©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the Type-II Phospholipase A in Alveolar Macrophages
DOWN-REGULATION BY AN INFLAMMATORY SIGNAL (*)

(Received for publication, April 18, 1995; and in revised form, May 17, 1995)

Daniel Vial (1)(§), Mario Seorale-Pose (2), Nathalie Havet (1), Laurence Molio (1), B. Boris Vargaftig (1), Lhousseine Touqui (1)

From the  (1)Unité de Pharmacologie Cellulaire, Unité Associée Pasteur, INSERM U285, the (2)Unité de Génétique et de Biochimie du Développement, Unité Associée Pasteur/CNRS URA 361, and Institut Pasteur, 25 rue Dr. Roux, 75015 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have shown previously that guinea pig alveolar macrophages (AM) synthesize a secretory phospholipase A (PLA) during in vitro incubation. Here, we report the molecular cloning of this enzyme and show that it has structural features closely related to all known mammalian type-II PLA. The mRNA and PLA activity were undetectable in freshly collected AM, but their levels increased dramatically to reach maximal values after 16 h of culture. Thereafter, the PLA activity remained constant with a parallel secretion in the medium, in contrast to mRNA level which returned to near basal values after 32 h. Incubation of AM for 16 h with the inflammatory secretagogue peptide f-Met-Leu-Phe (fMLP) markedly reduced the PLA activity and mRNA levels. This inhibition was prevented by preexposure of AM to pertussis toxin, an inhibitor of G-protein. In contrast, when AM were first cultured for 16 h and then incubated with fMLP, no significant change was observed in their PLA activity. In conditions where the type-II PLA was completely abrogated by fMLP, the latter did not alter the lipopolysaccharide-induced accumulation of tumor necrosis factor mRNA or the release of arachidonic acid induced by the subsequent addition of the calcium ionophore A23187. These studies show that the inflammatory peptide fMLP down-regulates the expression of the type-II PLA by AM through a process mediated by G-protein. A possible negative control of the type-II PLA expression during AM activation is suggested.


INTRODUCTION

Phospholipases A (PLA,()phosphatide 2-acylhydrolase, EC 3.1.1.4) are widely distributed enzymes (1) abundant in pancreatic juice and in the venoms of snakes and bees, in which they serve digestive functions. They are present in trace amounts in mammalian cells and are involved in the turnover and remodeling of membrane phospholipids. These enzymes catalyze the hydrolysis of ester bonds at the sn-2 position of membrane phospholipids and play a key role in the production of arachidonic acid (AA) and platelet-activating factor(2) . Mammalian PLAs are generally divided into two major groups, the secretory or low molecular mass (14-18 kDa) forms, termed sPLA and the cytosolic or high molecular mass (85 kDa) form, termed cPLA. The sPLA are divided into two different types: the pancreatic (type-I) PLA and the non-pancreatic (type-II) PLA(3, 4) . Molecular cloning and expression of the type-II PLA has been reported in various mammalian cells and tissues(3, 4) . Previous studies have suggested that PLAs may play an important role in acute lung inflammation (5) , but the type and cell origin of the PLA involved in this process have not been established. AM, the first line of defense against infectious agents and toxic particles in the airways, are suitably positioned to participate in allergic and inflammatory reactions in the lung. In vivo, these cells are source and target for a variety of inflammatory mediators produced during lung inflammation.

We have shown previously that cultured guinea pig AM synthesize and release a PLA with characteristics similar to those of known sPLA(6) . Here, we report the molecular cloning and expression of this enzyme in AM and its regulation by the inflammatory peptide fMLP. The latter is known to stimulate bronchoconstriction and to trigger rapid release of AA and metabolites from guinea pig AM both in vivo and in vitro(7) . We show that fMLP down-regulates the expression of this enzyme through a G-protein-mediated process. This effect is due to a decrease of sPLA mRNA level and was not accompanied by an alteration of AA release.


