©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Monocyte Chemotaxis to C-C Chemokines by Antisense Oligonucleotide for Cytosolic Phospholipase A(*)

(Received for publication, October 16, 1995; and in revised form, December 4, 1995)

Massimo Locati (1)(§) Giuseppe Lamorte (1) Walter Luini (1) Martino Introna (1) Sergio Bernasconi (1) Alberto Mantovani (1) (2) Silvano Sozzani (1)(¶)

From the  (1)Istituto di Ricerche Farmacologiche ``Mario Negri,'' via Eritrea 62, 20157 Milan, and the (2)Section of Pathology and Immunology, Department of Biotechnology, University of Brescia, 25123 Brescia, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Monocyte chemotactic protein (MCP)-1, a member of the C-C (or beta) branch of the chemokine superfamily, at chemotactic concentrations, induced a rapid release of [^3H]arachidonic acid but not of [^14C]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(2) (cPLA(2)). To address the role of cPLA(2) 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(2) expression, as evaluated by Western blot analysis and a nearly complete inhibition (81.8 ± 4.2%; n = 3) of [^3H]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(2) 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-1alpha/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(2) plays a major role in [^3H]arachidonic acid release by MCP-1 in human monocytes and provide direct evidence for the involvement of cPLA(2) in C-C chemokine-induced monocyte chemotaxis.


INTRODUCTION

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 alpha) chemokines, active on neutrophils and T lymphocytes (4, 5, 6, 7) , and the C-C (or beta) 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 (^1)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(2)) 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 [^3H]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-1alpha/LD78)(18, 19, 20) . In parallel, also the chemotactic response to MCP-1, MCP-3, RANTES, and MIP-1alpha/LD78 were increased(18) . These results, together with the finding that PLA(2) 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(2)(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(2) (cPLA(2)) 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(2) 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 [^3H]arachidonic acid; no detectable release of [^14C]oleic acid was observed. This effect paralleled the phosphorylation of cPLA(2) evaluated by Western blot analysis. In addition, by the use of a specific antisense oligonucleotide, we show that cPLA(2) plays a crucial role in the chemotactic response of human monocytes to C-C chemokines.


EXPERIMENTAL PROCEDURES

Chemoattractants

Human recombinant MCP-1 and RANTES were from PeproTech Inc. (Rocky Hill, NJ). Human recombinant MCP-3 and MIP-1alpha/LD78 were kind gifts from Drs. A. Minty (Sanofi Elf Bio Recherches, Labège, France) and L. Czaplewski (British Bio-Technology Limited, Cowley, UK), respectively. Human recombinant C5a was a generous gift from Dr. H. S. Showell (Pfizer Central Res., Groton, CT). Recombinant products were endotoxin free as assessed by Limulus Amebocyte Lysate assay (Bio Whittaker, Walkersville, MD). fMLP and platelet-activating factor were from Sigma.

Human Monocytes Purification

Human monocytes were obtained from buffy coats of normal blood donors through the courtesy of Centro Trasfusionale Ospedale Sacco (Milan, Italy) and Centro Trasfusionale Ospedale Caduti Bollatesi (Bollate, Italy) as described previously (18) . To reduce platelet contamination, monocytes were isolated according to the procedure described by Pawlowski et al.(29) with minor modifications(18) . Briefly, anticoagulated whole blood was diluted 1:4 with cold phosphate-buffered isotonic saline without Ca and Mg (PBS; Life Technologies, Inc.) and centrifuged at 150 times g at 4 °C for 20 min. The supernatant was discarded, and cell pellet was washed in PBS in the same conditions. Cells were resuspended in PBS containing 0.3 mM EDTA (Merck, Darmstadt, Germany), layered on top of Ficoll (Biochrom, Berlin, Germany), and centrifuged at 800 times g at room temperature for 25 min. Mononuclear cells were recovered, diluted, and washed twice in PBS at 4 °C. To remove platelets specifically adherent to monocytes, mononuclear cells were resuspended in fetal calf serum (FCS; Hyclone, Logan, UT) containing 5 mM EDTA and subjected to two sequential incubations (15 min) at 37 °C. Platelet-free mononuclear cells were recovered by centrifugation at 400 times g at room temperature for 15 min. Monocytes were further purified (>90% pure) by centrifugation at 600 times g on a 46% isoosmotic Percoll (Pharmacia Biotech Inc.) gradient, as described previously(30) . The monocyte preparation obtained did not release [^3H]arachidonic acid when challenged with 10 units/ml thrombin (Sigma).

