(Received for publication, April 17, 1997, and in revised form, May 22, 1997)
From the IgE-dependent and -independent
activation of mouse bone marrow-derived mast cells (BMMC) elicited
rapid and transient production of platelet-activating factor (PAF),
which reached a maximal level by 2-5 min and was then degraded
rapidly, returning to base-line levels by 10-20 min. Inactivation of
PAF was preceded by the release of PAF acetylhydrolase (PAF-AH)
activity, which reached a plateau by 3-5 min and paralleled the
release of Mast cells are highly specialized effector cells of the immune
system which, when activated, release various biologically active
molecules including histamine, proteoglycans, and proteases through
exocytosis, arachidonic acid-derived mediators such as eicosanoids
through activation of the cyclooxygenase and 5-lipoxygenase pathways,
and preformed and newly expressed cytokines (1). IgE-dependent activation of mast cells has also been
reported to elicit production of platelet-activating factor
(PAF),1 a lipid mediator with a
glycerophosphocholine backbone (2). PAF has been implicated in a number
of physiological and pathological processes, particularly allergy and
inflammation, affecting the respiratory, vascular, digestive, and
reproductive systems (3, 4). Its accumulation is usually tightly
regulated at the biosynthetic and degradative levels to avoid the
inappropriately high accumulation observed in many diseases. PAF
production often parallels immediate eicosanoid generation (5). In the
remodeling pathway for PAF biosynthesis proposed to occur in
inflammatory cells, Ca2+-dependent
phospholipase A2 may play a role in the production of both
lysoPAF and arachidonic acid from
1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine, then
acetyltransferase is thought to introduce an acetyl residue from
acetyl-CoA to the sn-2 position of lysoPAF leading to the formation of biologically active PAF (3-5). In turn, PAF is degraded and inactivated by PAF acetylhydrolase (PAF-AH) enzymes, a particular group of Ca2+-independent phospholipase A2s
that remove the acetyl moiety at the sn-2 position (6).
Whereas the molecular mechanisms of arachidonic acid metabolism which
lead to eicosanoid generation in mast cells have been well studied
(7-15), those of PAF metabolism remain largely unknown.
Recent studies have revealed that mammalian PAF-AHs can be classified
into intracellular and extracellular types (6). Intracellular PAF-AH
type I (PAF-AH-I) is a heterotrimer complex composed of 45-, 30-, and
29-kDa subunits (16). The 45-kDa subunit, which is not essential for
catalytic activity, exhibits striking homology (99%) with a protein
encoded by the causal gene (LIS-1) for Miller-Dieker lissencephaly, a human brain malformation manifested by a smooth cerebral surface and abnormal neural migration (17). The 30- and 29-kDa
subunits, which are highly homologous with each other, belong to a
novel type of serine proteases, and a sequence of ~30 amino acids
adjacent to their active serine residues exhibits significant
similarity to the first transmembrane region of the PAF receptor (18,
19). PAF-AH type II (PAF-AH-II) is a monomeric 40-kDa protein that
exhibits broader substrate specificity than PAF-AH-I in that PAF-AH-II
hydrolyzes oxidized phospholipids as effectively as PAF, whereas
PAF-AH-I is more specific for PAF (20, 21).
A secreted form of PAF-AH, which is abundantly present in plasma as a
lipoprotein-associated form (22), is believed to regulate base-line
circulating PAF levels and may be critical in resolving inflammation.
The cDNA for this enzyme encodes a 44-kDa secretory protein that
contains a typical signal sequence and a serine esterase consensus
motif GXSXG (23, 24). Plasma-type PAF-AH displays significant homology (~40%) with intracellular PAF-AH-II, but not
PAF-AH-I, over the whole sequence (21, 23). In agreement with this
similarity, plasma-type PAF-AH catalyzes hydrolysis of PAF and
structurally related oxidized phospholipids (23, 24). Pretreatment of
animals with recombinant plasma-type PAF-AH has been shown to block
PAF-induced inflammation (23), revealing its anti-inflammatory
function. Interestingly, deficiency of plasma-type PAF-AH is an
autosomal recessive syndrome that is associated with severe asthma in
Japanese children (25), in which a point mutation of exon 9 of the
plasma-type PAF-AH gene results in production of inactive protein (26).