EXPERIMENTAL PROCEDURES

Materials

Male Hartley guinea pigs were obtained from Elevages Saint Antoine (Pleudaniel, France). RPMI 1640 culture medium, fetal calf serum (FCS), and HBSS without Ca and Mg were from Life Technologies, Inc. Hepes, fatty acid-free bovine serum albumin (BSA), leupeptin, aprotinin, L-glutamine, 2-mercaptoethanol, fMLP, and phenylmethylsulfonyl fluoride were from Sigma. Sodium pentobarbital was from Sanofi Laboratories (Montpellier, France). LPS Escherichia coli 055:B5 was from Difco Laboratories. Fluorescent phospholipid (1-palmitoyl-2-(10-pyrenedecanoyl)-sn-glyceromonomethylphosphatidic acid, PA) was from Interchim (Montluon, France). [H]Arachidonic acid ([H]AA, 80-135 Ci/mmol), nylon membranes and DNA labeling kit (random priming) were from Amersham Corp. Products for staining cytocentrifuge smears (modified May-Grünwald-Giemsa) were from Diff-Quik (Duedingen, Switzerland). [P]dCTP was from ICN Biochemicals France (Orsay, France).

Methods

Cell Isolation and Incubation Procedures

Alveolar Macrophages

Male Hartley guinea pigs weighting 600-1000 g were anesthetized by the intravenous injection of sodium pentobarbital (20 mg/kg). Twenty successive bronchoalveolar lavages were performed sterilly with 5-ml aliquots of saline, containing 25 µg/ml streptomycin and 25 units/ml penicillin, which were injected with a plastic syringe through a polyethylene cannula inserted into the trachea. The cell suspensions were centrifuged at 475 g for 10 min at 25 °C, and the pellets were washed twice with saline containing 25 µg/ml streptomycin and 25 units/ml penicillin. The washed cell pellets were resuspended in RPMI 1640 culture medium containing 50 µg/ml streptomycin, 50 units/ml penicillin, 2 mML-glutamine, 10 mM Hepes, 0.4% BSA (w/v), and 10% FCS (v/v), pH 7.2, and adjusted at 3 10 cells/ml. Differential counts were made on modified May-Grünwald-Giemsa-stained cytocentrifuge smears. The composition of the major cell types in the bronchoalveolar lavages fluids comprised 85.7 ± 6.3% alveolar macrophages, 8.6 ± 2.3% eosinophils, and 5.7 ± 3.4% lymphocytes.

The cells were adjusted at 3 10 cells/ml and allowed to adhere in 35-mm culture dishes during 1 h at 37 °C in 5% CO, 95% air. At this step, the cell population of adherent cells consisted of 95-99% macrophages after the first hour of adhesion. The plates were then washed three times with medium (37 °C) and incubated with RPMI 1640 containing 3% FCS. AM were incubated for different time periods with or without fMLP (1 µM, otherwice stated). In certain experiments, AM were first allowed to adhere for 16 h and then incubated with fMLP. When appropriatly, PTX was incubated with AM for 3 h in the presence of 10% FCS. The cells were washed twice and reincubated with PTX in the presence or in the absence of 1 µM fMLP for 16 h.

Peritoneal Macrophages

Peritoneal lavages were performed sterilly with 5-ml aliquots of saline containing 20 units/ml heparin used to prevent cell aggregation. The cell suspensions were centrifuged at 475 g for 10 min at 25 °C, and the pellets were washed twice with saline containing 25 µg/ml streptomycin and 25 units/ml penicillin. After this step, PM were isolated by adhesion using the same procedure as for AM.

Peripheral Blood Monocytes

Twenty milliliters of arterial blood were collected in a tube containing 2 ml of acid citrate dextrose as an anticoagulant (2.5 mg of citrate trisodium + 1.4 g of citric acid + 2 g of glucose/ml). Blood was mixed in sterile conditions with 4 ml of 0.9% NaCl supplemented with 6% dextran (molecular weight 295,000; B-512, Sigma) and allowed to sediment for 30 min at room temperature. The leukocyte-rich plasma was aspirated and mixed with an equal volume of RPMI and carefully layered over 3 ml of Ficoll-Paque (Pharmacia) in 15-ml plastic siliconized tubes and centrifuged at 475 g for 40 min at room temperature. The white cell-rich fraction, which settled at the Ficoll-Paque surface, was removed and resuspended in RPMI medium containing 10% FCS. The cells were adjusted to 4 10 cells/ml and allowed to adhere for 1 h in the presence of 5 units/ml heparin to avoid cell aggregation. The plates were then washed three times with medium and incubated for 16 h with RPMI in the presence of 10% FCS in 35-mm culture dishes.

