1. No category

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1988 71: 1100-1107
Platelet-activating factor primes neutrophil responses to agonists: role in
promoting neutrophil-mediated endothelial damage
GM Vercellotti, HQ Yin, KS Gustafson, RD Nelson and HS Jacob
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Platelet-Activating
Factor
Promoting
inflammation
exposed
to
ment,
and
kallikrein,
stimuli
only
(1012
to
directly
factor
ineffectual.
PAF
generation.
them
subsequent
priming
dependent
within
during
subsequent
agonists.
but
priming
inhibited
by the
and
and
0
PAF
and
hum
in diverse
minutes.
by
PAF
cold
inflammatory
of
and
do
is
not
with
and
such
and
detaching
damage
that
can
this
PMN
marginated
lipid
occurs
such
name,
in mechaendothe-
particularly
and lysosomal
endothelium.
polymorphonuclear
cell
in the
PAF
effects,
because
coagulation
inflammation.
PAF
a lipid
is a common
activation
activated
by
feature
themselves,7’9
was
and
of activated
a variety
monocytes,
endothelial
initially
degranulate
cells,
cells
and
recognized
by
its
platelets
in vitro.
by Grune
& Stratton,
iioo
Inc.
has
our
the
studies
inhibitors
in
Inc.
more important
far lower than
neutrophils
release,
actions
needed
in inflamfor platelet
causes
chemotaxis,
degranulation
IOI2;
induces
pulmonary
sequestration
of
bronchoconstriction,
and increased
in vivo
PAF
have
may
begun
play
and
and
a critical
searches
potential
antiinflammatory
PAF analogues
or PAF
for
role
PAF
in inflammation,
antagonists
to act
agents.
Several
receptor
antagonists-are
as
such--either
now avail-
Many
of the latter
have
been
isolated
from
herbal
medicinals
used by the Chinese
for centuries
to relieve chest
able.
Shen has
kadsurenone,
a similar
is a furanoid,
investigators
described
from
receptor
a powerful
PAF
receptor
the Chinese
herbal
plant
antagonist
prepared
by this
L652-73
code-numbered
have
from
isolated
seeds
1 .‘
and
In addition,
leaves
of
the
Ginkgo
biloba
tree or Ya-chiao-pan
another
potent
PAF
receptor
antagonist,
BN5202l,’5’8
which
has been found
to
inhibit
all biologic
activities
of PAF so far studied.
including
neutrophils
The
enhancement
or
capacity
to
response
to a given
Belying
its
the
to
cell
to ARDS,
1 988
so.
PAF
of
to
permeability.
group
of severe
From the Departments
of Medicine
and Surgery.
University
of
Minnesota
Medical
School,
Minneapolis.
Submitted
September
8, 1987; accepted
December
1 1. 1987.
Supported
in part by National
Institutes
of Health Grants AM
0/387,
HL 33793,
HL 28935,
and HL 19725.
Address
reprint requests
to G.M. Vercellotti,
MD, FACP,
Box
480
UMHC.
University
of Minnesota
Hospital.
Harvard
Street at
East River Rd. Minneapolis,
MN 55455.
The publication
costs ofihis article were defrayed
in part by page
charge payment.
This article
must therefore
be hereby marked
“advertisement”
in accordance
with 18 U.S.C.
§1734 solely
to
indicate this fact.
0006-4971/88/7104-0027$3.OO/O
If
for
of
that
activators
damaging
itself.
role
surfaces
suggest
states.
hypotension,
tumor
of
amplification
stimulus
is due
that
different
agonist
has
Several
biologic
mediators,
including
necrosis
endotoxin,’9
factor2’
have
a
to prior
demonstrated
to prime
responses.
Others”22’23
have reported
enhanced
to stimuli
after preexposure
to PAF. Because
PMN
responses
is generated
by
both
cells24
inflammatory
of
to as
relevant
(IL-I)2#{176}and
interleukin-1
been
neutrophil
exposure
been
referred
presumably
a
“priming.”
©
on
trivial
cells-
antagonists.
we
become
therapeutic
& Stratton,
PAF
neutrophils,
discomfort.
antagonist,
Haifenteng’3;
(I -O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine),
produced
basophils,
aggregate
of stimuli
produced
by
their activities.
In fact,
stimulatory
effects
on
of
moreover,
others
kallikrein,
arachidonate
metabolites,
monobacterial
peptides.
Recent
reports
that endotheexposed
to thrombin,
synthesizes
platelet-activat(PAF)
prompted
our interest
in this compound
complement,
kines, and
hum, when
ing factor
PAF
exhibited
they
with
effective
endothelial
otherwise
activates
superoxide
Because
neutrophils
function
(PMN)
and
more
by coagulation.
that
PAF probably
at concentrations
mation;
proteases
In condi-
as polytrauma,
are presumably
flooded with a variety
damaged
tissues
that might modulate
we and others
have reported
potent
stimulated
are
by the
endothelium
by Grune
vascular
ARDS
human
and
of agonists
enhanced
release
toxic
that, in turn,
in which
cultured
inflammatory
aggregation,
tions
PAF
inhibited
calcium
addition
esters
potentiate
a possible
e 1988
with
by 0KM-
intracellular
or phorbol
be
so
identified
in
subsequent
perturbed
PAF-synthesizing
adult
respiratory
distress
syndrome
(ARDS).”3
Neutrophils
stimulated
by inflammatory
mediators
such as activated
complement
adhere
to the endothelial
surface;
adherent
cells
oxygen
metabolites
can damage
vascular
also
PMNs
excessive
PMN
the
rise
is synthesized
may
suggest
glycoprotein
primed
in lysing
need
required
to
cells
in
Jacob
enhanced
PMNs
PAF
H.S.
adhesive
F-Met-leu-Phe
endothelial
also
and
an
as FMLP.
