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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 3037-3043
Human Eosinophils Express, Relative to Other Circulating
Leukocytes, Large Amounts of Secretory 14-kD
Phospholipase A2
By
M. Blom,
A.T.J. Tool,
P.C. Wever,
G.J. Wolbink,
M.C. Brouwer,
J. Calafat,
A. Egesten,
E.F. Knol,
C.E. Hack,
D. Roos, and
A.J. Verhoeven
From the Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service and Laboratory of Experimental and Clinical
Immunology, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands; the Clinical and Laboratory Immunology
Unit, Department of Internal Medicine, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands; the Netherlands
Cancer Institute, Amsterdam, The Netherlands; and the Division of
Hematology, Department of Medicine, Lund Hospital, Lund, Sweden.
 |
ABSTRACT |
Human eosinophils perform several functions dependent on
phospholipase A2 (PLA2) activity, most notably
the synthesis of platelet-activating factor (PAF) and leukotriene
C4 (LTC4). Several forms of PLA2 have been identified in mammalian cells. In the present study, the
14-kD, secretory form of PLA2 was detected in human
eosinophils by immunocytochemical staining with the specific monoclonal
antibody (MoAb) 4A1. In contrast, preparations of neutrophils,
monocytes, lymphocytes, and basophils did not show detectable staining.
With two MoAbs in a sandwich enzyme-linked immunosorbent assay (ELISA), large amounts of sPLA2 were detected in lysates of
eosinophils, that were 20-fold to 100-fold higher than in the other
circulating leukocytes (ie, neutrophils, basophils, monocytes, and
lymphocytes). In addition, with a commercially available
sPLA2 activity assay kit, we were able to show high
activity of sPLA2 in human eosinophils relative to
neutrophils. Investigations at the ultrastructural level showed that
sPLA2 in eosinophils is mainly located in specific granules. Immunoelectron microscopy also visualized sPLA2
within phagosomes after addition of opsonized particles to the
eosinophils. However, sPLA2 was not detected in the
cell-free supernatants of activated eosinophils, in contrast to
eosinophil-cationic protein (ECP), which colocalizes with
sPLA2 in resting eosinophils. These findings warrant
further studies into the role of sPLA2 in eosinophil function.
| |
INTRODUCTION |
AN IMPORTANT FEATURE of the pathogenesis
of asthma is the accumulation of eosinophils in the lungs, a phenomenon
that is correlated with destruction of lung tissue.1,2
However, until now it is not clear why eosinophils preferentially
accumulate in the lungs and what induces their degranulation and
release of the lipid mediators platelet-activating factor (PAF) and
leukotriene C4 (LTC4).3-5
Eosinophil granule proteins can be cytotoxic for respiratory epithelial
cells and pneumocytes,6,7 and PAF and LTC4 can
induce bronchoconstriction and bronchial
hyperresponsiveness.8 In a previous study, we observed that
PAF synthesis by human eosinophils after in vitro activation with STZ
(serum-treated zymosan) is completely blocked by the phospholipase
A2 (PLA2)-inhibitor mepacrine.9 Mepacrine also inhibits LTC4 secretion from eosinophils
activated with the tripeptide fMLP
(formyl-methionyl-leucyl-phenylalanine).10 Furthermore, the
release from eosinophils of preformed mediators, such as eosinophil
cationic protein (ECP) and eosinophilic peroxidase (EPO), has been
shown to be influenced by the enzymatic activity of
PLA2.10 Because PLA2 activity plays
such an important role in the release of various eosinophil products,
we undertook further studies to characterize PLA2 activity
in human eosinophils.