Cloning of Guinea Pig Type-II PLAby RACE-RT-PCR

Total RNA was prepared after 16-h adherence of AM by using the Chomczynski and Sacchi method(8) . Then, 5` and 3` RACE-RT-PCR (rapid amplification of cDNA ends-reverse transcription-polymerase chain reaction) techniques were used to generate the full-length guinea pig type-II PLA cDNA from alveolar macrophages as described by Frohman(9) . First strand cDNA was synthesized from AM total RNA with an adaptor-d(T) primer 5`-AACCCGGCTCGAGCGGCCGC(T) (primer 1) and Superscript RT as described by the manufacturer (Life Technologies, Paisley, Scotland). PCR amplifications were in 100 µl of buffer containing 1 mM MgCl, 200 µM dNTPs, 10 mM Tris-HCl, pH 8.3, and 3 units of Taq polymerase (Perkin-Elmer). The primers used to clone the 3` extremity were primer 1 and human type-II PLA-specific primer, 5`-CGTCTGGAGAAACGTGGATGT. PCR amplifications were done using 40 cycles at 94 °C for 15 s, 43 °C for 30 s, 72 °C for 30 s. To clone the 5` extremity, single-strand cDNA was synthesized with primer 1 as above. After RNA hydrolysis by RNaseH, a dC tail was added with deoxynucleotidyl terminal transferase. The tailed cDNA was amplified by PCR with oligodeoxynucleotides 5`-GCGCCCAGTGTGCTGGCTGCA(G) (primer 2) and guinea pig type-II PLA-specific primer cDNA 5`-TGACAGGAGTTCTGCTTTAC, followed by two additional amplifications with primers 2 and 5`-TGCAGGTGATCGAGCTCC. PCR amplifications were done using 40 cycles at 94 °C for 15 s, 43 °C for 15 s, 72 °C for 60 s.

Full-length transcript contains 760 nucleotides and was subcloned into the EcoRV site of the pBluescript SK vector (Stratagene, La Jolla, CA).

Transient Expression of Guinea Pig Type-II PLAin COS Cells

COS-1 kidney SV40-transformed African green monkey cells (ATCC CRL 1650) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Paisley, Scotland) supplemented with 10 mM Hepes (pH 7.5) and 10% FCS. Full-length PLA cDNA was excised from the pBluescript SK vector (Stratagene, La Jolla, CA) with EcoRI and HindIII sites and subcloned into the same sites of the pcDNA 1 eucaryotic expression vector (In Vitrogen BV, Leek, The Netherlands). Approximately 8 10 cells were transfected with 10 µg of purified supercoiled plasmid DNA in 400 µl of Dulbecco's modified Eagle's medium, 10% FCS, 10 mM Hepes, 150 mM NaCl by electroporation (260 V and 960 microfarads).

Cells were removed after 48 h of incubation at 37 °C/5% CO, washed in PBS buffer, and lysed as indicated above before the measurement of PLA activity.

Analysis of PLAmRNA Levels

Cells were isolated and cultured as indicated above. Total RNA (10 µg/lane) was electrophoresed on a 1% agarose gel with the formaldehyde method (10) and then transferred onto nylon membranes. The blots were hybridized at 68 °C overnight as described by Church and Gilbert(11) , using a P-labeled (random priming) full-length guinea pig type-II PLA cDNA as a probe, and washed in 3 SSC and 5% SDS, followed by 1 SSC and 1% SDS washes. Blots were washed off and rehybridized with rat -actin cDNA at 65 °C, as internal control (1 SSC = 0.5 M NaCl, 0.015 M sodium citrate; SDS = sodium dodecyl sulfate).

Analysis of TNF mRNA Levels

AM were incubated with 10 µg/ml LPS in the presence or in the absence of 1 µM fMLP. After a 16-h incubation, total RNA was extracted and electrophoresed as indicated above. The blots were hybridized at 62 °C overnight with a P-labeled fragment of murine TNF cDNA corresponding to amino acids 83-184.

Preparation of Cell Lysates

At the end of the incubations, culture supernatants were harvested and centrifuged at 1500 g during 5 min at 4 °C to remove detached cells, and aliquots of 200 µl were stored at -20 °C until use. The culture dishes were kept in an ice bath, and adherent cells were washed and scrapped with a rubber policeman in 0.5 ml of cold HBSS containing 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 2 mM EDTA. Cells were then lysed by ultrasonication and kept at -20 °C until use.

