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Release
of Platelet-Activating
Cells
Factor
Following
F. Bussolino,
Platelet-activating
factor (PAF). a phospholipid
mediator
of
anaphylaxis.
is also known
to be released
in vitro from
both phagocytic
polymorphonuclear
neutrophils
(PMN) and
in response
to a variety
of stimuli.
The
fact
that
human
myeloid
cells of the HL-60
line can be made to
differentiate
in vitro
into macrophage-like
cells by 12O-tetradodecanoylphorbol-1
3-acetate
(TPA)
prompted
us
to investigate
the generation
and release
of PAF during
this transformation.
Both passive release of PAF at pH 9.5.
and active
release,
following
phagocytosis
of C3b- and
C3d-opsonized
yeast
spores.
and stimulation
with
C5a
anaphylatoxin
from
untreated
and TPA-treated
HL-60
P
LATELET-ACTIVATING
FACTOR
(PAF)
phospholipid
mediator
derived
from
cytes that causes
aggregation
of platelets
the release
reaction’
by a mechanism
distinct
(ADP)
from
those
and arachidonic
of
sensitized
rabbit
hexadecyl(
choline.8
Initially
phosphatidylcholine,67
identified
PAF
basophils
as
obtained
from
this
source,’9
PAF
proteins,
along
with basophil
some resemblance
from a pure human
ration.’2
from
cationic
While
a mixed
the
role
human
has
since
In the rat and
is recovered
mastocytes.”
mixed
leukoC5a anaphy-
and
the
leukocyte
in PAF
suspension
release
remains
to
be substantiated,
a similar
release
from polymorphonuclear
neutrophils
(PMN)
and monocytes
has been
demonstrated
by several
workers.’3’6
Studies
on purified
human
blood
and
peritoneal
Leukemic
and
L. Pegoraro
cells, PMN, and plastic-adherent
normal human monocytes
were
studied.
It was found
that after
3 days of TPA
treatment.
HL-60 cells released
PAF following
phagocytosis of C3b- and C3d-opsonized
yeast spores.
Inhibition
of
PAF release
by a selective
inhibitor
of phospholipase
A2
and labeling of PAF with sodium ‘4C-acetate
indicated
that
PAF generation
is a two-step
process:
(1 ) release
of PAF
precursor
from cell membranes
and (2) its acetylation.
A
model for the in vitro study of mechanisms
and metabolic
events
involved
in PAF generation
and release
could
perhaps
be built on these findings.
populations
have
PMN
in response
have
and
(C3b),
C5a anaphylatoxin,
or by monocytes
during
spores.’6
made
it clear that
monocytes
exhibits
by
opsonized
and neutroand peritoneal
phagocytosis
or opsonized
In this article,
HL-60
human
is released
either
as large phagocyta-
complement
spores,
proteins,
macrophages
plexes
shown
that PAF
to such stimuli
complexes,
baker’s
yeast
phil cationic
of
immune
Moreover,
corn-
Clark
et al.
PAF from human
neutrophils
the same
physicochernical
we study
promyelocytic
PAF.’4
established
the
So
far, PAF
animal
or
release
of PAF from
cell line established
the
by
Collins
et al.’7 These
cells can be induced
to differentiate
terminally
along
their
own
series
by polar
compounds’8
and into macrophage-like
cells by phorbol
specific
degranulation.9
A factor
to PAF
has recently
been
leukemic
basophil
prepa-
of basophils
F. Ghezzo,
characteristics
as rabbit-derived
has not been
obtained
from
human
cell lines.
1 0%)-2-acetyl-sn-glyceryl-3-phosphoryl-
neutrophil
antigen,
bearing
derived
(90%)/
Human
Differentiation
ble immune
and
IgE-
from
i-0-octadecyl
been demonstrated
in many animals.’#{176}”
mouse,
contrary
to expectations,
PAF
from macrophages
and not from purified
In man,
PAF has been obtained
from a
cyte suspension
following
challenge
with
latoxin,
is a
rabbit
leukoand triggers
that is quite
both
adenosine
diphosphate
acid.23’5
It is thought
to be an
alkyl
ether
analog
of
Hanahan
et al. have
HL-60
Macrophage-Like
By G. Camussi,
monocytes
From
diesters,’9
particularly
bol- 1 3-acetate
tumor
promoters.