Recently, it has become clear that PLA2 enzymes can be
classified in several groups. In man, four proteins have been
sequenced: an intracellular or cytosolic 85-kD protein (group IV) and
three, so-called secretory, 14-kD PLA2s (belonging to group
I, II en V, respectively).11,12 Group I PLA2 is
produced by pancreatic acinar cells, functions as a digestive enzyme
within the intestinal lumen, and is therefore also referred to as
pancreatic PLA2.11 Group II PLA2
(sPLA2) can be purified from human synovial
fluid,13 platelets, and can be detected in chondrocytes and
Paneth cells.14 During septic shock and inflammation, the
concentration of sPLA2 in serum is enhanced, but the
cellular source of sPLA2 in serum is
unknown.15-18 Group V PLA2 is present in
several human tissues and in murine P388D1
macrophages19 and murine mast cells.20 With
antibodies raised against the group II PLA2, we show in
this study that human eosinophils express secretory PLA2
(sPLA2) at levels much higher than found in other
leukocytes.
| |
MATERIALS AND METHODS |
Formyl-methionyl-leucyl-phenylalanine (fMLP), cytochalasin B (cyto B),
phorbol myristate acetate (PMA), and calcium ionophore A 23187 (Sigma
Chemical Co, St Louis, MO) were dissolved in dimethyl sulfoxide (DMSO)
at 1,000 times the final concentration for cell incubations. PAF
(Sigma) was dissolved in phosphate-buffered saline (PBS) containing
human-serum albumin (HSA) (0.5% vol/vol). Interleukin-5 (IL-5) was
obtained from Amersham Corp (Amersham, Birmingham, UK). STZ was
prepared as described previously.9 Serum-opsonized Sephadex
particles (SOS) were prepared by incubating swollen Sephadex G15 beads
(150 mg) in 1 mL of human AB serum for 30 minutes at 37°C followed
by three washes in 0.9% (wt/vol) NaCl. Washed beads were resuspended
in incubation medium (see below) to a concentration of 150 mg/mL. The
monoclonal antibodies (MoAbs) 4A1 and 10B2 raised against
sPLA2 were kindly provided by Dr F.B. Taylor, Jr (Oklahoma Medical Research Foundation, Oklahoma City, OK). The polyclonal anti-sPLA2 rabbit antiserum was purchased from Cayman
Chemical Co (Ann Arbor, MI). All other chemicals were reagent grade.
Incubation medium for the cell incubations contained 132 mmol/L NaCl,
6.0 mmol/L KCl, 1.0 mmol/L CaCl2, 1.0 mmol/L
MgSO4, 1.2 mmol/L potassium phosphate, 20 mmol/L HEPES, 5.5 mmol/L glucose and 0.5% (wt/vol) human albumin, pH 7.4.
Isolation of granulocytes and neutrophils.
Blood was obtained from healthy volunteers. Granulocytes were isolated
from the buffy coat of 500 mL of blood anticoagulated with 13 mmol/L
trisodium citrate (pH 7.4) essentially as described before.21 In short, the mononuclear cells were removed by
centrifugation of the blood over isotonic Percoll (1.077 g/mL, pH 7.4).
After isotonic lysis of the erythrocytes in the pellet with an ice-cold solution containing 155 mmol/L NH4Cl, 10 mmol/L
KHCO3, and 0.1 mmol/L EDTA (pH 7.2), the granulocytes were
washed twice in PBS and suspended in incubation medium. Where
indicated, neutrophils were further purified by depletion of the
eosinophils with CD9 MoAbs and magnetic Dynabeads, coated with
sheep-antimouse antibodies. The resulting preparations always contained
less than 0.2% eosinophils.
Purification of eosinophils.
Eosinophils were purified as previously described.21 In
short, granulocytes were incubated for 30 minutes at 37°C to
restore the initial density of the cells. After this incubation period, the cells were washed and resuspended in PBS supplemented with HSA
(0.5% wt/vol) and trisodium citrate (13 mmol/L). After preincubation of the granulocytes for 5 minutes at 37°C, fMLP (10 nmol/L) was added to the cell suspension, and the incubation was continued for 10 minutes. Subsequently, the eosinophils were purified by centrifugation
(20 minutes at 800g) over isotonic Percoll (1.082 g/mL, pH
7.4), washed and resuspended in incubation medium. This suspension
contained for >98% eosinophils.