Measurement of sPLAActivity

The measurement of sPLA activity was carried out using the fluorometric assay described by Radvanyi et al.(12) and shown to be selective for the sPLA type(6) . The fluorescent PA was dried under nitrogen and suspended in ethanol at a concentration of 0.2 mM. Vesicles were prepared by mixing the ethanol solution of the fluorescent phospholipid with a buffer solution containing 50 mM Tris-HCl, 100 mM NaCl, 1 mM EGTA (pH 7.5). After 2 min of vigourous agitation, 980 µl of substrat solution were mixed with 10 µl of 10% fatty acid-free BSA. Macrophage homogenates were maintained in an ice-cold bath throughout the experiment and aliquots (10 to 50 µl) were introduced into the cuvettes and allowed to equilibrate at 37 °C for 1 min. The reactions were then initiated with 10 µl of CaCl at a 10 mM final concentration in 4 10 mm disposable plastic cuvettes. The fluorescence measurements were performed with a Jobin et Yvon JY3D spectrofluorometer equiped with a xenon lamp and monitored using excitation and emission wavelengths of 345 and 398 nm, respectively, with a slitwidth of 4 nm.

Measurement of

AA Release

After 16 h incubation, AM were washed twice with RPMI 3% FCS to remove nonadherent cells and then incubated with 1 µCi/ml [H]AA (final concentration) for 2 h in RPMI containing 3% FCS and 0.4% BSA, at 37 °C in 5% CO, 95% air. The plates were washed three times with the medium (37 °C) to remove the unincorporated [H]AA and reincubated in serum-free medium containing 0.4% BSA. Aliquots (50 µl) were taken off from the supernatant before cell washing, to determine the extent of [H]AA incorporation. Labeled AM were stimulated with the calcium ionophore A23187 (1 µM) or its vehicle MeSO (0.1%) for different time periods (1-60 min) at 37 °C. The radioactivity in 50 µl of the supernatant was determined by liquid scintillation spectrophotom-etry, with ACS II as a scintillation liquid. Control experiments showed that the vehicle MeSO had no effect on AA release.

Control of Cell Viability

Cell viability was checked by the trypan blue dye exclusion test. Cell lysis was controlled by measuring the release of lactate dehydrogenase activity in the medium using a commercial kit from Boehringer (Mannheim, Germany).

Calculations and Statistical Analysis

Data are expressed as mean ± S.E. of separate experiments, and statistical analyses were performed using Student's t test (*, p < 0.05;, p < 0.01).


RESULTS

Cloning of the Guinea Pig Type-II PLA

The type-II PLA was cloned by RACE-RT-PCR from AM as described under ``Methods.'' The deduced amino-acid sequence of the guinea pig protein has all the mammalian type-II PLA characteristics. The mature protein consists of 125 amino acids and shows 70% sequence identity with human type-II PLA and 35% with guinea pig type-I PLA (Fig. 1).


Figure 1: Sequence of the type-II PLA from guinea pig AM: comparison with human and rat type-II PLA and guinea pig type-I PLA. The guinea pig (gp) type-II PLA sequence is compared to that of human (hum; (22) and (23) ) and rat type-II PLA(24) and guinea pig (gp; (25) ) type-I PLA. indicates cysteine residues, whose positions are conserved in all primary structures of type-II PLA.



To further characterize the PLA cloned from AM, the cDNA was transfected into COS cells as described under ``Methods.'' COS cells transfected with the PLA construct expressed > 150 times more PLA activity than cells transfected with the vector alone. The PLA activity produced by transfected COS cells had characteristics similar to those of guinea pig AM (6) and other mammalian type-II PLA(3, 4, 5) . Indeed, the enzyme hydrolyzed preferentially negatively charged phospholipids PA > PG and, at much lower rate, PC, with optimal activity being observed at pH 7-8 and 10 mM calcium.

Expression of the Type-II PLAin AM, PM, and PBM

The type-II PLA activity and mRNA were undetectable when freshly collected AM were allowed to adhere for 1 h in the presence of 3% FCS. However, both PLA activity and mRNA level increased dramatically to reach maximal values after 16 h of incubation of AM. The PLA activity remained at maximal level over 32 h incubation with a parallel secretion in the medium, in contrast to mRNA level, which returned to near basal level after 32 h of incubation (Fig. 2, a and b). The expression of the type-II PLA activity and mRNA did not require the presence of serum in the medium and was not due to contaminating endotoxin (data not shown), thus confirming our previous findings(6) .