Oligonucleotide Treatment

A 16-base-long antisense oligonucleotide (Duotech, Milan, Italy) was designed originating just downstream from the initiation site of the cPLA(2) cDNA (26, 27) : 5`-GTGCTGGTAAGGATCTAT-3`. As control, the same oligonucleotide with four mismatched bases (5`-GTGCTCCTAAGTTTCTAT-3`) or an unrelated c-myb antisense oligonucleotide (5`-GCAGCGCGTCCGCCGAGA-3`) (31) were used. Human monocytes (0.7 times 10^6/ml) were cultured in Petriperm dishes (Haereus, Vienna, Austria) in the presence of different concentrations of oligonucleotides in RPMI 1640 (Biochrom, Berlin, Germany) for 4 h. A final concentration of 5% FCS was then added, and monocytes were kept in culture for additional 24-40 h(31) . Oligonucleotide treatment and culture conditions were not toxic for monocytes as assessed by trypan blue dye exclusion (cell viability, >95%).

Immunoblot Analysis of cPLA(2)

Control and activated monocytes (2 times 10^7/ml) in Hanks' balanced salt solution (Biochrom, Berlin, Germany) or oligonucleotide-treated cells were resuspended in lysis buffer (100 mM Tris-HCl, pH 7.4, 2 mM EDTA, 100 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 100 µM leupeptin, 2000 units/ml aprotinin, 50 µM pepstatin A, 200 µM sodium orthovanadate, 200 µM NaF). After the addition of sample buffer (0.625 M Tris-HCl, pH 6.8, 0.5 M 1,4-dithiothreitol, 20% SDS), samples were boiled for 5 min, and the proteins were separated on 8% SDS-polyacrylamide gel electrophoresis (20 mA/gel) using a 60:1 ratio of acrylamide:bisacrylamide. Electrophoresis was stopped 4 h after the tracking dye had left the gel as described previously(32) . Samples were transferred to nitrocellulose paper (BA 83, Schleicher & Schuell) by electroblotting (120 mA overnight at room temperature). The quality of the transfer was evaluated by staining the nitrocellulose paper with Ponceau S (0.5% in 5% trichloroacetic acid). Nitrocellulose paper was incubated for 1 h in TNE (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% Nonidet P-40) containing 5% nonfat dry milk and then incubated for 1 h with rabbit anti-human cPLA(2) (1:2000 dilution) kindly donated by Dr. L. Marshall (SmithKline Beecham Pharmaceuticals, King of Prussia, PA) and Dr. J. D. Clark (Genetics Institute Inc., Cambridge, MA). The two antisera were used with identical results. Immunoreactivity was detected by ECL (Amersham Corp.) and quantitated by densitometric analysis (IBAS 2 Kontron, Milan, Italy). In some experiments, monocytes were resuspended in extraction buffer (10 mM HEPES, pH 7.4, 0.25 M sucrose, 2 mM EGTA, 2 mM 1,4-dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 100 µM leupeptin, 2000 units/ml aprotinin, 50 µM pepstatin A, 200 µM sodium orthovanadate, 200 µM NaF) and disrupted by sonication. Sonicated cells were centrifuged at 800 times g for 10 min, and the supernatants were further centrifuged at 150,000 times g at 4 °C for 1 h. Supernatants and pellets obtained with this procedure were used as cytosol and membrane fractions, respectively(33) .