Purification of monocytes, lymphocytes, and basophils.
Monocytes and lymphocytes were isolated from the upper fraction of the
Percoll gradient (1.077 g/mL) (the mononuclear cells) by countercurrent
centrifugal elutriation as described previously by De Boer and
Roos.22 The purities of both cell fractions were > 90%.
Basophil purification was performed by depletion of residual contaminating cells (mainly lymphocytes and a few monocytes) from the
last elutriation fraction, with antibodies against these cells (CD2,
CD14, CD16, CD19) and magnetic Dynabeads as described.23 The purity of the basophils in this preparation was > 92%. The mononuclear cell fractions were kept on ice to prevent activation.
Immunocytochemical staining methods.
Human granulocytes (containing about 5% eosinophils), and purified
eosinophils, basophils, lymphocytes, and monocytes were centrifuged on
microscope slides and fixed in acetone for 10 minutes. All incubation
steps were performed at room temperature. The preparations were
preincubated with normal goat serum (1:10 diluted in PBS, containing
1% [wt/vol] bovine serum albumin [BSA]) for 15 minutes and were
subsequently incubated for 1 hour with the anti-sPLA2 MoAb
4A1 (25 µg/mL). Control preparations were incubated with PBS/BSA
without MoAb 4A1 or with irrelevant MoAb of the appropriate subclass.
After each incubation step,the preparations were washed with PBS. Next,
the preparations were incubated with 0.3% (vol/vol) H2O2, 0.1% (wt/vol) sodium azide in PBS for 20 minutes to block endogenous peroxidase activity. Subsequently, the
preparations were incubated for 30 minutes with biotinylated rabbit
antimouse-Ig F(ab)2 fragments (Dakopatts, Glostrup,
Denmark) 1:200 diluted in PBS containing 10% (vol/vol) normal human
serum (NHS). The preparations were washed and incubated for 30 minutes
with horseradish peroxidase (HRP)-labeled streptavidin-biotin(AB)
Complex (Dakopatts), diluted 1:200 in PBS-NHS. HRP activity was
detected by using 3-amino-9-ethylcarbazole (Sigma) in the presence of
0.03% (vol/vol) H2O2 yielding a red color.
Preparations were fixed in paraformaldehyde (PFA; 4% vol/vol), counterstained with Mayer's hematoxylin and mounted with
glycerolgelatin. The preparations were examined by light microscopy, to
determine the localization and intensity of staining of
sPLA2. The specificity of this procedure was confirmed by
the absence of staining of pancreas tissue preparations (described to
contain PLA2 type I14) and positive staining of
Paneth cell preparations (described to contain
sPLA2,14 results not shown).
Enzyme-linked immunosorbent assay (ELISA) for sPLA2.
Concentrations of sPLA2 in cell lysates or cell
supernatants were determined with an ELISA modified from Smith et
al.24 MoAb 10B2 and 4A1 against sPLA2 were used
as catching and detecting antibody, respectively. In some experiments,
the polyclonal anti-sPLA2 rabbit antiserum was used as
catching antibody. The amount of sPLA2 was assessed by
comparison with a (sigmoidal) calibration curve obtained with purified
recombinant human sPLA2 (kindly provided by Dr C. Schalkwijk, Centre for Biomembranes and Lipid Enzymology, University of
Utrecht, Utrecht, The Netherlands). The sensitivity of the measurements
in this assay amounted to 0.2 to 5 ng/mL. Lysis of the different cell
types (2 × 107 cells/mL) was performed for 30 minutes
at 4°C in incubation medium with the following additions: 1% NP-40
(vol/vol), phenylmethyl sulfonyl fluoride (PMSF; 100 µg/mL),
tosyl-leucyl chloromethylketone (TLCK; 1 mmol/L), leupeptin (0.1 mmol/L), soybean trypsin inhibitor (SBTI; 20 µg/mL) (Sigma), and EDTA
(5 mmol/L).
Measurement of sPLA2 activity.