These observations, together with the finding that PAF receptor
transgenic mice are more susceptible to methacholine-induced bronchial
hypersensitivity than normal littermates (27), imply that PAF and
plasma-type PAF-AH are involved in propagating and terminating allergic
reactions, respectively.
Although circulating blood is rich in plasma-type PAF-AH, the source of
this enzyme in the extravascular space is poorly understood. Plasma-type PAF-AH activity increased dramatically in the peritoneal cavities of guinea pigs with endotoxic shock (28), raising the possibility that PAF-AH accumulating at inflamed sites is not only
exudated from plasma but is also produced by tissue cells. Macrophages
are likely to be one of the main sources of extravascular plasma-type
PAF-AH, since monocytes have been shown to produce plasma-type PAF-AH
during differentiation into macrophages (23). Here we report that mast
cells are a rich source of plasma-type PAF-AH. Plasma-type PAF-AH
released from mast cells activated by various stimuli degrades PAF
produced by these cells, revealing an anti-inflammatory property of
mast cells during allergic and non-allergic inflammation.
Mouse recombinant c-kit ligand (KL),
interleukin (IL)-3, and IL-10 were expressed by baculovirus-infected
Sf9 cells, as described previously (7-9). Mouse IL-1 Bone marrow cells from male BALB/cJ mice were cultured for
up to 10 weeks in 50% enriched medium (RPMI 1640 supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, and 0.1 mM non-essential amino acids) and 50% WEHI-3
cell-conditioned medium as a source of IL-3 (7-9). After 3 weeks,
>98% of the cells in the culture were BMMC, as assessed by staining
with toluidine blue or Alcian blue and safranin. After washing twice
with enriched medium, the cells were cultured at 1 × 106 cells/ml in enriched medium containing 5 ng/ml IL-1 CTMC
were obtained from the peritoneal cavities of Wistar rats (Nippon
Bio-Supply Center) as described previously (29). Briefly, rats (male,
weighing > 350 g) were injected intraperitoneally with 50 ml
of Hanks' balanced salt solution containing 0.1% BSA, and the
peritoneal cells were harvested. After centrifuging these cells with
Hanks' balanced salt solution containing 38% BSA, CTMC were collected
from the bottom of the tube. The purity and viability of the cells were
assessed by staining with toluidine blue and trypan blue, respectively,
and CTMC with > 95% purity and viability were used for the
subsequent studies.
In typical experiments, mast cells
at a concentration of 1 × 106 cells/ml in Tyrode's
gelatin buffer were sensitized for 30 min with 1 µg/ml IgE anti-TNP
and then activated for various periods at 37 °C using 1-100 ng/ml
TNP-BSA as an antigen (7). Alternatively, the cells were stimulated
with 200 ng/ml KL or 1 µM A23187. For rat CTMC
activation, 1 µM lysophosphatidylserine, an activation cofactor, was added together with the above stimulators (30). After
activation, PAF-AH activity was measured as
described previously (16). Briefly, a total volume of 250 µl of
sample was incubated in the standard incubation system for assaying
PAF-AH, comprising 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 20 µM
[acetyl-3H]PAF
(1-O-hexadecyl-2-[acetyl-3H]sn-glycero-3-phosphocholine;
5 µCi/nmol) (NEN Life Science Products) for appropriate periods at
37 °C. The reaction was stopped by adding 2.5 ml of
chloroform:methanol (4:1) and 0.25 ml of water. Aliquots (600 µl) of
the aqueous phase were subjected to radioactivity measurements to
determine the amount of [3H]acetic acid liberated. When
1-O-[3H]octadecyl-PAF (Amersham) was used as a
substrate, the reaction products were extracted by the method of Bligh
and Dyer (31), spotted onto thin layer chromatography plates (Merck),
and then developed with a solvent system of chloroform:methanol:acetic acid:water (50:25:8:4 v/v). Lipid spots were visualized by exposing the
plates to iodine vapor. The spots corresponding to PAF and lysoPAF were
identified by comparison with authentic PAF (Cayman Chemical) and
lysoPAF (Avanti) standard and scraped off. The radioactivities recovered were quantified.
