, 3) ). Chemoattractants
play a pivotal role in this process by promoting integrin-mediated
leukocyte-endothelial cell interaction, leukocyte shape change, and
inducing the shedding of L-selectin from leukocyte
membrane(2, 4) . Since the close interaction among
leukocytes, endothelial cells, and chemotactic factors occurs in
conditions of lateral shear stress, it is very likely that chemotactic
agonists can best accomplish their function if anchored on the surface
of the endothelial layer(5) . Chemotactic signals, locally
produced in response to inflammatory agonists, promote the directional
migration of leukocytes. Classical chemotactic factors are formylated
peptides, of which fMLP ()is the prototype, products of
complement activation cascade (C5a) and a number of cytokines including
the members of the recently discovered family of
chemokines(6, 7, 8, 9) . In addition
to these factors, a number of lipids with chemotactic activity have
been reported. These include arachidonic acid and products related to
the arachidonic acid cascade, such as leukotriene B4, platelet
activating factor, and lysophosphatidylcholine and
diacylglycerols(6, 10, 11, 12, 13) .
Phosphatidic acid (PA) is a simple phospholipid which plays a
crucial role in lipid biosynthesis(14) . Recent studies have
focused attention on the possible role of PA as a second messenger (see
Refs. 15, 16 for reviews). PA can be produced through the hydrolysis of
choline-containing phosphoglycerides by the action of phospholipase D
(PLD) in a number of cell types including human
phagocytes(15, 16) . PA can be converted to
1-radyl-2-acyl-glycerols (DG) by the enzyme PA phosphohydrolase (17, 18, 19) or act directly as a second
messenger(6, 15, 16, 20) . In
neutrophils (21, 22, 23, 24, 25) and
monocytes, ()chemotactic factors, such as fMLP, C5a,
leukotriene B4, interleukin-8, and MCP-1 (monocyte chemotactic
protein-1) induce activation of PLD and a number of reports have linked
PA formation with the regulation of the oxidative burst (20, 25, 26, 27, 28, 29, 30) and
granule release(24, 31, 32) . More recently,
PA accumulation was implicated in neutrophil chemotaxis both in
vitro and in vivo(33) . In addition, exogenously
added PA and lysoPA were shown to induce a number of biological
responses including DNA
synthesis(34, 35, 36) , invasion of hepatoma
and carcinoma cells into monolayers of mesothelial cells(37) ,
actin stress fibers assembly(38, 39) , and to activate
effector enzymes, such as PLD(40, 41) , phospholipase
A2(34, 42) , and phosphorylation of focal adhesion
kinase(43) , a tyrosine kinase present at the focal adhesion
where stress fibers originate. LysoPA was also reported to induce
chemotaxis of Dictyostelium discoideum amoebae(44) .
At least part of these actions appear to be mediated by a putative
specific Bordetella pertussis toxin-sensitive GTP-binding
protein-coupled membrane receptor(45, 46) . These
findings prompted us to investigate whether PA and lysoPA could also
play a role as chemotactic factors for human mononuclear phagocytes.
In this study, we report that PA and lysoPA, bound to polycarbonate filters, are able to induce directional migration of human monocytes. In addition, micromolar concentrations of PA activates actin polymerization and calcium transients. The effect was restricted to PA and lysoPA, since other phospholipids and lysophospholipids were ineffective, and was inhibited by Bordetella pertussis toxin and suramine. Since lysoPA is produced in large amounts (1-5 µM in serum) by activated platelets during blood clotting(47, 48) , it is possible that these lipids can play an important role in the regulation of phagocyte recruitment in inflammation and wound healing.
Haptotaxis was assessed using the microchamber technique
and polycarbonate filters as described above. Portion of the filter (4
cm) were coated on the lower side with the chemoattractant
by floating in a solution (1 ml of RPMI, 1% FCS) containing different
concentrations of the agonists in 6-well plates (3046 Falcon, Becton
Dickinson, Milan, Italy) at 37 °C for 30 min (51, 52) . The filters were then extensively washed in
assay medium, blotted on filter paper, and mounted in the chemotaxis
chamber. The assay was then performed and evaluated exactly as
described above. For some experiments, filters were coated with 50
µg/ml fibronectin (Sigma) overnight(53) . Then, the filters
were washed and coated with phospholipids as described above.