Human eosinophils or neutrophils (depleted for eosinophils) were
resuspended (108 cells/mL) in PBS containing 0.34 mol/L
sucrose, 10 mmol/L HEPES, 100 µg/mL PMSF, 1 mmol/L TLCK, 0.1 mmol/L
leupeptin, and 5 mmol/L EDTA (final pH 7.0), sonicated at 21 kHz and 8 µm peak-to-peak amplitude for 3 × 15-second intervals at
4°C and stored at  80°C until use. sPLA2
ELISA measurements of these sonicates show identical amounts of
sPLA2 protein compared with lysis in 1% NP-40 as described above (data not shown). sPLA2 activity measurement was
performed with a commercially available assay system (Cayman Chemical
Company) exactly as recommended by the manufacturer. The assay uses the 1,2-dithio analog of diheptanoyl phosphatidylcholine, which serves as a
substrate for most PLA2 with the exception of group IV
cytosolic PLA2. On hydrolysis of the thio ester bound at
the sn-2 position by PLA2, free thiols are detected by
reaction with 5,5 -dithio-2-nitrobenzoate (DTNB). The
hydrolysis of DTNB was quantified by measuring the absorbance at 405 nm
at different time points and determination of the slope, using 10.0 mmol 1 · L+1 · cm1 as
the adjusted (for pathlength) molar extinction coefficient.
ELISA for ECP.
ECP in cell supernatants was measured with a slightly modified sandwich
ELISA previously described by Reimert et al.25 Specific polyclonal rabbit-antihuman ECP antibodies were obtained by
immunization of rabbits with highly purified ECP.26 Plates
were coated overnight (o/n) with rabbit-antihuman ECP (3 µg IgG/mL). After blocking with BSA (2%, wt/vol), samples and
standard (purified ECP) were incubated in various dilutions for 90 minutes at 37°C in a buffer (pH 7.5) containing 0.1 mol/L NaCl, 50 mmol/L NaH2PO4, 0.1% Tween-20, 0.1%
CTAB, 0.2% BSA and 20 mmol/L EDTA. Bound ECP was detected after incubation with biotin-conjugated rabbit-antihuman ECP by adding
a mixture of avidin and biotinylated alkaline phosphatase according to
the manufacturer's description (ABC-Complex Dakopatts). Enzymatic
activity was determined with phosphatase substrate tablets (Sigma 104, Sigma). The range of the measurements was 0.10 to 10 ng/mL.
Immunoelectron microscopy.
Eosinophils were prewarmed for 10 minutes, primed with IL-5
(10-10 mol/L) for 30 minutes, and incubated with STZ for 10 minutes. All cells were fixed for 2 hours at room temperature in a
graded (2% to 8%) PFA series in PBS to preserve the antigenicity of
sPLA2 and were pelleted in 10% gelatin in PBS. This
fixation procedure was found to be optimal for staining with antibodies
directed against sPLA2 (see below). The gelatin slab was
kept at 4°C in PBS containing 1% PFA. Small pieces of gelatin were
infiltrated with 2.3 mol/L sucrose in PBS for 4 hours, mounted on
copper pins at 4°C, and frozen in liquid nitrogen. About 80 nm
cryopreparations were made on an MT-7 ultracryomicrotome (Research and
Manufacturing Co, Tucson, AZ) and incubated at room temperature with
mouse MoAb anti-sPLA2 10B2 (dilution 1/50) followed by
incubation with rabbit antimouse (RAM)-IgG and goat-antirabbit
(GAR)-IgG conjugated to 10 nm gold (both at dilution 1/40) (Amersham).
As a control, anti-sPLA2 was replaced by a nonrelevant
murine IgG. All incubations lasted 1 hour. After immunolabeling, the
cryopreparations were embedded in a mixture of methylcellulose and
uranyl acetate. All preparations were examined with a Philips CM10
electron microscope (Eindhoven, The Netherlands).
| |
RESULTS |
Immunocytochemical staining of leukocytes with MoAbs against
sPLA2.