The total lipids of cells and
supernatants, extracted by the method of Bligh and Dyer (31), were
developed on thin layer chromatography plates with a solvent system of
chloroform:methanol:acetic acid:water (50:25:8:4, v/v) and visualized
by exposing the plates to iodine vapor. PAF was then extracted from the
scraped silica gel powder by the method of Bligh and Dyer (31) and
reconstituted in Tyrode's buffer. Rabbit platelets, prelabeled with
0.25 µCi/ml [14C]serotonin (NEN Life Science Products)
for 20 min at room temperature, were resuspended in Tyrode's buffer at
5 × 108 cells/ml. A 100-µl portion of the platelet
suspension was mixed with 10 µl of extracted sample or authentic PAF
standard and incubated for 2 min at room temperature. Platelet
activation was stopped by adding 150 mM formaldehyde, and
the [14C]serotonin released into the supernatant was
quantified by BMMC or RBL-2H3
cells (5 × 106 cells) were preincubated for 10 min
with 25 µCi/ml [3H]sodium acetate (NEN Life Science
Products) and then activated for various periods with IgE/antigen or
A23187 in the continued presence of [3H]sodium acetate.
After stopping the reaction by adding 0.1% SDS, the lipids contained
in the cells and/or supernatants were extracted by the method of Bligh
and Dyer (31) and developed on thin layer chromatography plates. The
spot corresponding to PAF was identified by comparison with an
authentic PAF standard and scraped off, and the radioactivity was
measured (32).
Total
RNA of BMMC, extracted using guanidinium thiocyanate with TRIzol (Life
Technologies, Inc.) according to the manufacturer's instructions, was
mixed with the oligo(dT) primer and avian myeloblastosis virus reverse
transcriptase (TaKaRa) and incubated for 30 min at 50 °C. The
resulting cDNA was subjected to PCR using a 21-mer sense primer,
5 Equal amounts (10 µg) of total RNA were
applied to each lane of 1.2% formaldehyde-agarose gels,
electrophoresed, and transferred to Immobilon-N (Millipore). The
resulting blots were then sequentially probed with mouse plasma-type
PAF-AH (obtained by reverse transcriptase-PCR as described above),
cyclooxygenase-2, and The
supernatants and pellets of the activated BMMC (1 × 107 cells/ml) were applied to 10% SDS-polyacrylamide gels
and electrophoresed under reducing conditions. The separated proteins
were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) using a semidry blotter (MilliBlot-SDE system; Millipore)
according to the manufacturer's instructions. The membranes were then
washed once with Tris-buffered saline (TBS) (pH 7.2) containing 0.1%
Tween 20 (TBS-T) and then blocked for 1 h in TBS-T containing 3%
skimmed milk. After washing the membranes with TBS-T, antibody against
guinea pig plasma-type PAF-AH was added at a dilution of 1:1,000 in
TBS-T and incubated for 2 h. Following three washes with TBS-T,
the membranes were treated for 1 h with horseradish
peroxidase-conjugated goat anti-rabbit IgG (Zymed) (1:10,000 dilution)
in TBS-T. After six washes, the protein bands were visualized with the
aid of an ECL Western blot analysis system.
cDNA for guinea pig
PAF-AH (28) was subcloned into pCRTM3 (Invitrogen) and
transfected into CHO-K1 cells (RIKEN Cell Bank) using CellFectin (Life
Technologies, Inc.) by a method described previously (33). Three days
after transfection, the PAF-AH activity released into the supernatants
was measured.
Data were analyzed by Student's
t test. Results are expressed as means ± S.E., with
p = 0.05 as the limit of significance.
The time course of PAF production by BMMC sensitized with
IgE anti-TNP and activated with TNP-BSA as antigen is shown in Fig. 1. In this experiment, mixtures of the supernatant and
the cell fraction were subjected to lipid extraction, and the PAF
produced was purified by thin layer chromatography and quantified by a rabbit platelet aggregation assay as described under "Experimental Procedures." Significant production of PAF occurred immediately after
antigen challenge, reaching a peak of approximately 1 nmol/106 cells by 2-5 min (Fig. 1A).
Thereafter, PAF disappeared rapidly and returned to the basal level by
10 min. The de novo synthesis of PAF was also assessed by
monitoring the incorporation of [3H]acetate into PAF
(32). As in the PAF bioassay (Fig. 1A), the level of
[acetyl-3H]PAF in A23187-stimulated BMMC,
which was barely detectable before cell activation, reached a peak by 2 min and declined thereafter, almost returning to the basal level by
10-20 min (Fig. 1B). PAF produced by BMMC was exclusively
associated with the cells and was not released into the
supernatants.