Figure 2: Time-dependent expression of the type-II PLA in AM. AM, adjusted to 3 10 cells/ml, were allowed to adhere for 90 min, then washed, and cultured for 1, 6, 16, and 32 h. Panel a, after the indicated times, culture supernatants were harvested and centrifuged to remove detached cells. Culture dishes were washed and macrophages lysates were prepared as indicated under ``Methods.'' The measurement of PLA activity was performed using aliquots of 10-50 µg of protein from cell lysates and 10-50 µl from supernatants. The results show PLA activity in cells () and cell-free supernatants () expressed in nmol/min/mg of protein. PLA activity in supernatants were reported to cell protein content of corresponding wells. The values are the mean ± S.E. of eight separate experiments. Panel b, type-II PLA mRNA expression. Total cellular RNA (10 µg) was extracted from AM at the indicated times. Northern blots were carried using guinea pig type-II PLA cDNA as a probe. Rat -actin cDNA was used as an internal control.



We also investigated the expression of the type-II PLA in guinea pig peritoneal macrophages (PM) and peripheral blood monocytes (PBM). These cells expressed the type-II PLA at much lower levels than AM. Indeed, very low or undetectable levels of PLA activity and mRNA were observed in PM and PBM cultured for 16 h in the same conditions (Table 1, Fig. 3). The low expression of PLA activity in PM and PBM may be due to the presence of heparin (used during PM and PBM isolation; see ``Methods''), which may interfere with the PLA enzymatic assay. This is not the case since addition of heparin (1-20 units/ml) directly to the cuvettes did not interfere with the PLA fluorometric assay. Furthermore, addition of heparin (20 units/ml) to the bronchoalveolar lavage or during the first hour of adhesion of AM failed to inhibit the expression of PLA activity of AM measured after a 16-h culture (data not shown).




Figure 3: differential expression of the type-II PLA in AM, PBM, and PM. Isolated AM, PM, and PBM were adjusted at 3 10 cells/ml and cultured for 16 h, after which type-II PLA mRNA levels were analyzed as indicated under ``Methods.''



Effect of fMLP on the Expression of the Type-II PLAin AM

Since AM are a target for a variety of inflammatory stimuli in the lung, we investigated the effects of the chemoattractant peptide fMLP on the expression of the type-II PLA in AM. Incubation of AM with 1 µM fMLP led to a marked and prolonged decrease in cell-associated PLA activity (Fig. 4a). This effect was concentration-dependent with a parallel decrease in the level of PLA activity in the medium (Fig. 5). FMLP had no effect on cell viability as assessed by lactate dehydrogenase release and trypan blue exclusion test (data not shown). Northern blot analysis showed that incubation of AM with fMLP led to a marked decrease of mRNA level (Fig. 4b). In addition, preincubation of AM for 3 h with PTX prevented almost totally the inhibitory effect of fMLP on the expression of the type-II PLA in AM. By itself, PTX had no significant effect on PLA activity and mRNA level (Fig. 6, a and b).


Figure 4: Effect of fMLP on the expression of the type-II PLA in AM. AM were allowed to adhere for 90 min, washed, and incubated in the presence or in the absence of 1 µM fMLP. Panel a, at the indicated times, the plates were washed and then cell-associated PLA activity was measured in control () and fMLP-treated AM (). Panel b, after a 16-h incubation with or without fMLP, type-II PLA mRNA expression was analyzed by Northern blot in control (C) and fMLP-treated AM (F).




Figure 5: Concentration-dependent effect of fMLP on cell- and medium-associated PLA activity. AM were incubated with increasing concentrations of fMLP for 16 h, and PLA activity was measured in cells () and cell-free () supernatants. *, p < 0.05; **, p < 0.01.




Figure 6: Supression by pertussis toxin of the inhibitory effect of fMLP on type-II PLA expression in AM. AM were allowed to adhere for 3 h in the presence or in the absence of PTX (50 ng/ml). The plates were washed and reincubated with or without fMLP (1 µM) in the presence or in the absence of PTX (50 ng/ml). After 16 h incubation, PLA activity (a) and type-II PLA2 mRNA (b) were analyzed as indicated under ``Methods.''