The MoAb 4A1 raised against recombinant human sPLA2 was
used to determine the presence of the 14-kD sPLA2 in
circulating leukocytes. As positive control for the histochemical
staining procedure, HEPG2 cells, which are known to express
sPLA2,27 were included in the experiments. The
few cells staining positive for sPLA2 in the granulocyte
fraction (Fig 1A) had the appearance of
eosinophils. Indeed, in cytospins of purified eosinophils, all cells
stained positive for sPLA2 (Fig 1B). In Fig 1B, it can be
seen that the sPLA2 present in the eosinophils is granular
distributed. The same pattern of staining was seen in the HEPG2 cells
(data not shown). The immunocytochemical preparations of neutrophils,
monocytes, lymphocytes, and basophils did not exhibit a detectable
staining (data not shown).
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| Fig 1.
Visualization of sPLA2 in (A) human
granulocytes and (B) purified eosinophils. Granulocytes (containing 5%
eosinophils) and purified eosinophils were centrifuged on microscope
slides, and sPLA2 was detected by immunohistochemical
staining with MoAb 4A1 as described in Materials and Methods.
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Measurement of the intracellular content and activity of
sPLA2.
To quantitate the level of sPLA2 in eosinophils, a
sandwich ELISA with MoAbs directed against sPLA2 was
applied. Previous studies have shown that this assay is specific for
the human sPLA2 enzyme24 and that a strong
correlation exists between PLA2 activities and
immunoreactive material in plasma samples of patients undergoing IL-2
therapy.28 Table 1 shows that
the content of sPLA2, present in the lysates of eosinophils
was 20-fold to 100-fold higher than that of the other circulating
leukocytes. Although sPLA2 was not visualized in
preparations of monocytes and neutrophils immunocytochemically stained
for sPLA2, lysates of monocytes and neutrophils (depleted for eosinophils) did contain a relative small amount of
sPLA2 (Table 1), whereas the results obtained with lysates
of purified basophils were on the borderline of the sensitivity of our
assay. The low signal in the neutrophil lysates could not be explained by the presence of contaminating eosinophils (being less than 0.2%).
In the lysates of lymphocytes, sPLA2 was not detected.
The pronounced difference between neutrophils and eosinophils was
confirmed with ELISA measurements in which the monoclonal 10B2 antibody
was replaced as catching antibody by a polyclonal anti-sPLA2 rabbit antiserum. The levels of
sPLA2 measured in this modified ELISA amounted to 1.4 ± 0.25 and 32.0 ± 5.8 (mean ± standard error of mean [SEM],
n = 5) ng sPLA2/107 cells,
respectively, for neutrophils and eosinophils. Measurement of
sPLA2 activity as described in Materials and Methods showed an activity of 14.6 ± 2.9 (mean ± SEM, n = 3)
nmol/min/108 cells in sonicates from human eosinophils and
1.6 ± 0.4 (mean ± SEM, n = 3) nmol/min/108 cells in
sonicates from neutrophils, again indicating that eosinophils contain
high amounts of sPLA2 relative to neutrophils. It should be
noted that for the sPLA2 activity measurement, we have used crude whole cell sonicates. These sonicates might have contained endogenous inhibitors of the sPLA2 enzyme.29
Immunoelectron microscopic studies on the localization of
sPLA2 in human eosinophils.
Investigation of the intracellular localization of sPLA2 in
unstimulated eosinophils was undertaken by immunoelectron microscopy (immuno-EM). Immunogold-labeled MoAb against sPLA2 was
detected in granules that contained the typical eosinophil crystals
(Fig 2). It is difficult to ascertain
whether the heterogeneity in the labelling in the granules is due to
heterogeneity in the localization of sPLA2 or to partial
destruction of the epitopes for the antibody due to the fixation
procedure used.
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| Fig 2.