The rapid
disappearance of PAF from IgE/antigen- or A23187-stimulated BMMC led us
to formulate the hypothesis that BMMC might contain a PAF-inactivating
enzyme. We found that when the supernatants of IgE/antigen-activated,
but not unstimulated, BMMC were incubated with
[acetyl-3H]PAF (Fig. 2) and
1-O-[3H]octadecyl-PAF (data not shown), there
was substantial and reproducible generation of
[3H]acetate and [3H]lysoPAF, respectively,
indicating that the supernatants of activated BMMC contained PAF-AH.
The release of PAF-AH activity from IgE/antigen-activated BMMC reached
a maximum of ~40% by 3-5 min then plateaued (Fig. 2A),
preceding the inactivation of PAF by these cells (Fig. 1A). Dose-response experiments revealed that the release of PAF-AH activity
peaked at 10-100 ng/ml antigen with an EC50 of ~1 ng/ml, whereas no appreciable release was observed without antigen challenge (Fig. 2B). The kinetics and dose dependence of PAF-AH
release showed close correlation with those of
Rat serosal CTMC also released PAF-AH (Fig. 3,
upper) as well as
When the
supernatant and remaining cell pellet of A23187-activated BMMC were
subjected to SDS-polyacrylamide gel electrophoresis/immunoblotting using antiserum against guinea pig plasma-type PAF-AH, a single protein
band with a molecular mass of ~58 kDa, which corresponds to the size
of glycosylated plasma-type PAF-AH (28), was detected (Fig.
4A). The intensity of the band visualized in
the cell pellet was three times as strong as that in the supernatant,
consistent with the distribution of PAF-AH activities in the
supernatant (~24%) and pellet (~76%) in this experiment. This
result suggests that the PAF-AH released from activated mast cells was
identical or immunochemically related to plasma-type PAF-AH.
To confirm that mast cells express plasma-type PAF-AH, reverse
transcriptase-PCR analysis was carried out using a set of primers based
upon the cDNA sequence of murine plasma-type PAF-AH (24). A single
~600-base pair fragment, consistent with the predicted size of
plasma-type PAF-AH cDNA (residues 653-1267), was specifically amplified from RNA obtained from BMMC (Fig. 4B). DNA
sequencing revealed that this PCR fragment indeed encoded the
corresponding portion of murine plasma-type PAF-AH (data not
shown).
To confirm that plasma-type PAF-AH
contributes to the rapid degradation of PAF produced by mast cells, the
effect of exogenous recombinant plasma-type PAF-AH expressed by CHO-K1
cells transfected with PAF-AH cDNA on PAF generation by activated
BMMC was examined. As shown in Fig. 1B, PAF production by
activated BMMC was markedly attenuated when recombinant plasma-type
PAF-AH was added to the medium, indicating that extracellular PAF-AH
has the capacity to inactivate BMMC-associated PAF.
When
BMMC were cultured for 2 days with KL in the continued presence of
IL-3, the expression level of plasma-type PAF-AH was similar to the
level expressed in BMMC maintained in IL-3 alone (Fig.
5A). Culture of BMMC with KL + IL-10
increased PAF-AH activity 1.6-fold, with a concomitant increase in
steady-state expression of the transcript for plasma-type PAF-AH (Fig.
5A, inset). Culture of BMMC with the cytokine
triad KL + IL-10 + IL-1
Plasma-type PAF-AH has potentially important physiological and
pathological roles because of its ability to abolish the diverse effects of PAF and oxidized phospholipids, including inflammation, shock, and thrombosis (6). Accumulating evidence suggests that decreased degradation of these biologically active lipid molecules results in pathological responses. In plasma, PAF-AH is associated with
lipoprotein particles and has been implicated in atherosclerosis, where
it functions as a scavenger of oxidized phospholipids in modified low
density lipoprotein (22). Acquired PAF-AH deficiency has been described
in patients with systemic lupus erythematosus (34) and septic shock
(35). Increased levels of PAF have been reported in children with acute
asthmatic attacks (36), and inherited plasma-type PAF-AH deficiency,
caused by a point mutation in exon 9 which leads to complete abolition
of catalytic activity (26), has been observed in the Japanese
population, especially in children with severe asthma (25). We have now
demonstrated that mast cells are a potent source of plasma-type PAF-AH.