In the experiments cited above, fMLP was added to AM before the synthesis of PLA took place. We next examined whether fMLP interferes with the expression of PLA when added to AM after enzyme synthesis. AM were first allowed to adhere for 16 h (incubation period leading to a maximum of type-II PLA synthesis) and then incubated with fMLP for an additional period. In these conditions, no significant change in the PLA activity was observed as compared to untreated AM (data not shown). This is not due to the failure of fMLP to stimulate 16-h-old AM, since the latter released substantial amounts of AA metabolites when incubated with fMLP(6) .

Effect of fMLP on TNF Expression and AA Release

These experiments were performed to investigate whether the inhibition of the type-II PLA expression is due to a nonspecific effect of fMLP. We first examined the effect of fMLP on the LPS-induced TNF expression. Fig. 7shows that incubation of AM with 10 µg/ml LPS for 16 h led to a marked TNF mRNA accumulation. Similar levels of TNF mRNA were observed when AM were incubated with combination of fMLP and LPS (Fig. 8). By itself, fMLP induced a weakly detectable accumulation of TNF mRNA.


Figure 7: Failure of AM incubation with fMLP to interfere with the LPS-induced TNF expression. AM were incubated simultaneously with 1 µM fMLP and 10 µg/ml LPS for 16 h. Total RNA was extracted, and then TNF mRNA was analyzed by Northern blot as indicated under ``Methods.''




Figure 8: Failure of AM pretreatment with fMLP to interfere with the ionophore-induced AA release. AM were incubated with or without fMLP (1 µM) for 16 h and incubated with [H]AA (1 µCi/ml, final concentration) at 37 °C for 2 h. Then, the plates were washed three times with the medium (37 °C) to remove the unincorporated [H]AA and stimulated with 1 µM calcium ionophore A23187 () or with its vehicle MeSO (). After 30 min of stimulation, the radioactivity in 50 µl of the supernatant was determined by liquid scintillation spectrophotometry, with ACS II as a scintillation liquid. The results show the release of AA expressed in cpm 10/ml and are the mean ± S.E. of four separate experiments.



In addition, we examined whether preincubation of AM with fMLP modifies their ability to release AA when stimulated with a second stimulus, the calcium ionophore A23187. AM were cultured for 16 h, labeled with [H]AA for 2 h and then stimulated with the calcium ionophore A23187. This led to a time-dependent release of AA which plateaued after 30 min (data not shown). This release was not altered when AM were preincubated with fMLP for 16 h before stimulation with the calcium ionophore (Fig. 8). No significant difference was observed in the levels of [H]AA incorporation between untreated- and fMLP-treated AM (62.5 ± 4.5 and 55.4 ± 5.9, n = 8, respectively, expressed as percent of total added [H]AA).


DISCUSSION

AM are cells of the mononuclear phagocyte system suitably positioned to participate in allergic and inflammatory reactions in the lung. They are a source and a target of numerous inflammatory mediators, such as cytokines and AA derivatives. The release of AA in activated cells is mainly catalyzed by PLA, although the type of PLA involved in this process varies with cell type and animal species. We have shown previously that guinea pig AM produce high level of a sPLA during in vitro incubation, although this enzyme was undetectable in freshly collected cells(6) . We suggested that adherence of AM to plastic dishes and/or the removal of inhibitory factors present in the lung may be involved in the induction of sPLA synthesis by isolated AM. Indeed, recent studies from our laboratory suggest that pulmonary surfactant may be one of these inhibitory factors.()In the present study, we have cloned this enzyme from AM and show that it has structural features similar to those of mammalian type-II PLA. The type-II PLA mRNA was undetectable in freshly collected AM, but its level increased progressively in parallel to PLA activity. Furthermore, the increase of PLA activity was prevented by actinomycin D or cycloheximide, suggesting a de novo synthesis of mRNA and protein(6) . Taken together, these results suggest that (i) in vitro incubation of AM induces type-II PLA gene expression and (ii) there is a direct correlation between mRNA level and PLA activity during the first 16 h of culture. However, the mRNA level reached its maximal level within 16 h and then returned to near basal values, in contrast to PLA activity which remained constant over 32 h of incubation. This suggests that (i) the enzyme is stored in stable form probably in granules as reported previously for other cell types (for review see (13) ) and (ii) there is active translational and/or post-translational regulations of the this enzyme in AM in parallel to the regulation of its mRNA expression.