Localization of sPLA2 in human eosinophils
isolated from peripheral blood. Ultrathin cryosections were incubated
with MoAb 10B2, rabbit antimouse IgG and goat antirabbit IgG conjugated to 10-nm gold particles. (A) Overview of an eosinophil showing labeling
mainly on the matrix of specific granules, although these granules do
show heterogeneity. Nucleus (n); bar = 500 nm. (B) Detail of the
cytoplasm showing dense labeling in some granules (closed arrows) and
absence of label in others (open arrows). Bar = 200 nm.
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Immunolocalization of sPLA2, after addition of STZ to
IL-5-primed eosinophils, was also studied with immuno-EM. After
binding, STZ particles were engulfed by the eosinophils with a lag time of about 2 minutes (unpublished data). The cells were fixed 10 minutes
after the addition of STZ for the preparation of sections suitable for
immuno-EM. Clearly, phagosomes had been formed and immunogold particles
depicting sPLA2 were present in these compartments (Fig 3A). Some background staining was
noted under these conditions (Fig 3B), but this staining was
considerably lower than the staining obtained with the
anti-sPLA2 antibody.
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| Fig 3.
Localization of sPLA2 in human eosinophils
after incubation of the cells with STZ for 10 minutes. (A) Ultrathin
cryosections were incubated as described in the legend to Fig 2. The
micrograph shows several STZ particles contained in a phagosome that
are strongly labeled for sPLA2. Nucleus (n); bar = 500 nm. (B) Micrograph showing some aspecific labeling of the STZ-treated
eosinophils incubated with rabbit antimouse IgG and goat antirabbit IgG
conjugated to 10-nm gold particles. Some specific granules (g) are seen
near the phagosomes. Bar = 500 nm.
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Detection of sPLA2 in the supernatant of stimulated
eosinophils.
Using the sPLA2 ELISA, we measured the sPLA2
content in supernatants collected from unstimulated eosinophils and
from eosinophils treated with PAF, fMLP, PAF-fMLP, cyto B, or cyto
B-fMLP. None of these treatments induced the secretion of
sPLA2 into the supernatant. Other stimuli, such as
Ca2+-ionophore A23187, PMA, or combinations of these
stimuli, also did not result in the release of sPLA2 in the
supernatant (data not shown).
In another set of experiments, eosinophils were stimulated with STZ
particles or serum-opsonized Sephadex (SOS). As shown in
Table 2, ECP was released, especially after
stimulation with the much larger SOS particles leading to frustrated
phagocytosis (Egesten et al, manuscript in preparation).
In agreement with the enhanced percentage of eosinophils able to bind
opsonized particles after IL-5 pretreatment,9 the release
of ECP was enhanced for both the STZ- and SOS-induced ECP release after
IL-5 pretreatment. However, the same supernatants did not contain
sPLA2 as determined by the sPLA2 ELISA (Table
2).
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Table 2.
Release of Granule Proteins From Untreated and
IL-5-Primed Eosinophils After Stimulation With Opsonized Particles
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DISCUSSION |
This study has shown that human eosinophils express high amounts of the
secretory 14-kD sPLA2, whereas neutrophils and monocytes express only a small amount. Our data indicate that in future studies
on the role of secretory 14-kD sPLA2 in monocytes or
neutrophils, contamination by eosinophils has to be excluded because of
the very high amounts of this enzyme in eosinophils. In lymphocytes, sPLA2 was not detectable by ELISA or by immunocytochemical
staining of cytospin preparations. In basophils, a small amount could
be present, the ELISA signal being on the borderline of our assay. A
preliminary report has appeared describing a possible role of secretory
PLA2 in LTC4 synthesis by human
basophils.30
Because we used for this study antibodies raised against the synovial
type of sPLA2 (belonging to group II of PLA2s),
it is most likely that the enzyme detected by us in human eosinophils is identical to this protein. However, it cannot be completely excluded
that another member of the sPLA2 gene family is highly expressed in eosinophils, eg, the recently cloned group V enzyme. Interestingly, this latter protein has recently been shown in murine
macrophages19 and mast cells20 to play an
important role in arachidonic acid release, a role previously assigned
to the group II PLA2 enzyme.