Release of Labeled Fatty Acids

Monocytes (10^6/ml) were labeled with 1 µCi/ml [^3H]arachidonic acid (200 Ci/mmol) and/or 1 µCi/ml [^14C]oleic acid (60 mCi/mmol) (Amersham) during the last 18 h of oligonucleotide treatment. Incubation did not affect cell viability (>95%, by trypan blue dye exclusion) nor the ability of monocytes to migrate in response to MCP-1. At the end of the incubation, cells were washed twice and resuspended in RPMI 1640 medium supplemented with 0.2% fatty acid free bovine serum albumin (Sigma). Monocytes (10^7/ml) were prewarmed at 37 °C for 5 min and then stimulated. The reaction was terminated by the addition of 2 ml of chloroform/methanol/formic acid (1:2:0.2, v/v/v) followed by agitation. Cell extracts were transferred to centrifuge tubes, and 1 ml of water and 2 ml of chloroform were added. Chromatographic separation of lipids was performed by evaporating the organic phase under a stream of nitrogen, redissolving the residue in chloroform, and loading the extract on silica gel G plates (Merck). Fatty acids were separated by thin layer chromatography using hexane/ethyl ether/formic acid (15:10:1, v/v/v) as a solvent system for 30 min as reported previously (18) . Free fatty acids position on TLC plates was determined as comigration with commercially available standards after exposure to iodine vapors. Quantitative determination was obtained by scraping portions of the silica gel into scintillation vials followed by liquid scintillation spectrometry. The results are expressed as the percentage of radioactivity in the fatty acid band on the total radioactivity recovered from each lane. For phospholipid analysis, TLC plates (silica gel H) were resolved with solvent system of chloroform/methanol/acetic acid/water (50:25:8:2, v/v/v) for 15 cm. Phospholipids were identified based on comigration with commercially available standards, and quantitative evaluation was performed by liquid scintillation spectrometry(18) .

Migration Assay

Monocyte migration was evaluated using a microchamber technique (34) as described previously(18, 30) . 27 µl of chemoattractant diluted in RPMI 1640 medium with 1% FCS were seeded in the lower compartment of the chemotaxis chamber (Nucleopore Corp., Pleasanton, CA), and 50 µl of cell suspension (1.5 times 10^6/ml) were seeded in the upper compartment. The two compartments were separated by a 5-µm pore size PVP membrane (Nucleopore). Chambers were incubated at 37 °C in air with 5% CO(2) for 90 min. At the end of the incubation, filters were removed, fixed, and stained with Diff-Quik (Baxter s.p.a., Rome, Italy). Migrated monocytes in five high power oil immersion fields were counted.

Enzyme-linked Immunosorbent Assay for MCP-1

MCP-1 levels in monocyte culture supernatants were evaluated by a specific sandwich enzyme-linked immunosorbent assay exactly as described previously(35) . The lower limit of sensitivity for this assay is 40 pg/ml.


RESULTS

Effect of MCP-1 on the Release of [^3H]Arachidonic Acid and [^14C]Oleic Acid from Labeled Human Monocytes

Initial studies were designed to examine the selectivity of MCP-1-induced PLA(2) activity(s) for arachidonic acid. Monocytes cultured in nonadherent conditions in the presence of both [^3H]arachidonic acid and [^14C]oleic acid for 18 h showed a comparable uptake of the two labels (47.4 ± 2.5 and 46.9 ± 1.9%, respectively; n = 4) with more than 90% of the labels incorporated in the phospholipid pools (data not shown). Distribution of the labels in phospholipids was: 60 ± 3.6 and 72 ± 2.6% phosphatidylcholine, 11 ± 2.0 and 9 ± 1.8% phosphatidylinositol/phosphatidyletanolamine, and 31 ± 4.7 and 12 ± 2.9% phosphatidylserine for [^3H]arachidonic acid and [^14C]oleic acid, respectively (n = 3). Maximal chemotactic activity of MCP-1 is observed at 50-100 ng/ml (6-12 nM; 30) and these concentrations were used throughout this study. As shown in Fig. 1, 100 ng/ml MCP-1 induced a rapid accumulation of [^3H]arachidonic acid that peaked between 3 and 10 min and, at 3 min, corresponded to 196 ± 18% (n = 4) of control group activity (Fig. 1). MCP-1 activation appeared to be specific for arachidonic acid because no detectable release of [^14C]oleic acid was observed up to 30 min of stimulation. In the same experimental conditions, 10M fMLP for 3 min resulted in the release of 577 ± 128% and 163 ± 11% of control cells for [^3H]arachidonic acid and [^14C]oleic acid, respectively (n = 4).


Figure 1: Effect of MCP-1 on the release of [^3H]arachidonic acid ([^3H] 20:4) and [^14C]oleic acid ([^14C] 18:1) from labeled human monocytes. Human monocytes were separated and labeled as detailed under ``Experimental Procedures.'' Cells (10^7/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 [^14C]oleic acid, the S.E. is contained within the symbol size.**, p < 0.01 by paired Student's t test.