Figure 1:
Ability of
various PAs to induce human monocyte migration. Human monocytes (1.5
10
/ml in PBMC) were tested for their ability to
migrate across a polycarbonate filter in response to different forms of
PAs. PAs were prepared by sonication in RPMI 1% FCS as detailed under
``Experimental Procedures'' and added to the lower
compartments of the chemotactic chamber. At the end of the incubation
(90 min), the number of monocytes in five high power
microscope-immersion fields was evaluated. Results are the average
numbers of two to five different experiments, each one performed in
triplicate, at the net of basal migration (against medium; 24 ±
3, n = 25). For each experimental point, the variation
between the experiments was less than 15%. In the same assay
conditions, net monocyte migration in response to an optimal
(10
M) concentration of fMLP was 65
± 5 (n = 25). PAs used were: didecanoyl PA (10:0-PA), dimyristoyl PA (14:0-PA), dipalmitoyl PA (16:0-PA), distearoyl PA (18:0-PA), dioleoyl PA (18:1-PA), and 1-stearoyl-2-arachidonyl PA (18:0,20:4-PA).
The ability of PA with various chemical structures to induce monocyte migration was evaluated (Fig. 1). All the different forms were active, though with a different relative potency and dose-response curves. Activation by saturated PAs decreased with increasing acyl chain length with 10:0-PA>14:0-PA = 18:0-PA>16:0-PA. Activation was restored by introducing acyl chain unsaturation in long chain phosphatidic acids (e.g. 18:1-PA versus 18:0-PA), with the most active being the naturally occurring 18:0,20:4-PA.
To clarify whether monocyte migration in response to 18:0,20:4-PA was dependent on the presence of a chemotactic gradient between the two compartments of the chamber, checkerboard experiments were performed using polycarbonate filters. As reported in Table 2, maximal migration occurred in the presence of a positive concentration gradient (higher concentration in the lower well). In the presence of a negative gradient (higher concentration in the upper well) or in the absence of gradient (equal concentration in the upper and lower wells) no enhanced migration occurred. Although the type of assay used does not formally exclude a chemokinetic component, these results strongly suggest that monocyte migration in response to 18:0,20:4-PA is chemotaxis (directional migration) rather than chemokinesis (activated random migration).
In order to evaluate if other lipids could mimic PA action on human monocytes, a number of different phospholipids and their corresponding lyso-derivatives were tested in the chemotaxis assay. LysoPAs (18:1-lysoPA and 16:0-lysoPA) stimulated monocyte migration. From a quantitative point of view, lyso-PAs were similar (16:0-LysoPA) or weaker (18:1-LysoPA) than the corresponding molecular species of PA (Fig. 2A). On the contrary, all the other phospholipids and lysophospholipids tested were not active (Fig. 2B).
Figure 2:
Ability of various phospholipids to induce
human monocyte migration. Human monocytes (1.5
10
/ml in PBMC) were tested for their ability to migrate
across a polycarbonate filter in response to a fixed concentration (300
µM) of different phospholipids. Phospholipids were
prepared by sonication in RPMI 1% FCS as detailed under
``Experimental Procedures'' and added to the lower
compartments of the chemotactic chamber. At the end of the incubation
(90 min), the number of monocytes in five high power
microscope-immersion fields was evaluated. Results are the average
numbers of three different experiments, each one performed in
triplicate, at the net of basal migration (against medium; 21 ±
4, n = 22). In the same assay conditions, net monocyte
migration in response to an optimal (10
M)
concentration of fMLP was 78 ± 6 (n = 22).
Phospholipids used were: panel A, 1,2-dioleylglycerol (18:1-DG), dioleoyl PA (18:0), oleoyl lysoPA (18:1-LPA), dipalmitoyl PA (16:0-PA), and palmitoyl
lysoPA (16:0-LPA); panel B, 1-stearoyl-2-arachidonyl
PA (18:0,20:4-PA), phosphatidylinositol (PI),
lysophosphatidylinositol (LPI), phosphatidylglycerol (PDG), lysophosphatidylglycerol (LPDG),
phosophatidylethanolamine (PE), lysophsophatidylethanolamine (LPE), phosphatidylserine (PS),
lysophosphatidylserine (LPS), phosphatidylcholine (PC), lysophosphatidylcholine (LPC).