(TPA),
We
1 2-0-tetradecanoylphorthe
now
most
present
efficient
evidence
of these
that TPA-
treated
HL-60
cells
release
PAF
under
phagocytic
stimuli.
The metabolic
events
involved
are compared
with those of the release
of PAF from normal
human
PMN
and monocytes.
MATERIALS
AND
METHODS
cell
Cells
HL-60,
From
logia.
the Laboratorio
di Immunopatologia.
Universit#{224} di Torino,
Universit#{224}di Torino,
Supported
and
Torino,
in part
by
Cattedra
the Istituto
di Nefro-
di Medicina
Interna.
Italy.
CNR.
Grants
80.00645.04
and
80.00602.04.
Submitted
Address
cina
Interna,
Janury
reprint
9. 1981;
requests
Torino,
Italy.
(c) J 982 by Grune
to Prof
Torino,
August
L. Pegoraro,
Corso
26. 1981.
Istituto
Polonia
di Medi14.
10126.
by Dr
lOtM.
Both
of 2.5
obtained
fuged
Inc.
TPA
in acetone,
density
from
on
adhere
in
Calif.)
at
KG-I
human
Wistar
Institute,
l640supplemented
incubator.
dissolved
and
Rovera,
in RPMI
clear-rich
& Stratton,
0006-4971/82/5901-0003$Ol.00/0
16
accepted
Universit#{224} di
grown
CO2
Rome.
K-562,
provided
control
x
and
105/ml.
human
Ficoll
Borchert,
added
fraction
was
35-mm
diameter
adjusted
90 mm,
collected
the
dishes
nonadherent
seeded
Vol. 59,
1 .6 x
at
cells
and
The
and
(Falcon,
cells
was
of
in EDTA,
106 cells/mI
plastic
Blood,
Minn.)
were
described.”
to 5 x
Petri
were
in 5%
concentration
mononuclear
previously
(kindly
calfserum
Prairie,
final
cells
blood,
as
lines
USA)
fetal
Eden
at the
TPA-treated
normal
37#{176}C.After
12%
Plastic-adherent
gradient,
cell
Philadelphia,
with
(Dr.
and
myeloid
were
the
were
centrimononu-
allowed
to
Oxnard,
removed
No. 1 (January),
1982
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PLATELET ACTIVATING
by
several
0.25%
washings
bovine
in
serum
Kankakee,
their
ability
for their
or
Ca
positivity
by
staining2’
coupled
sodium
fluoride.22
Only
completely
abolished
phage-like
cells,
mononuclear
esterase
were
and
and
phagocytosis.
described’6
mentation,
osmotic
Ca
Mg
basophils
80%
by
shock,
Human
98%
HL-60
cells
PMN
were
and
3 times
minimal
staining
than
centrifugation,
platelet
stainable
plastic-adherent
Negligible
of
PAF
with
were
positive
as
sedi-
TTBSA
without
cell
blue9
buffer,
indomethacin
washed
twice,
NaCI.
Complement
obtained
and
by
human
resuspended
for
ment
serum
obtained
(Sigma)
l
were
U/mI
or EDTA-treated
containing
in
PAF
the
medium
and
inactivator.
extensively
An
was
used
as extensively
and
MOller-Eberhard.26
analytical
technique
ileum.27
C3d
antigen
reported,”
according
Only
one
and
and
as the
surface
in
to the
technique
of Vallota
was
activity
pun-
observed
by
electrophoresis,
was
concentration
tested
was
on guinea
2.3
graphic
pig
challenged
at 37#{176}C
with
C5a
(0.2
.tg/ml)
an
alkaline
using
to
evaluate
sun-Yvette,
the
(56
as follows:
,Ci/ml
challenge
of
with
for
by
after
of
Merck,
sodium
PAF
l0
cells
‘4C-acetate;
at
in
divided
into
extracted
substrate,
37#{176}Cwith
l0
at room
the
supernatants
with
6:4 methanol-water
centnifugation
phospholipid-nich
fraction
water(0.8).2t
layer,
at
After
the
was
recovery
material
and
was
and
the
phased
in
evaporation
TTBSA;
1’l’BSA
precipitated
nedissolved
in 0.5
lower
ml TTBSA
of a 10
exhibited
the
BSA
was
of
(Packard
NaOH
Instruments,
liquid.
dried
that
basis
of its physico-
Kodak,
before
Illinois),
using
on TLC
I -0-octadecyl-2-acetyl-3-GPC
0.03
N
N
(3)
lipids;
HCI,
in
0.03
scraped
in
Plates
chromatography.