Effect of MCP-1 on cPLA(2) Phosphorylation

Agonist-triggered cPLA(2) activation, including that mediated by seven transmembrane domain receptors, is associated with increased phosphorylation of the protein on serine residues resulting in a stable 3-4-fold increase of cPLA(2) catalytic activity(36, 37, 38, 39) . cPLA(2) phosphorylation correlates with the appearance of a more slowly migrating electrophoretic form of the protein. In agreement with previous reports (40, 41) , Western blot analysis of resting human monocytes shows that in these cells cPLA(2) migrates as a doublet (Fig. 2). Three min of stimulation with 100 ng/ml MCP-1 caused a decrease of the intensity of the faster migrating band and an increase in the intensity of the slower migrating species. Similar results were obtained with fMLP (Fig. 2) and with MCP-3 (data not shown). A short (1 min) preincubation of monocytes with 100 nM platelet-activating factor, before MCP-1 stimulation, resulted in the complete loss of the faster migrating band (Fig. 2). These results parallel the synergism observed between platelet-activating factor and MCP-1 in terms of [^3H]arachidonic acid release from prelabeled cells(18, 20) . The relative changes in the two immunoreactive bands could be quantified by densitometry and expressed as the ratio of the slower over the faster migrating band: 1.5 ± 0.2 and 4.1 ± 0.6 for control and MCP-1-stimulated cells, respectively (n = 3; p < 0.05 by paired Student's t test). Fig. 3shows that a shift in the ratio of cPLA(2) immunoreactive bands was present in both cytosolic and membrane fractions. The effect was time-dependent, detectable 1 min after stimulation, reaching statistical significance in both cytosol and membrane between 3 and 5 min and declining to basal levels thereafter (data not shown).


Figure 2: Effect of MCP-1 stimulation on the electrophoretic mobility of cPLA(2) in human monocytes. Monocytes (2 times 10^7/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 10M fMLP for 1 min. Whole cell lysates were prepared in the presence of 1% Nonidet P-40 and analyzed by immunoblotting with specific cPLA(2) rabbit antisera as described under ``Experimental Procedures.''




Figure 3: Time course of MCP-1 stimulation on cPLA(2) 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.



Inhibition of cPLA(2) Expression by Antisense Oligonucleotide

Because of the lack of specific inhibitors, it is difficult to correlate cell functions to activation of different PLA(2) forms. Antisense technology provides a unique approach to this problem and was successfully used in monocytic cells(42, 43) also to inhibit PLA(2) isoforms(23, 43) . Table 1reports that monocytes treated with 10 µM cPLA(2) antisense oligonucleotide for 48 h showed a marked decrease of the immunoreactive bands detected by quantitative Western blot, when compared with cultured untreated cells or with monocytes exposed to a similar concentration of control oligonucleotide in the same experimental conditions. Table 1also shows that [^3H]arachidonic acid release was almost completely blocked in antisense oligonucleotide-treated monocytes challenged with 100 ng/ml MCP-1. Inhibition was not due to a toxic effect of the treatment because cell viability was higher than 90% (data not shown), and inhibition was not the result of homologous desensitization by MCP-1 released in the culture medium, because at the end of the incubation MCP-1 levels in untreated, antisense, and control oligonucleotide-treated cultures were similar (0.37 ± 0.15, 0.44 ± 0.15, and 0.39 ± 0.12 ng/ml, respectively; n = 3; p > 0.05 of oligonucleotide-treated versus untreated groups).



Effect of cPLA(2) Antisense Oligonucleotide on Chemotaxis

The effect of cPLA(2) antisense oligonucleotide treatment on monocyte chemotactic response to MCP-1 was investigated. fMLP was used as reference chemoattractant. Fig. 4shows that antisense oligonucleotide treatment (0.3-3 µM for 48 h) did not significantly alter the spontaneous migration of monocytes when compared with cells treated with control oligonucleotide or to untreated cultured monocytes. On the contrary, the number of cells migrated across polycarbonate filters in response to 50 ng/ml of MCP-1 was inhibited in a concentration-dependent manner (IC = 1.9 ± 1.1 µg/ml; n = 3), with complete inhibition observed between 3 and 10 µM antisense oligonucleotide (Fig. 4, upper panel, and data not shown). Control oligonucleotide did not affect basal or activated cell migration. Inhibition of MCP-1 response could partially be overcome by the use of higher concentrations of the agonist (Fig. 5A). In parallel experiments, the same monocyte population migrated normally in response to both optimal (10M) and suboptimal (10-10M) chemotactic concentrations of fMLP, indicating that inhibition was not the result of toxicity (Fig. 4, lower panel, and 5B).