Since AM derive from the differentiation of blood monocytes, we investigated whether other mononuclear phagocyte cells are able to produce the type-II PLA when cultured in the same conditions as AM. Our results show that this enzyme is produced at much lower levels in PBM and PM, suggesting that this process is linked to cell differentiation and associated specifically with airway macrophages.

In a subsequent step, we investigated the regulation of the type-II PLA expression in fMLP-stimulated AM. The assumption was that an inflammatory stimulus such as fMLP would increase the activation and/or secretion of sPLA, as described for cytosolic PLA products. Indeed, fMLP is known to induce a rapid release of AA by macrophages from different animal species, including guinea pig AM(7) . However, and most surprisingly, incubation of AM with fMLP led to an almost total repression of synthesis and secretion of the type-II PLA. The inhibitory effect of fMLP involved a G-protein-mediated process, since it was prevented when AM were pretreated with PTX before the addition of fMLP. Moreover, this inhibition occurred at mRNA level expression, since it was accompanied by a dramatic decrease of the type-II PLA mRNA accumulation. It is unlikely that incubation of AM with fMLP led to proteolytic degradation of the PLA protein, since no alteration was observed in the PLA activity when AM were first cultured for 16 h (to allow maximal accumulation of the enzyme) and then incubated with fMLP for additional periods. However, further investigations are required to examine whether PLA undergoes post-translational modifications in fMLP-stimulated AM. On the other hand, our results clearly show that the fMLP-induced inhibition of the type-II PLA expression is not due to a nonspecific effect of fMLP. Indeed, fMLP failed to interfere with LPS-induced TNF expression or with the release of AA induced by the subsequent addition of the calcium ionophore A23187. This also indicates that inhibition of the type-II PLA synthesis is not accompanied by an alteration of the ability of AM to release AA, suggesting that this enzyme may not mediate AA release induced by the calcium ionophore A23187. However, the role of this enzyme in the release of AA is still controversial and seems to be dependent on cell types and animal species(14, 15, 16, 17, 18, 19, 20) .

In summary, the present studies show, for the first time, cloning and expression of the type-II PLA in a cell involved in lung inflammation. The pathophysiological relevance of these studies is related to the use of guinea pig as an experimental model for human allergic disease, particularly immediate bronchoconstriction and delayed bronchopulmonary hyperresponsiveness(21) . Moreover, there is compelling evidence for a central role of sPLA in the pathogenesis of acute lung injury(4, 5) . We also report that the expression of sPLA in AM is down-regulated by an inflammatory stimulus, suggesting a possible negative control of the expression of this enzyme during AM activation. This down-regulation involves a G protein-mediated process. The mechanisms and intracellular messengers linking G-protein activation and sPLA2 down-regulation are under investigation.


FOOTNOTES

*
This work was supported by a grant from the Association Franaise de Lutte contre la Mucoviscidose. 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.

§
To whom correspondence and reprint requests should be addressed. Tel.: 40-61-31-30; Fax: 45-68-87-03; touqui{at}pasteur.fr

The abbreviations used are: PLA, phospholipase A; AA, arachidonic acid; AM, alveolar macrophages; FCS, fetal calf serum; fMLP, formyl-methionyl-leucyl-phenylalanine; LPS, lipopolysaccharide; PBM, peripheral blood monocytes; PM, peritoneal macrophages; PTX, pertussis toxin; sPLA, secretory PLA; TNF, tumor necrosis factor; BSA, bovine serum albumin; PA, phosphatidic acid; PCR, polymerase chain reaction; RACE-RT-PCR, rapid amplification of cDNA ends-reverse transcriptase-PCR.

L. Touqui, R. Hidi, Z. Lahona, and B. B. Vargaftig, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Prof. F. Rougeon for allowing us to perform molecular biology experiments in his laboratory. Murine TNF cDNA was a kind gift of Dr Jean-Hervé Colle. We gratefully thank Dr. Nolle Doyen and Martine Fanton d'Andon for help and advice for COS cell transfection. We thank Dr. Isabelle Rosinski-Chupin for providing us with the rat -actin probe. We also acknowledge Dr. Brid Laoide and Dr. Catherine Rougeot for critical reading and comments of the manuscript.