I
standard
not
affect
lipids.
sq
cm
counter
as
for
7 days
using
were
for
the
at
x-ray
immersed
3 hr
Preliminary
did
in
sodium
(Packard)
flurography33
in ether
(4)
mg/mI
with
by
N
by
water;
experiments
exposed
N.Y.).
and
lysoleci-
(0.03
lnstagel
( I)
IgE.
inactivation
NaOH
lipids
(Packard)
2,5-diphenyloxazolone
from
in a scintillation
plates
Rochester
A2:
PAF
(Sigma),
were
counted
by
as reference
5 mm,
plates
its ability
phospholipase
M NH4OH;
Acetate-labeled
detected
(Eastman
showed
on the
0.5
from
was
biologi-
unaffected
or as
was
phase
the
was
at 37#{176}C).In the
radioactivity
30
10 mm,
both
(Sigma)
and
g for
and
sphingomyelin
0.5
and
After
chloroform
with
for
were
tubes,
chloroform-methanol-
A2 (Behningwerke)
chromatographic
2,5-diphenyloxazolone
vacuum
methanol
8 for 60 mm
20%
chloro-
and
plates
which
and
in water
to
(60F254
as PAF
treatment
phospholipase
films
(0.95)used
in
by
70#{176}C
were
the
plastic
in TTBSA,2’
aggregation,
acid,
gel
at 2500
The
phosphatidylcholine
M periodic
silica
chloroform-methanol-
I : I :0.9
characterized
to boiling
submitted
( I :2:0.8).
to
using
retained
were
PAF,
into
system.
basophils,28
and
scintillation
-
with
centnifugation
and
acetone-
which
fractions
to a
chromato-
chloroform,
using
scavengers,
applied
Mo.)
(1:4),
resuspended
was
ADP
rabbit
and
was
with
l-0-octadecyl-2-acetyl-3-GPC,3#{176}
‘4C-acetate,
from
and
as
pH
squares,
volume
chloroform
of the
N
with
or without
and
platelet
and
0.5
inactivation
One
effective
PAF)
Louis,
scraped
brought
characteristics2’
Rf
presence
extracted
g for 20 mm.
3000
chemical
0.03
were
were
indomethacin
NaOH,
during
lipids
by
PAF.29
microliters
fraction
recover
squares,
and
material
resistance
at 2000
removed
1000.
8 U/mI
U/mI
St
To
in a biphasic
rabbit
(2)
fugation
supernatant
as
x
of PAF
Germany),
cm
dried,
active
(Sigma),
p-bromo-diphenacyl-
were
to 100
precoated
solvent.2’
temperature
induce
thin
Italy).
After
incubation,
the
x 103M),
followed
by centni-
The
milliliter
minimal
Ten
2 ml chloroform-methanol-water
aspirated,
sensitized
(PBDB)
(C. Erba,
Milano,
was stopped
with EDTA
(5
10 mm.
determined.
per
Benveniste;
“M).
eluted
plates
West
resulting
at
bromide
reaction
g for
10
pooled
on glass
1 sq
mm
in the
with
0.1
of PAF-
was
about
chloroform-methanol
as
with
In
Gif-
suspension
2 washings
as phagocytable
5 mm
molecules
30 mm
Dr.
sequentially
Darmstadt,
(65:35:6)
same
overnight
(CEA,
TLC
in
of incubation).
‘4C-acetate
at 22#{176}C
for
and
releasable
incubation
by
or
PAF
30 mm
sodium
cells
incubation
C3b-BYS
preincubation
9.5
were
or C3d-BYS,
times.
mCi/mM)
adherent
106
processed
C3b-BYS
obtained
(pH
uptake
France)
phagocytosis,
200
10.6
in I ml ofTTBSA
incubation
was
pH
BYS,
106
different
environment
20#{176}C
in TTBSA,
order
5 x
or adherent
by
5 x
PAF-containing
water
to
(5 x lOs)
amount
units
with
chloroform-rich
and
activity.’
cally
in suspension
platelet
I -0-octadecyl-2-acetyl-3-GPC3#{176}
4, Mallinckrodt,
and
(1:1),
water,
x lO’#{176}M.
Release
Cells
g/lOO
by adding
The
Characterization
ARCC
column,
then
PAF
(Silic
preparatory
and
band
15.25
aggregation
results
and
(CPK)
aggregation.