Figure 4: Effect of cPLA(2) 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 times 10^6/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 (10M). 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 times 10^6/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.



Effect of cPLA(2) Antisense Oligonucleotide on Chemotaxis to C-C Chemokines and C5a

Because of the discrepancy of effect of cPLA(2) antisense oligonucleotide treatment on monocyte chemotaxis to MCP-1 and fMLP, other C-C chemokines and a second classical chemoattractant were tested in the chemotaxis assay. All the agonists were used at their optimal chemotactic concentrations. In the same experimental conditions, all the agonists tested induced the release of arachidonic acid from human monocytes(18, 19) . (^2)Chemotactic response to the four C-C chemokines was significantly inhibited (p < 0.05 by paired Student's t test) by the antisense oligonucleotide treatment (10 µM, 48 h) with the percentage of inhibition of 86.7 ± 9.5 (n = 19), 70.3 ± 15.6 (n = 5), 75.1 ± 5.3 (n = 3), and 79.3 ± 15.1 (n = 3) for MCP-1, MCP-3, MIP-1alpha/LD78, and RANTES, respectively (Fig. 6). On the contrary, the chemotactic responses to C5a and fMLP were only minimally affected (20.2 ± 17.6; n = 7 and 13.5 ± 11.2; n = 21, respectively), and inhibition never reached statistical significance (p = 0.152 and p = 0.123 by paired Student's t test for C5a and fMLP, respectively; Fig. 6).


Figure 6: Effect of cPLA(2) 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 times 10^6/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-1alpha/LD78; 70 ± 8, n = 3, 100 ng/ml RANTES; 122 ± 24, n = 7, 50 ng/ml C5a; 135 ± 24, n = 21, 10M 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'').




DISCUSSION

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(2) activation represents the most direct and the main mechanism of arachidonic release from the sn-2 position of membrane phospholipids. Thus, activation of PLA(2) 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 [^3H]arachidonic acid release and phosphorylation of cPLA(2) 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(2) 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(2) 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(2) 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(2) 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(2) antisense oligonucleotide-treated monocytes released only a minute fraction (<20% of control oligonucleotide-treated cells) of [^3H]arachidonic acid when challenged with MCP-1 (Table 1). Taken together, these data indicate that cPLA(2) 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(2) 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(2) 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(2), 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(2) molecules. fMLP and C5a receptors could also induce the required levels of free arachidonic acid through the activation of other types of PLA(2) that are not efficiently coupled to chemokine receptors. Alternatively, fMLP and C5a receptors could bypass cPLA(2) 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(d) value of MCP-1 receptors(47, 54) . A higher degree of receptor occupancy could activate residual cPLA(2) 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(2)-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-beta(61) , and fMLP (62) were shown to activate cPLA(2) 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(2) 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.


FOOTNOTES

*
This work was supported in part by the National Research Council (Italy) project ACRO, by Strategic Project Cytokines, and by NATO Grant CRG 910243. The generous contribution of the Italian Association for Cancer Research is gratefully acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Italian Association for Cancer Research.

To whom correspondence should be addressed: Istituto di Ricerche Farmacologiche ``Mario Negri,'' via Eritrea 62, 20157 Milan, Italy. Tel.: 2-390141; Fax: 2-3546277; SOZZANI{at}IRFMN.MNEGRI.IT.

(^1)
The abbreviations used are: fMLP, formyl-methionyl-leucyl-phenylalanine; MCP, monocyte chemotactic protein; MIP-1alpha/LD78, macrophage inflammatory protein-1alpha; PLA(2), phospholipase A(2); cPLA(2), cytosolic PLA(2); PBS, phosphate-buffered saline; FCS, fetal calf serum; RANTES, regulated on activation normal T cells expressed and secreted.

(^2)
M. Locati and S. Sozzani, unpublished results.


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