(Received for publication, April 18, 1995; and in revised form, May 17, 1995)
From the
We have shown previously that guinea pig alveolar macrophages
(AM) synthesize a secretory phospholipase A
Phospholipases A
We have shown previously that cultured
guinea pig AM synthesize and release a PLA
The cells were adjusted at 3
Full-length transcript contains 760 nucleotides and was
subcloned into the EcoRV site of the pBluescript
SK
Cells were removed after 48 h of incubation at 37
°C/5% CO
After 16 h incubation, AM were washed twice with RPMI 3% FCS to
remove nonadherent cells and then incubated with 1 µCi/ml
[
Figure 1:
Sequence of the type-II
PLA
Figure 2:
Time-dependent expression of the type-II
PLA
Figure 3:
differential expression of the type-II
PLA
Figure 4:
Effect of fMLP on the expression of the
type-II PLA
Figure 5:
Concentration-dependent effect of fMLP on
cell- and medium-associated PLA
Figure 6:
Supression by pertussis toxin of the
inhibitory effect of fMLP on type-II PLA
Figure 7:
Failure of AM incubation with fMLP to
interfere with the LPS-induced TNF
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
[
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
Since AM derive from the differentiation of blood
monocytes, we investigated whether other mononuclear phagocyte cells
are able to produce the type-II PLA
In a subsequent step, we investigated the regulation of
the type-II PLA
In summary, the present studies show, for the first time, cloning
and expression of the type-II PLA
We thank Prof. F. Rougeon for allowing us to perform
molecular biology experiments in his laboratory. Murine TNF
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(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.
(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 PLA
s 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
PLA
s 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.
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.
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.
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 PLA
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 PLAby RACE-RT-PCR
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.
vector (Stratagene, La Jolla, CA).
Transient Expression of Guinea Pig Type-II
PLA
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 PLAin COS Cells
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).
, washed in PBS buffer, and lysed as indicated
above before the measurement of PLA
activity.
Analysis of PLA
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 mRNA Levels
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
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 mRNA Levels
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 sPLA
The measurement of sPLAActivity
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
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 Me
SO (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
Me
SO 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).
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).
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 PLA
The type-II PLAin AM, PM,
and PBM
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) .
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).
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 PLA
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 PLAin AM
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).
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).
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.
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
These experiments were performed to investigate whether
the inhibition of the type-II PLA Expression and AA
Release
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.
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.''
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
Me
SO (
). 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).
, 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.
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.
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 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.
, 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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.