PAF-containing
acid
PAF
source
Teknika,
BYS
silic
and
methanol
generated
polyacrylamide-gel
effective
The
were
was
detectable
Biologic
minimal
the
and
aggregation
about
thrombin
Purification
comple-
(Organon
on
of maximal
Mo.),
normal
inactivated,
BYS
serum
anaphylatoxin
described.25
The
C3d
50%
platelets.
used
Iris-
presence
Louis,
determined
in arbitrary
PAF, kindly given
(corresponding
CrP
thrombin-induced
we
in the
St.
840):
zl of
ADP-dependent
was
reproducible
substance
solution
Ehlenberger
with
gives
reference
platelets,
300
gelatin,
and
for 50%
50%
ng/ml
TTBSA,’6
heat
for
in 0. 1 5 M
an anti-,C//3,A
C3d-BYS
of
TLC
(ELVI
in
Co.,
g/lOOl
expressed
aggregation
with
either
adsorbed
to detect
electrophoresis
as previously
method
serum,
anti-human
C5a
fled,
the
human
before
phosphokinase
acid
necessary
was
PAF
0.25%
to stirred
on platelet
were
with
Binding
using
Germany).
with
rabbit
rpm
Chemical
aggregation
(Sigma)
concentration
washed
900
with
(31.25
dose
of fresh
at
arachidonic
Maximal
l/Ml
of
(CrP)-creatinine
30 mm,
for
(C3b-BYS)
I ml
buffer.
4C-
3 times
in an aggregometer
stirred
system
block
thrombin
prepa-
3 washings
same
by
normal
immunofluorescence.
form
to
aggregation.3
released
concentration
with
sodium
washed
4C-acetate
aggregation
(Sigma
enzymatic
CPK)
or PMN
boiled
BYS
of BYS
at 37#{176}C
after
C3b-BYS
using
Denmark)
initial
by immunofluorescence,
Mannheim,
West
from
Nussenzweig,24
of C3b
mg
in I ml of the
was assessed
(Behringwerke,
were
activated
20
20 mm
resuspended
mg/mI)
at the
(C3b)
incubating
serum
20
with
was
excess
described,9
phosphate
(synthetic
(BYS,
experiments
phase
supplemented
creatinine
pure,
0.1%
Stimuli
spores
the
remove
were
of l05M
As
yeast
to
by
platelets
Tyrode’s
This
of
l0
follows:
Baker’s
detected
as previously
x
the
PMN
90%-98%
Less than
were
2-5
supernatants.
and
( I :I )
was
prepared
prepared
toluidine
histamine23
In
chloroform
Assay
PAF
adherent
contamination.
mononuclear
amounts
was
gelatin
with
lower
characterization.
as monocytes/macro-
differential
characterization.
the
methanol-water
for
of esterase
esterase
regarded
PAF
described,#{176}
with
May-GrUnwald-Giemsa
were
lymphomononuclear
contaminants.
in either
rations.
when
washed
for
acetate,
assayed
inhibition
L#{246}ffier.22More
metachromatically
present
were
as previously
with
TPA-treated
to ensure
smears
stained
with 2%-I0%
cells
to
cells
previously
and
the
according
with
Lab,
Plastic-adherent
cells
beads,
esterase
(Miles
Mgt’6
HL-60
latex
supplemented
V (TTBSA)
and
IPA-treated
to phagocytize
17
buffer
fraction
without
cells
and
Tris-Tyrode’s
albumin
USA)
mononuclear
for
FACTOR FROM HL-60
and
in
then
experiments
the
behavior
of
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
18
CAMUSSI
100
RESULTS
PAF
release
was assayed
untreated
and treated
with
days;
from
K-562
and KG-
from
HL-60
cells,
both
TPA
for I hr and for 3
1 cells,3233
both untreated
and
3 days;
treated
with
peripheral
(Table
1).
HL-60
PAF.
48
TPA
blood
for
mononuclear
and
from
normal
cells
and
PMN
II.
a,
U.
0
cells
treated
with
Morphological
hr almost
all
cells
TPA
for
3 days
UI
(1)
released
examination
showed
that
after
became
attached
to the plastic
UI
-I
UI
surface
and stopped
proliferating.’9
By the third
day,
they exhibited
intense
(>90%)
phagocytosis
of inert
latex particles,
and strong
(>80%)
a-NAE
positivity.