The fact that the main effector cell of allergic inflammation, the mast
cell, has the capacity to secrete this anti-inflammatory enzyme into
the surrounding microenvironment provides new insight into the
regulation of local and systemic allergic responses and is an
unexplored anti-inflammatory aspect of mast cells.
IgE-dependent and -independent activation of BMMC elicited
immediate PAF generation (Fig. 1), which occurred in association with
the other two well characterized immediate responses,
The rapid inactivation of PAF by activated BMMC led us to the
unequivocal finding that mast cells release PAF-AH upon activation. PAF-AH release occurred in parallel with the release of
Unlike liver cells (37) and macrophages (23), which spontaneously
secrete plasma-type PAF-AH into their culture supernatants, PAF-AH
secretion from mast cells is principally regulated by the receptor-coupled signal transduction pathway coupled with
degranulation. The observations that the release percentages of PAF-AH
activity were similar to those of Limited information is currently available on the transcriptional
regulation of PAF-AH enzymes. An anti-inflammatory glucocorticoid increased PAF-AH levels in the plasma of rats (38) and increased the
secretion of PAF-AH activity from macrophage-like differentiated HL-60
cells (39). An increase in plasma-type PAF-AH transcript has been
reported during differentiation or maturation of monocytes into
macrophages (23). Here we showed that, among the cytokines tested,
IL-10, when combined with KL, significantly increased PAF-AH at the
transcript level and that the addition of IL-1 In conclusion, a detailed analysis of PAF metabolism by activated mast
cells revealed that plasma-type PAF-AH is exocytosed by these cells and
contributes to autocrine degradation of PAF. Furthermore, its
expression level in mast cells increases during the late phase of cell
activation after inflammatory stimulus. In view of the potential
anti-inflammatory properties of this enzyme, we speculate that mast
cells contribute not only to the initiation of inflammation related to
allergy by releasing a wide variety of inflammatory mediators, but also
to its termination by sequestering the PAF produced by mast cell
themselves and other effector cells at inflamed sites.
We thank Drs. J. Arm and H. Katz
(Harvard Medical School) for the IgE anti-TNP and TNP-BSA; Dr. J. Trzaskos (Merck DuPont) for the cyclooxygenase-2 cDNA
probe; and Drs. K. Inoue, H. Arai, and J. Aoki for the intracellular
PAF-AH cDNA probes.
Department of Health Chemistry,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-hexosaminidase, a marker of mast cell exocytosis.
Immunochemical and molecular biological studies revealed that the
PAF-AH released from activated mast cells was identical to the
plasma-type isoform. In support of the autocrine action of exocytosed
PAF-AH, adding exogenous recombinant plasma-type PAF-AH markedly
reduced PAF accumulation in activated BMMC. Furthermore, culture of
BMMC with a combination of c-kit ligand, interleukin-1
and interleukin-10 for > 24 h led to an increase in
plasma-type PAF-AH expression, accompanied by a reduction in
stimulus-initiated PAF production. Collectively, these results suggest
that plasma-type PAF-AH released from activated mast cells sequesters
proinflammatory PAF produced by these cells, thereby revealing an
intriguing anti-inflammatory aspect of mast cells.
Materials
was purchased
from Genzyme. Lysophosphatidylserine was purchased from Avanti. A
cDNA probe and rabbit antiserum for guinea pig plasma-type PAF-AH
were prepared as described previously (28). IgE anti-trinitrophenyl
(TNP) and TNP-conjugated bovine serum albumin (TNP-BSA) were provided
by Drs. J. P. Arm and H. Katz (Harvard Medical School, Boston). A
cDNA probe for mouse cyclooxygenase-2 was provided by J. Trzaskos,
Merck DuPont. Prostaglandin D2 radioimmunoassay kit and ECL
Western blotting kit were purchased from Amersham Corp. A23187 was
purchased from Sigma.
,
100 units/ml IL-3, 100 units/ml IL-10, or 100 ng/ml KL, either alone or
in combination, as required for the experiments.
-hexosaminidase release (8), PAF-AH release and PAF
production (see below) was assessed.
-scintillation counting. To correct any differences in
lipid extraction efficiency between samples, a trace of
[acetyl-3H]PAF was added to each sample before
extraction, and the recovery of radioactivity in the PAF fraction after
the final extraction was monitored.