Figure 3:
Ability of PA and lysoPA to induce
monocyte migration by haptotaxis. Human monocytes (1.5
10
/ml in PBMC) were tested for their ability to migrate in
response to filter-bound 18:0,20:4-PA (PA) and 18:1-lysoPA (LPA) (haptotaxis) or filter-bound + soluble PA
and LPA (chemotaxis). Filters were coated on one side with
different concentrations of PA and LPA for 30 min, washed, and
assembled in the chemotactic chamber with the coated side facing the
lower compartment (positive gradient, +) or with the coated
surface facing the upper compartment (negative gradient, -). At
the end of the incubation (90 min), the number of monocytes in five
high power microscope-immersion fields was evaluated. Average number of
triplicate determinations at the net of basal values (24 ± 2) of
one experiment representative of three is
shown.
Figure 4:
Homologous and heterologous
desensitization of monocyte migration by PA, lysoPA, and MCP-1.
Monocytes (1.5 10
/ml in PBMC) were preincubated
with 1 mM 1-stearoyl-2-arachidonyl-PA (PA), 1 mM oleoyl lysoPA (LPA) or 6
10
M MCP-1 at 37 °C for 30 min. The cells were then
washed and assayed for their migration toward homologous or
heterologous stimuli. Results are expressed as percent of inhibition
with respect to relative control group (cell preincubated with medium
and tested against the three single agonists). The mean numbers
(±S.E.) of four separate experiments performed in triplicate are
reported. * p < 0.05,** p < 0.01 with respect
to PA. °p < 0.05 and °°p < 0.01
with respect to LPA, by Student's t test.
Figure 5:
Effect of PTox and CTox pretreatment on
monocyte migration. Monocytes (1.5 10
/ml in PBMC)
were incubated at 37 °C with different concentrations of the toxins
for 90 min. At the end of the incubation, cells were washed twice,
resuspended in RPMI 1% FCS, and tested in the migration assay.
Phospholipids tested were 1-stearoyl-2-arachidonyl PA (PA),
1,2-dioctanoylglycerol (DG), and oleoyl lysoPA (LPA)
at the concentration of 1 mM, or 6
10
M MCP-1. Panel A, results are average numbers
± S.D. of triplicate determinations of one of two experiments at
the net of basal migration (45 ± 5). Panel B, monocytes
were incubated with 1 µM PTox and then tested in the
chemotaxis assay. The mean values ± S.D. of 5 (PA, DG, and MCP-1) and 3 (LPA) different
experiments performed in triplicate are reported. Results are expressed
as percent of inhibition. Migration values of cells incubated in the
absence of the toxins in response to the different agonists were
assumed as 100%. *p < 0.01 against respective control group
(no PTox).
Figure 6:
Effect of PA and lysoPA on F-actin
content. Percoll-purified human monocytes (10/ml) were
incubated with 1-stearoyl-2-arachidonyl PA (PA) for 30 s.
Staining of the cells with NBD-phallacidin and determination of F-actin
content was performed as described under ``Experimental
Procedures.'' Data are the mean values ± S.D. of three to
six independent experiments. The results are expressed as the relative
F-actin content calculated as the ratio of the fluorescence intensity
of stimulated over that of unstimulated cells. All the points showed
are statistically significant (p < 0.05 by Student's t test, with respect to control (untreated
cells).
Figure 7:
Effect of PA, lysoPA, and DG on
[Ca]
. Percoll-purified
monocytes (10
/ml) were incubated with Fura-2 acetoxymethyl
ester (1 µM) at 37 °C for 15 min, washed, and then
exposed in cuvette (5
10
/ml) to different
concentrations of the agonists (1-stearoyl-2-arachidonyl PA (PA), 1,2-dioctanoylglycerol (DG), lyso oleoyl-PA (LPA) or MCP-1). One experiment representative of at least
four is shown. Results are expressed as ratio of fluorescence at two
excitation wavelengths (340 and 380 nm) and emission at 487
nm.
Figure 8:
Effect
of Suramine on PA-, lysoPA-, DG-, and MCP-1-induced monocyte migration.
Human monocytes (1.5 10
/ml in PBMC) were tested for
their ability to migrate across a polycarbonate filter in response to a
fixed concentration (1 mM) of 1-stearoyl-2-arachidonyl PA (PA), 1,2-dioctanoylglycerol (DG), lyso oleoyl-PA (LPA), or 6
10
M MCP-1.