By contrast,
HL-60
cells untreated
or treated
for 1 hr
were unable
to ingest
latex
beads,
were
negative
for
ct-NAE,
and
amounts.
increased
released
PAF
only
passively
and
in small
The TPA-treated
control
cells also exhibited
adherence,
though
only
the
KG-I
line
mlnut#{149}s
became
phagocytic
(80%)
and a-NAE
staining
positive (50%).
No PAF
release
was detected
from either
treated
or untreated
cells.
Release
of PAF
from
treated
HL-60
cells
was
comparable
to that of normal
peripheral
mononuclear
adherent
cells and PMN
(Fig.
I ). Both groups
release
PAF
passively
at pH 9.5 and actively
during
BYS
phagocytosis,
but
C3d-BYS
ET AL.
the
challenge.
ent cells release
toxin
challenge.
latter
did
Conversely,
not
PMN
PAF actively
Both
PMN
respond
and
to
Kinetics
1.
TPA-treated
9.5.
activity
migrated
coincidentally
PAF activity
was absent
in the
the
nonadher-
following
CSa
and adherent
clear
cell
preparations
contained
metachromatically
staining
basophils,
mine was detected
in the supernatants;
of the release of PAF at 37’C from C3b-BYS
HL-60 cells (U). mononuclear
plasticadherent cells (A). and PMN (#{149})
(5 x iO cells/mI).
PAF release is
expressed as the percentage
of the total PAF releasable
at pH
Fig.
phagocytic
contained
anaphylamononu-
The
free
PAF
than
0.1%
and no histathis amount
of
same biologic
aggregation
Rf 0.22 only.
phase,
which
‘4C-acetate.
and
biolabeled
same
as synthetic
sensitized
basophils
less
with the
methanol
PAF
obtained
PAF
and PAF
in four respects
derived
(Table
activity
on washed
rabbit
was ADP
and arachidonic
were
the
from
IgE2): ( 1 ) the
platelets,
since
acid indepen-
basophils
is insufficient
for PAF
release.’3’6
The failure of mononuclear
cells in suspension
to respond
to
C3b-BYS
suggests
that
they can only produce
PAF
dent;
namely
under
phagocytic
Fluorography
particularly
in the presence
of methanol;
(3) inactivation by phospholipase
A2 treatment;
in the case
of
labeled
PAF,
this is accompanied
by loss of >90%
spot
of
stimuli
when they become
of the TLC
plates
showed
radioactivity
PMN
and
tants.
Conversely,
found
for
(Rf
0.22)
plastic-adherent
was
detectable
mononuclear
cell
2 spots
TPA-treated
(Rf
HL-60
Table 1 . Release of PAF (in U/mI)
Stimuli
adherent.
that only
From
0.22
and
cells
(Fig.
HL-60.
K-562.
Plastic-Adherent
periodic
1
from
superna-
0.76)
2).
and
band
PAF
choline.
KG-i Cells Untreated
Cells and PMN Under
HL-60
(6)t
HL-60 +
TPA i hr
(6)
K-562
(4)
acid,
radioactivity;
were
HL-60
+
iRA
3 Days
(6)
(2)
similar
acid-stable
but
(4)
between
physicochemical
and unaffected
rapidly
the
inactivated
same
sphingomyelin
and Treated
With
Various
Stimuli
by
TPA.
TLC
characteristics,
weak
base
by strong
pattern,
and
and From
KG-i
(2)
K-562
+
TPA 3 Days
(4)
KG-i
+
WA 3 Days
(2)
PMN
(10)
with
a sharp
lysophosphatidyl-
Human
Mononuclear
Mononuclear
Plastic-Adherent
Cells
hO)
16
170
17
0
0
0
0
100
180
C3b-BYS
0
95
0
0
0
0
0
95
104
C3d-BYS
0
73
0
0
0
0
0
0
92
C5a
0
0
0
0
0
0
0
52
0
BYS
0
0
0
0
0
0
0
0
5
pH9.5
. For
each assay.
tNumber
5 x 1 O cells
of experiments.
in suspension
or adherent
were
used.
in
base,
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PLATELET
ACTIVATING
FACTOR
FROM
HL-60
19
As shown
with
PBDB,
in Fig. 3, when
cells were preincubated
PAF
release
could
be detected
during
phagocytosis,
and
reduced.