-AGACAAATCTGCATCGGCAAC-3
and an antisense primer 5
-TTGGTGAGGTCGATGGCTACTC-3
, which correspond to nucleotides 653-673
and 1246-1267 of mouse plasma-type PAF-AH cDNA, respectively (GenBank/EMBL Data Bank accession number U34277) (24). Thirty amplification cycles were performed at 94 °C for 1 min, 55 °C for
1 min, and 72 °C for 2 min with exTaq polymerase
(TaKaRa), after which a major product with an estimated size of
approximately 600 base pairs was resolved in 1.5% (w/v) agarose gel,
purified with a gel extraction kit (QIAGEN), subcloned into the
pCRTM3 cloning vector (Invitrogen), sequenced using a
Taq cycle sequencing kit (TaKaRa) according to the
manufacturer's instructions, and analyzed using an automated DNA
sequencer (DSQ-1000L; Shimadzu).
-actin cDNA probes labeled with
[32P]dCTP (Amersham) using random priming kit (TaKaRa).
All hybridizations were carried out at 42 °C for 16 h in 50%
formamide, 0.75 M NaCl, 75 mM sodium citrate,
0.1% SDS, 1 mM EDTA, 10 mM sodium phosphate (pH 6.8), 5 × Denhardt's solution (Sigma), 10% dextran sulfate (Sigma), and 100 µg/ml salmon sperm DNA (Sigma). The blots were washed three times at room temperature with 150 mM NaCl, 15 mM sodium citrate, 1 mM EDTA, 0.1% SDS, and 10 mM sodium phosphate (pH 6.8), for 5 min each, followed by
two washes at 55 °C with 30 mM NaCl, 3 mM
sodium citrate, 1 mM EDTA, 0.1% SDS, and 10 mM sodium phosphate (pH 6.8) for 15 min each. The blots were visualized by
autoradiography with Kodak X-OMAT AR films and double intensifying screens at
80 °C for 2 days.
Production and Subsequent Inactivation of PAF by Activated Mast
Cells
Fig. 1.
PAF production by activated mouse BMMC.
Panel A, IgE-sensitized BMMC were activated for the
indicated period with 10 ng/ml TNP-BSA, and PAF produced was quantified
by a bioassay using rabbit platelets. A representative result from
three independent experiments is shown. Panel B, BMMC were
preincubated with [3H]acetate and then activated for the
indicated period with 1 µM A23187. The amount of
[3H]PAF accumulated within the cells (closed
circles), released into the supernatant (open circles),
and generated in cells activated in the presence of recombinant
plasma-type PAF-AH (closed squares) is shown. Values are
expressed as means ± S.E. of three independent experiments.
*p < 0.05 versus before cell
activation.
[View Larger Version of this Image (16K GIF file)]
-hexosaminidase
release (Fig. 2, A and B). When BMMC were
stimulated with another mast cell secretagogue, KL, release of PAF-AH
activity also occurred in parallel with that of
-hexosaminidase,
reaching a maximum of ~20% by 3~5 min (Fig. 2C). A23187
stimulation of BMMC also resulted in both PAF-AH and
-hexosaminidase
release, which reached 20~40% within 2 min (data not shown). This
close correlation between PAF-AH and
-hexosaminidase release
strongly suggests that PAF-AH is stored in the secretory granules of
BMMC and is exocytosed following cell activation.
Fig. 2.
Release of PAF-AH from activated mouse BMMC.
Panel A, IgE-sensitized BMMC were activated for the
indicated period with 10 ng/ml TNP-BSA. Panel B,
IgE-sensitized BMMC were activated for 10 min with various
concentrations of TNP-BSA. Panel C, BMMC were activated for
the indicated period with 200 ng/ml KL. Results are expressed as the
percentage releases (mean ± S.E.; n = 5) of
PAF-AH (top) and -hexosaminidase (bottom)
calculated by the formula (S/(S + P)) × 100, where S and P are the contents of equal portions of each supernatant and cell pellet, respectively.
*p < 0.05 versus without cell
activation.
[View Larger Version of this Image (26K GIF file)]
-hexosaminidase (Fig. 3,
lower) in response to IgE/antigen or KL in the presence of
lysophosphatidylserine added as a activation cofactor (30). This
observation implies that PAF-AH release from activated mast cells is
not limited to a particular mast cell phenotype but reflects a general
phenomenon. However, in contrast to BMMC, in which the release
percentages of PAF-AH and
-hexosaminidase were almost equal (Fig.