Although eosinophil degranulation and synthesis of PAF and
LTC4 are dependent on PLA2 activity, the role
of the (secretory) sPLA2 in various eosinophil responses
can only be speculated on. The fact that mepacrine has been suggested
to specifically inhibit sPLA231 and is able to
completely block STZ-induced PAF synthesis in human
eosinophils9 might indicate an important role for
sPLA2 activity in eosinophil activation. In addition, the
presence of sPLA2 in the eosinophil phagosomes may indicate
a function for this PLA2 in the killing of microorganisms.
Mammalian sPLA2 is able to directly degrade phospholipids
of Gram-negative bacteria.32
Despite significant degranulation (as indicated by the release of ECP),
sPLA2 did not appear to be released from eosinophils on
activation in vitro. sPLA2 is clearly lipolytic and
catalyzes a reaction at the water/lipid interface.11 Hence,
it is not unlikely that directly after secretion and exposure to mmol/L Ca2+ concentrations, the enzyme rebinds to the membrane.
However, immunoelectron microscopy did not yield an indication for the presence of sPLA2 on the outer membrane after eosinophil
activation. Likewise, we did not detect sPLA2 on the plasma
membrane by indirect immunofluorescence with the MoAbs 4A1 or 10B2
(data not shown). Possibly, insertion of the enzyme into the lipid
bilayer prevents binding of the antibodies. Cell fractionation followed
by detergent lysis of the various fractions may be helpful to resolve
this issue. Bach et al33 have shown that quantification of
release of eosinophil granular proteins is hampered by destruction on eosinophil activation. Measurement of remaining eosinophil-derived neurotoxin (EDN) and EPO in activated eosinophils showed
a recovery of only 40% in the cell pellet of activated eosinophils,
without apparent release of these proteins (<2%). These
investigators could not explain this low recovery. Also, we have found
a low recovery of sPLA2 protein (as determined by ELISA
measurements of sPLA2 in the supernatant and the remaining
amount of sPLA2 in the cell pellet of activated
eosinophils) of 51% ± 8% and 66% ± 6% (mean ± SEM, n = 3) in PAF/STZ- and PMA-stimulated human eosinophils respectively,
compared with a recovery of 95% ± 5% in unstimulated eosinophils.
The cause of this poor recovery is currently under investigation.
Previously, Rosenthal et al34 have shown the presence of a
14-kD PLA2 in phagosomes of neutrophils on activation with
serum opsonized S. aureus. Under certain conditions, this enzyme can be
found in the supernatant of activated neutrophils.34,35 Our
study shows that the level of sPLA2 is about 20-fold higher in eosinophils than in neutrophils, but also that under in vitro conditions the enzyme is not readily released from the eosinophils. Although we were not able to show release of sPLA2 protein
from eosinophils in vitro, sPLA2 might be released from
eosinophils in vivo. Bowton et al36 have recently described
enhanced sPLA2 activity in bronchoalveolar lavage (BAL)
fluid after antigen challenge in asthmatic patients. These
investigators did not examine the cellular source of the
sPLA2, although they suggested the mast cell as a possible
candidate. However, our results, while not refuting mast cells as an
important source of sPLA2, might suggest eosinophils as
another cellular source of sPLA2 in BAL fluids. Released
sPLA2, eg, in the lungs, may act on other cell types present in the lung: addition of sPLA2 to mast cells
results in the release of histamine, one of the agents responsible for
the symptoms during an asthmatic attack.37
| |
FOOTNOTES |
Submitted September 29, 1997;
accepted December 2, 1997.
Supported by The Netherlands Asthma Foundation (Project No.
14.89.25).
Address reprint requests to A.J. Verhoeven, PhD, Central
Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Prof. H. van den Bosch (University of Utrecht, The
Netherlands) for advice on sPLA2 activity measurements.
| |
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Abstract
Introduction
Abstract