By
leased
from
the
complete
Rf
0.22
peak
labeled
TPA-treated
HL-60
was
lipid
cells
markedly
material
was
re-
unaffected.
DISCUSSION
PAF release
gered
from
by phagocytic
tor-dependent
results
indicate
small
amounts
human
leukocytes
or soluble
stimuli
or independent
that the HL-60
when
stimulated
independent
mechanism,
10.6. Following
treatment
PAF releasable
increased,
and
Fig.
2.
Fluorography
“C-acetate
on TIC
(1)
Sodium
TPA-treated
HL-60
cells.
was recovered.
Arrow
indicates
where
“C-acetate.
PAF
mechanisms.’6’34
Our
cell line only releases
with
a receptoras incubation
at
TPA,
the amount
receptor-dependent,
recently
been
trigrecep-
is greatly
mediator
stimuli
demonstrated
in
pH
of
is now
vitro
that
HL-60
cells can be induced
by TPA
to differentiate
into macrophage-like
cells and that myeloid
cells from
normal
subjects
and leukemic
patients
respond
in the
biolabeled lipids from the supernatants
of C3b-BYS
(2) PMN. (3) plastic-adherent
mononuclear
cells. (4.5)
phagocytic
activity
plates.
has
be
in an alkaline
environment
release
in response
to the
under
phagocytic,
possible.
It
such
with
may
through
biologic
same
way2#{176}
by displaying
several
morphological
functional
changes:
glass
adhesion,
phagocytosis
inert
particles,
secretion
of lysozyme,
appearance
0. origin; SF. solvent front.
monocytic
Table 2. Characterization
cytochemical
of PAF-Active
markers.3536
K-562
and
of
of
and
KG-
Material
Source of PAF
Mononuclear
Plastic-Adherent
mc
Rabbit
gE-Sensitized
Basophils
0.22
WA-Treated
Synthetic
PAF
0.22
Cells
0.22
HL-60 Cells
0.22
100#{176}C,Smin,inH2O
100
100
100
100
0.03 N
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
(Rf)
Pattern
Sensitivity
to
physicochemical
and enzymatic
treatmentst
HCI
O.O3NNaOH,inH2O
0.5
M periodic
acid
0.5MNH4OH
0.03
0.5
N NaOH.
N NaOH.
in H2O
Phospholipase
Biologic
A2
pIateIets
CP/CPK
+ Indomethacin
Solvent
system:
‘Rf of lysolecithin,
tTLC-rurified
A2 from porcine
are expressed
Twenty
0
0
0
0
0
0
0
0
0
0
0
0
activity
On rabbit
+
in methanol
CHCI3/CH3OH/H2O,
sphingomyelin
80
80
80
80
81
78
80
77
77
78
80
79
65:35:6.
and phosphatictylcholine:
0.15,
0.30,
and 0.46.
respectively.
and synthetic PAF. adjusted to a concentration
of 100 U PAF/mI,
were submitted
to physiocochemical
pancreas) treatments,
and the factor activity was compared with the initial activity (nontreated
controls).
as the percentage
microliters
of PAF
for 3 mm at 37 #{176}C.
Results
of activity
adjusted
are expressed
recovered
to 1 00
in treated
U PAF/mI
in arbitrary
units
were
and enzymatic
(phospholipase
Results of a typical experiment
samples.
tested
as millimeters
on rabbit
of chart
platelets,
distance.
with
or without
preincubation
with
CP/CPK
or indomethacin
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
20
CAMUSSI
PAF
A
in the
same
way
as C3b-bearing
such as adherent
mononuclear
suggests
that
C3b
receptors
Moreover,
the release
C3d-BYS
of PAF
receptor
cells and
are induced
were
both
mediated
stimulus
phagocytosed
and
from
TPA-treated
and
induces
the
0:
U
released
was
lated
IgE
indistinguishable
rabbit
acetyl-3-GPC
platelets,
from
basophils
in terms
physicochemical
and
of
its
This
TPA.
triggered
HL-60
which
which
do
receptor-
release
from PMN
but not from plastic-adherent
It was also ineffective
on TPA-treated
indicating
the absence
of C5a
receptors.
*
cells,
PMN.
by
cells
and
from
plastic-adherent
monocytes,
possess
C3d receptors,24
but not from PMN,
not.24
C5a
anaphylatoxin
is a soluble
80
ET AL.
of
PAF
monocytes.