2), the amount of PAF-AH released from rat CTMC was about half of that
of
-hexosaminidase after each stimulation (Fig. 3).
Fig. 3.
Release of PAF-AH from activated rat serosal
CTMC. IgE-sensitized rat CTMC were activated for 10 min with 10 ng/ml TNP-BSA or 200 ng/ml KL in the presence or absence of 1 µM lysophosphatidylserine, and the amount of PAF-AH
(top) and -hexosaminidase (bottom) released into the supernatants was assessed. Values are expressed as means ± S.E. of five independent experiments. *p < 0.05 versus without cell activation (control).
[View Larger Version of this Image (26K GIF file)]
Fig. 4.
Detection of plasma-type PAF-AH in mouse
BMMC. Panel A, equal portions of the supernatant (lane
1) and cell pellet (lane 2) of A23187-activated BMMC
were subjected to immunoblot analysis using antiserum against guinea
pig plasma-type PAF-AH. Panel B, total RNA obtained from
BMMC was subjected to reverse transcriptase-PCR using mouse plasma-type
PAF-AH primers. The sample was applied to 1.2% (w/v) agarose gel and
stained with ethidium bromide (lane 2). Lane 1 shows molecular mass markers. bp, base pairs.
[View Larger Version of this Image (57K GIF file)]
, which has previously been shown to induce
several genes related to inflammatory responses in BMMC (7, 12),
increased plasma-type PAF-AH expression further, to approximately
3-fold (Fig. 5A). Expression of
-actin transcript used as
a control did not change appreciably (data not shown). The increase in
PAF-AH mRNA expression after culture with KL + IL-10 + IL-1
became evident 1 day after the start of culture, delayed compared with
the induction of proinflammatory proteins such as cyclooxygenase-2,
which reached a peak at 2 h and disappeared by 10 h (Fig.
5B), and was accompanied by concomitant increase in PAF-AH
activity (data not shown). After rapid PAF-AH release initiated by KL
within a few minutes as shown in Fig. 2C, only a small
(<5% of the total PAF-AH activity in the cells) amount of PAF-AH was
gradually released into the culture medium during 24-48 h of culture
with KL + IL-10 + IL-1
(data not shown). These KL + IL-10 + IL-1
-treated BMMC released severalfold more PAF-AH activity (6~10
nmol/min/106 cells) than the cells maintained in IL-3
(2~3 nmol/min/106 cells) following 10-min stimulation
with 1 µM A23187. In accordance with increased PAF-AH
expression and secretion, A23187-stimulated accumulation of PAF in
BMMC, which had been cultured for 2 days with KL + IL-10 + IL-1
, was
significantly less than that in replicate cells maintained in IL-3
(Fig. 5C).
Fig. 5.
Effects of various cytokines on the
expression of PAF-AH in mouse BMMC. Panel A, BMMC were
cultured for 2 days with the indicated cytokines, and PAF-AH activities
in the cell lysates were measured. Inset, total RNAs
obtained from these cytokine-treated BMMC were subjected to RNA
blotting with mouse plasma-type PAF-AH cDNA obtained by reverse
transcriptase-PCR as shown in Fig. 4B. Panel B,
time course of changes in cyclooxygenase-2 (COX-2) and plasma-type PAF-AH transcripts in BMMC after culture with KL + IL-10 + IL-1. Panel C, BMMC cultured for 2 days with KL + IL-10 + IL-1
(open circles) or maintained in IL-3 (closed
circles) were activated for the indicated period with 1 µM A23187, and PAF accumulation in these cells was
assessed. A representative result from four independent experiments
(panels A and B) and means ± S.E. of three
independent experiments (panel C) are shown. *p < 0.05 versus before cell
activation.
[View Larger Version of this Image (18K GIF file)]
-hexosaminidase exocytosis and eicosanoid generation (8, 9).
Temporal and spatial differences exist in the evolution of PAF and
eicosanoid production; the accumulation of PAF was transient,
disappearing by 10 min after stimulation (Fig. 1), and remained
cell-associated (Fig. 1B), whereas prostaglandin
D2 generation reaches a plateau lasting for several hours
and is released predominantly into the supernatant (8, 9). The
cell-associated property of PAF has been commonly observed in a wide
variety of cell types (6, 32). The amount of PAF produced by activated
BMMC reached nearly 1 nmol/106 cells, which is comparable
to that produced by endothelial cells (32), and is therefore likely to
be biologically significant. In the early 1980s, it was shown that PAF
was released from activated BMMC into the supernatant, but the amount
was about 500 times less than that observed in the present study (2).