REFERENCES
  1. Van den Bosch, H., (1980)Biochim. Biophys. Acta 604, 191-246 [Medline] [Order article via Infotrieve]
  2. Braquet, P., Touqui, L., Shen, T. Y., and Vargaftig, B. B.(1987)Pharmacol. Rev. 39, 97-120 [Medline] [Order article via Infotrieve]
  3. Dennis, E. A. (1989)J. Biol. Chem. 269, 13057-13060 [Free Full Text]
  4. Glaser, K. M., Mobilio, D., Chang, J. Y., and Senko, N.(1993)Trends Pharmacol. Sci. 14, 92-98 [CrossRef][Medline] [Order article via Infotrieve]
  5. Vadas, P., Browning, J., Edelson, J., and Pruzanski, W.(1993)J. Lipid Med. 8, 1-30
  6. Hidi, R., Vargaftig, B. B., and Touqui, L.(1989)J. Immunol. 151, 5613-5623 [Abstract/Free Full Text]
  7. Boukili, A. M., Bureau, M. F., Lellouch-Tubiana, A., Lefort, J., Simon, M. T., and Vargaftig, B. B.(1989)Br. J. Pharmacol. 98, 61-70 [Abstract]
  8. Chomczynski, P., and Sacchi, N.(1987)Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  9. Frohman, M. A., Dush, M. K., and Martin, G. R.(1988)Proc. Natl. Acad. Sci. U. S. A. 85, 8998-9002 [Abstract]
  10. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  11. Church, G. M., and Gilbert, W.(1984)Proc. Natl Acad. Sci.U. S. A. 81, 1991-1995 [Abstract]
  12. Radvanyi, F., Jordan, L., Russo-Marie, F., and Bon, C.(1989)Anal. Biochem. 177, 103-109 [Medline] [Order article via Infotrieve]
  13. Mayer, R. J., and Marshall, L. A.(1993)FASEB J. 7, 339-348 [Abstract/Free Full Text]
  14. Mounier, C., Faili, A., Vargaftig, B. B., Bon, C., and Hatmi, M.(1993)Eur. J. Biochem. 216, 169-175 [Abstract]
  15. Barbour, S. E., and Dennis, E. A.(1993)J. Biol. Chem. 268, 21875-21882 [Abstract/Free Full Text]
  16. Murakami, M., Kudo, I., and Inoue, K.(1993)J. Biol. Chem. 268, 839-844 [Abstract/Free Full Text]
  17. Pfeilschifter, J., Schalkwijk, C., Briner, V. A., and van den Bosch, H.(1993) J. Clin. Invest. 92, 2516-2523 [Medline] [Order article via Infotrieve]
  18. Mounier, C., Vargaftig, B. B., Franken, P. A., Verheij, H., Bon, C., and Touqui, L. (1994)Biochim. Biophys. Acta 1214, 88-96 [Medline] [Order article via Infotrieve]
  19. Kishino, J., Ohara, O., Nomura, K., Kramer, R., and Arita, H.(1994)J. Biol. Chem. 269, 5092-5098 [Abstract/Free Full Text]
  20. Roshak, A., Sathe, G., and Marshall, L. A.(1994)J. Biol. Chem. 269, 25999-26005 [Abstract/Free Full Text]
  21. Pretolani, M., and Vargaftig, B. B.(1993)Biochem. Pharmacol. 45, 791-800 [CrossRef][Medline] [Order article via Infotrieve]
  22. Kramer, R. M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow, E. P., Tizard, R., and Pepinski, R. B.(1989)J. Biol. Chem. 264, 5768-5775 [Abstract/Free Full Text]
  23. Seilhamer, J., Pruzanski, W., Vadas, P., Plant, S., Miller, J. A., Kloss, J., and Johnson, L. K.(1989)J. Biol. Chem. 264, 5335-5338 [Abstract/Free Full Text]
  24. Komada, M., Kudo, I., Mizushima, H., Kitamura, N., and Inoue, K.(1989)J. Biochem.(Tokyo)106,545-547 [Abstract]
  25. Ying, Z., Tojo, H., Nonaka, Y., and Okamoto, M.(1993)Eur. J. Biochem. 215, 91-97 [Abstract]

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