HL-60
cells,
The
PAF
antigen-stimu-
l-0-octadecyl-2-
biologic
characteristics,
activity
on
and
TLC migration.
cm
The
require
gy.
B
metabolic
events
involved
the presence
of extracellular
Release
to membrane
tion, while
of PAF
from
in PAF
cations
phagocytes
has
and phagocytic
stimuli
membrane
phospholipase
Polonsky
et al.38 have demonstrated
cytes
in an alkaline
environment
precursor
have
*
that
the
cm
3.
biolabeled
Distribution
of radioactivity
after TIC
of “C-acetate
lipids
extracted
from
the supernatants
of 10’ (A)
HI-60
cells, (B) mononuclear
plastic-adherent
cells
TPA-treated
preincubated
with
sodium
“C-acetate.
Cells
incubated
with
(#{149})
and without
(U) i0 C3b-BYS.
Effect
of preincubation
with PBDB
(A). Arrows
indicate
where
PAF biologic
activity
was recovered.
Migration
distances
on the abscissa.
Reproducible
results
were
obtained
in experiments
performed
on 5 separate
occasions.
I, however,
are
not
able
to release
PAF.
The
increase
in passively
releasable
PAF and its active
release
from
macrophage-like
HL-60
cells
during
phagocytosis
seem to be related
to TPA-induced
differentiation
and
not
to an
aspecific
effect
of TPA
on cellular
membrane.
In fact, PAF was not released
from HL-60
KG-I
HL-60
points
A2.
that
release
1-0-alkyl-2-lyso-GPC.38
suggested
‘4C-acetate
by PAF
PMN,
plastic-adherent
cells
related
to acti-
hog leukothe PAF
Demopoulos
PAF
molecule
et al.6
contains
an
acetyl
group
in position
2 and
that
this
group
is
required
for its biologic
activity.
An acetyltransferase
that
utilizes
l-0-hexadecyl-2-lyso-GPC
as substrate
has also
been
observed
in rat spleen.39
Uptake
of
U
Fig.
been
stimulation
rather
than to particle
ingesinhibition
by PBDB
of PAF release
induced
by both soluble
vation
of a cell
§
generation
and ener-
treated
for
I hr with
TPA,
and
from
cells treated
for 3 days with TPA.
cells
phagocytosed
C3b-BYS
K-562
and
TPA-treated
and
released
released
during
mononuclear
treated
HL-60
indicates
its biosynthesis.
Like plastic-adherent
TPA-treated
the PAF
that
phagocytosis
cells, and
acetylation
mononuclear
cells
HL-60
cells
incorporate
molecule,
but unlike
normal
also
incorporate
different
TLC
it in
migration
This
is in agreement
that
‘4C-acetate
another
pattern
with
uptake
into
part
and
fraction
no PAF
acids and neutral
lipids was increased
inhibits
PAF release
and incorporation
in
they
with
activity.
et al.,4#{176}
who
membrane
of
PMN,
‘4C-acetate
leukocytes,
lipid
and
Cabot
cell
forms
of
TPA-
free
a
found
fatty
by TPA.
PBDB
of ‘4C-acetate
into
its molecule,
but does
not prevent
‘4C-acetate
labeling
of the Rf 0.76 fraction,
in other words,
it does
not interfere
with acetylation.
In addition,
such inhibition
suggests
0-alkyl-2-lyso-GPC,
that
a suitable
substrate,
such
as
which
is derived
from phospholi-
pase
A2 activity
cell’s
ability
ability
to release
to display
two
on plasma
membranes,
PAF may
sequential
is needed.
1A
thus be related
to its
enzymatic
steps:
(1)
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
PLATELET
ACTIVATING
hydrolysis
FACTOR
FROM
21
HL-60
of I -0-alkyl-2-acetyl-GPC
phospholipids
(2) acetylation
from
affected
by phospholipase
at position
2 by an acetyl
membrane
granulocytic
A2 activation;
transferase.
The HL-60
cell line has so far been extensively
as a model
for induction
of differentiation
along
or
the
that
it releases
results
indicate
used
the
mechanisms
generation
monocytic
lineage.
PAF
when
that it may
and metabolic
release.
and
By
showing
treated
with
TPA,
our
also serve in the study
of
events
involved
in
PAF
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1982 59: 16-22
Release of platelet-activating factor from HL-60 human leukemic cells
following macrophage-like differentiation
G Camussi, F Bussolino, F Ghezzo and L Pegoraro
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