Phospholipids were prepared as detailed under ``Experimental
Procedures'' and added to lower compartments of the chemotactic
chamber. At the end of the incubation (90 min), the number of monocytes
in five high power microscope-immersion fields was evaluated. Suramine
was dissolved in RPMI 1% FCS and added to the cell suspension just
before use. Results are the average numbers ±S.E. of four
different experiments (PA and DG) or the average number of two
different experiments (LPA and MCP-1) each one performed in triplicate.
Numbers represent percent of inhibition of chemotactic activity. Cell
migration in the absence of suramine at the net (100%) of basal values
(45 ± 87) was 62 ± 5, 62 ± 2 (n =
4), 47, and 65 (n = 2) for PA, DG, LPA, and MCP-1,
respectively. *p < 0.05 by Student's t test
of PA versus DG.
Exogenously added PA and lysoPA were shown to produce numerous effects, including calcium mobilization(62, 63, 64) , activation of MAP kinases(65, 66) , induction of actin polymerization(38, 39) , and activation of focal adhesion kinase (43) in several cell types. These events are known to be implicated in the induction of leukocyte chemotaxis(6, 59) .
The present study shows that PA
was able to activate directional migration of human monocytes in a
polycarbonate filter assay with an efficacy (peak percentage migration)
comparable to that of other chemotactic factors, such as fMLP, MCP-1,
leukotriene B4, and DG (Table 1). Monocyte migration was PTox
sensitive (Fig. 5) and was dependent on a positive gradient of
the chemotactic agonist, as evaluated by checkerboard-type analysis ( Table 2and Fig. 3). Interestingly, PA and LPA act as
membrane-bound haptotactic agonists. Other physiological chemotactic
agonists were found to induce cell migration by haptotaxis, such as
casein(67) , C5a(68) , and
interleukin-8(51, 52) . In addition, membrane-bound
molecules, such as platelet-activating factor(69) , MIP-1,
and RANTES (regulated upon activation, normal T expressed) (70, 71) where shown to promote cell adhesion and
migration. Chemotactic factors play a crucial role in the regulation of
leukocyte-endothelial cells interaction by the induction of leukocyte
shape change and modulating adhesion
molecules(2, 4, 5) . In normal conditions of
lateral shear stress, it is unlikely that a soluble gradient of
chemotactic factors can accomplish this role. Thus, migration to an
immobilized gradient of chemotactic agonists present on the surface of
endothelial cells or extracellular matrix components represents a
physiological condition(5) .
Studies in other cell types
have shown that exogenous PA can act by itself, via a specific
cell-surface receptor(35, 36, 72) , while
others have attributed this effect to lysoPA contaminating the
commercial preparation of PA(64) . In addition, there is
evidence that in some cell types PA can be converted to lysoPA by the
action of a PA-hydrolyzing phospholipase
A2(47, 48, 73) , raising the possibility that
lysoPA is the effector molecule responsible for the biological action
of PA. In our study, among the different phospholipids and
lysophospholipids tested, lysoPA was indeed the only phospholipid that
could substitute for PA in inducing monocyte migration. In this study,
lysoPA showed, at best, an identical concentration curve as PA ( Fig. 2and Fig. 3), and in cross-desensitization
experiments, PA and lysoPA induced only a partial (30%)
heterologous desensitization (Fig. 4). Taken together, these
data suggest that contamination of the PA preparation with lysoPA
cannot account for the present observation.
There is evidence that
some cell types(17, 18) , including neutrophils (19, 74, 75) , possess a membrane-bound PA
phosphohydrolase which is able to convert PA to DG. Thus, it is
possible that the effect of PA on chemotaxis is not direct but mediated
by PA-derived DG. Several findings argue against this hypothesis and
support a direct role of PA in the induction of monocyte activation:
(i) in chemotaxis assays, PA induced a nearly complete (88 ± 5%)
homologous desensitization and only a partial (38 ± 3)
cross-desensitization with DG (Fig. 4); (ii) PA-induced monocyte
chemotaxis was much more sensitive to the effect of suramine than DG
(IC = 0.35 and 1.52 mg/ml, respectively; p < 0.05; Fig. 8); (iii) PA and DG induced an increase of
[Ca
]
with different kinetics.