The rather low level of PAF detected in this earlier study might have
been due to failure to extract most of the cell-associated PAF,
since absolute ethanol, which is not an efficient extractor of
phospholipids, was used to extract cell-associated PAF (2).
Alternatively, the different conditions under which BMMC were cultured
might have affected their capacity to produce PAF.
-hexosaminidase with identical time course, dose, and stimulus
specificity criteria (Figs. 2 and 3), implying that PAF-AH is stored in
mast cell secretory granules. Immunochemical and molecular biological
studies revealed that this secretory PAF-AH is identical to the
plasma-type enzyme (Fig. 4). The involvement of plasma-type PAF-AH in
degrading mast cell-associated PAF is supported by the following three
lines of evidence. First, PAF-AH release, which reached a peak by 2 min
(Fig. 2), preceded the degradation of PAF, which became evident after 5 min (Fig. 1). Second, adding recombinant plasma-type PAF-AH markedly
reduced the accumulation of PAF in BMMC (Fig. 1B). Third, increased expression of plasma-type PAF-AH in BMMC after culture with
KL + IL-10 + IL-1
led to decreased A23187-induced PAF production (Fig. 5), even though the expression of cytosolic and secretory phospholipase A2s, which are implicated in PAF
biosynthesis, increases in BMMC after such cytokine treatment (8, 12).
Nonetheless, the ability of extracellular PAF-AH to quench
cell-associated PAF suggests that the PAF produced by BMMC is located
predominantly in the plasma membranes of the activated cells.
-hexosaminidase in BMMC (Fig. 2),
that the distribution of immunoreactive PAF-AH protein in the
supernatants and pellets of activated BMMC correlated with that of
PAF-AH activity (Fig. 4A), and that the changes in PAF-AH
activity and PAF-AH mRNA levels after cytokine treatment occurred
in parallel (Fig. 5A) suggest that the plasma-type isozyme
is the dominant PAF-AH isoform expressed in BMMC. This conclusion is
further supported by our recent preliminary study that the transcripts
for the intracellular PAF-AH were barely detectable in BMMC even after
culture with KL + IL-10 + IL-1
under the experimental conditions
shown in Fig. 5.2 In contrast, the fact
that the release percentages of PAF-AH activity were consistently about
half of those of
-hexosaminidase in rat CTMC (Fig. 3) may reflect
either granule heterogeneity or the presence of other PAF-AH isozymes
in this mast cell phenotype.
to KL + IL-10
increased its expression further (Fig. 5A). It is notable
that the cytokine triad, KL + IL-10 + IL-1
, represents a potent
inflammatory stimulus for BMMC, inducing proinflammatory proteins such
as secretory type II phospholipase A2 (12),
cyclooxygenase-2 (7), IL-1
(40), and IL-6 (41). However, the
induction of these proinflammatory proteins occurred within 2-10 h
after the initiation of culture, whereas PAF-AH induction, which
occurred after 1 day of culture (Fig. 5B), lags behind the
induction of proinflammatory mediators. This relatively late induction
of plasma-type PAF-AH, a presumptive anti-inflammatory enzyme, might
reflect a signal for the termination of inflammatory reactions that
mast cells initiate.
*
This work was supported by grants-in-aid for scientific
research from the Ministry of Education, Science, and Culture of Japan, and by a grant from the Human Science Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-3-3784-8196; Fax: 81-3-3784-8245; E-mail:
kudo{at}pharm.showa-u.ac.jp.
1
The abbreviations used are: PAF,
platelet-activating factor; PAF-AH, PAF acetylhydrolase; KL,
c-kit ligand; IL, interleukin; TNP, trinitrophenyl; BSA,
bovine serum albumin; TNP-BSA, TNP-conjugated bovine serum albumin;
BMMC, bone marrow-derived mast cells; CTMC, connective tissue mast
cells; PCR, polymerase chain reaction; TBS, Tris-buffered saline.
2
K. Nakajima, M. Murakami, and I. Kudo,
unpublished observation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.