The effect of PA was rapid and biphasic, while that of DG was
monophasic and slower (Fig. 7); (iv) propranolol (up to 60
µM), a PA phosphohydrolase
inhibitor(17, 74) , did not alter monocyte chemotaxis
to PA at concentrations able to inhibit the conversion of
intracellularly formed PA to DG(19, 20, 74) ,
(data not shown). However, it is possible that the membrane-bound and
the intracellular forms of PA phosphohydrolase have a different
sensitivity to the effect of inhibitors(74, 75) ; (v)
finally, and most convincing, PA-induced monocyte chemotaxis was
inhibited in a concentration-dependent manner by PTox, while in the
same assay conditions the action of DG was not affected (Fig. 5).
Actin polymerization is known to be an important
step in several biological processes, including cell
motility(59) . Recently, it was reported that PA induces
F-actin formation in fibroblasts (39) and that PA is one of the
second messengers of lysoPA-induced actin polymerization(41) .
Also in monocytes, PA induced a rapid activation of actin
polymerization in monocytes (Fig. 6). The kinetics of the effect
was slightly slower than that of MCP-1, a classical chemotactic factor,
but was more sensitive than haptotaxis to the action of PA (EC = 9.5 and 115 µM for F-actin and haptotaxis,
respectively).
At higher concentrations (0.3-1 mM),
PA and lysoPA induced a rapid (within seconds) increase of
[Ca]
, in monocytes (Fig. 7). These concentrations are more than one log higher than
those able to induce the same biological response in other cell types,
including Rat-1 fibroblasts, platelets, PC12 cells, and Xenopus
laevis oocytes(35, 36) . In these cells, lysoPA
induces calcium transients at concentrations in the nanomolar
range(64) . Nanomolar concentrations are compatible with the
reported affinity of the putative lysoPA receptor (45, 46) that also binds PA although with 100-fold
lower affinity(46) . However, concentrations in the micromolar
range are required to induce DNA
synthesis(45, 61, 76) , platelet activation (77) , integrin activation(78) , and to activate
effector enzymes, such as phosphatidylinositol-specific phospholipase
C(45, 64, 77, 79) , phospholipase
D(40) , and phospholipase A2(42, 45) . Thus,
it is possible that more than one receptor is responsible for PA and
lysoPA activity or, alternatively, that PA can act directly as a second
messenger(29, 42, 80, 81, 82, 83, 84) .
Phospholipids can cross the lipid bilayer
membrane(85, 86) , and there is evidence that
exogenous PA is incorporated into the outer leaflet of lipid bilayer
and internalized into cells(85, 87) . The data
reported here suggest that monocyte activation by PA is mediated, at
least in part, by the activation of a PTox-sensitive GTP-binding
protein-coupled membrane receptor. Monocyte migration in response to PA
is inhibited by PTox treatment, and PA can induce complete homologous
desensitization. However, inhibition by PTox was only partial
(
50%) and in preliminary experiments, PA-induced actin
polymerization was not sensitive to the action of the toxin. (
)Thus, it is possible that in monocytes, exogenously added
PA also may bypass the activation of a surface receptor. PA is produced
by phagocytes in response to chemotactic
stimuli(21, 22, 23, 24, 25) .
It has been calculated that PA concentration can reach micromolar
levels in receptor-stimulated neutrophils (21, 88) and
millimolar levels in stimulated rat hepatocytes in
vivo(80) . Platelet activation during coagulation of whole
blood results in serum concentrations of 1-5 µM lysoPA(48) . The results reported here raise the
intriguing possibility that PA leaking out of some cell types and
interacting with specific receptors may amplify the action of
chemotactic agents. A recent report has shown that lysoPA enhances
fibronectin binding and assembly to cultured fibroblastic cells (89) implicating a role for this lipid in extracellular matrix
deposition. PA and lysoPA were also reported to induce the release of
transforming growth factor
in mouse skin(90) . Thus, PA
and lysoPA could act at multiple levels in the regulation of leukocyte
infiltration, acting directly as an attractant signal, regulating
extracellular matrix component deposition, and inducing the release of
chemotactic factors, such as transforming growth factor
.
In conclusion, we report that immobilized PA and lysoPA are able to induce haptotactic migration of human monocytes in vitro and that this action resembles, on several aspects, that of classical chemotactic factors. Since PA is produced in monocytes after chemotactic receptor stimulation and released by activated platelet in vivo it is possible that this simple phospholipid may play an important role in regulating monocyte infiltration into tissues.