Role
Damage
in response
Because
1-652.
prime
both
been
interested
damage
vascular
syndromes,
.
of the
1 antibody
time
is
BN 52021
molecule
is associated
has
neutrophils
PAF
exhibition
methoxy-PAF
priming
UR LABORATORY
nisms by which
five
antagonists
intact
superoxide
is
to Agonists:
Nelson,
expression
either
PMNs
aggregation
inhibited
receptor
An
lyso-PAF
responses.
is
PAF
kadsurenone.
because
the
PAF
be otherwise
including
and
is maximal
be
that
R.D.
levels
quantities
is, permits
would
of responses
release.
report
that
that
are
comple-
in miniscule
as well,
Gustafson,
monokines,
We
but
present
not
Responses
Endothelial
K.S.
(PMNs)
metabolites,
stimuli
elastase
Yin,
activated
(PAF).
PMNs
“primes”
to
cells
acid
activates
mol/i)
respond
H.Q.
including
arachidonic
platelet-activating
not
Vercellotti,
polymorphonuclear
agonistic
Neutrophil
Neutrophil-Mediated
By G.M.
During
Primes
and
PMN
PAF
endothelial
cells227
in response
to diverse
mediators
such as thrombin,
we reasoned
that PAF might
prime
responses
of marginated
PMNs
to otherwise-weak
neutrophil
agonists
and thereby
promote
wondered
neutrophil-mediated
whether
PAF
enhanced
endothelial
this
hypothesis
phil
superoxide
yates
PMN
endothelial
antagonists
damage.
The
by demonstrating
production
adhesion
that
and
damage.
If so, we
might
inhibit
such
present
PAF
elastase
to endothelium
studies
amplifies
release
and
Blood, Vol 71, No4(April),
validate
neutroand
subsequent
1988:
aggraendo-
pp 1100-1107
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
PAF
PRIMED
thelial
PMNs
injury.
AUGMENT
ENDOTHELIAL
PAF
Furthermore,
amplified
responses,
excessive
inflammatory
which
DAMAGE
antagonists
suggests
1101
inhibit
a role for these
these
agents
in
states.
removed
METHODS
-
experiments
the
cells
were
after
centrifugation
for
the
measurement
of cytochrome
c
reduction
Materials.
PAF (DL-a-phosphatidylcholine,
$-acetyl--y-O-hexadecyl or L-a-phosphatidylcholine,
$-acetyl--y-O-alkyl:
P1402 and
P9525),
phorbol myristate
acetate
(PMA),
DL-a-lysophosphatidylcholine, -y-O-hexadecyl
(lyso-PAF),
ferricytochrome
C (horse heart
type), f-Met-Leu-Phe
(FMLP),
superoxide
dismutase,
dimethylsulfoxide
(DMSO),
and bovine serum albumin
(BSA) were purchased from Sigma Chemical
Co (St Louis). Hanks’ balanced
salt
solution
(HBSS)
and medium
199 were obtained
from GIBCO
(Grand
Island, NY). Sodium
5’Cr and “In oxine were obtained
from Amersham
Corp (Arlington
Heights,
IL). Fura-2 was obtained
from Molecular
Probes
(Eugene,
OR).
L652-73l
(L-652)
and
kadsurenone
were a kind gift of Dr John Chabala
(Merck,
Sharp,
and Dohme,
Rahway,
NJ). BN52021
and methoxy-PAF
were kind
gifts of Dr Pierre Braquet (Institut
Henri Beaufour,
Paris). 0KM-I
antibody
was obtained
from Ortho Diagnostic
Systems
(Westwood,
MA).
All materials
were endotoxin
free as tested by the limulus
amebocyte
assay.
Preparation
of PMNs.
Human
volunteer
blood
samples
(40
mL) were drawn
(after
receiving
informed
consent
under
the
guidelines
of the Committee
on the Use of Human
Subjects
in
Research
at the University
of Minnesota)
into a plastic syringe
containing
20 mL hydroxyethylstarch
(Hespan;
American
Hospital
Supply Corp, Irvine, CA) and 200 units preservative-free
heparin
(Upjohn
Co, Kalamazoo,
MI). The mixture was allowed to sediment
at room temperature
and the supernatant
collected
and centrifuged
at 400 g for five minutes. The pellet was then resuspended
in 0.2 mL
ice-cold
HBSS containing
100 mg/dL
glucose.
Residual
erythrocytes were lysed in I 5 mL ice-cold
water, and after 25 seconds
isotonicity
was reconstituted
by the addition of 5 mL of 3.6% sodium
chloride.
The suspension
was centrifuged
in the cold at 400 g for five
minutes and the pellet resuspended
in 5 mL HBSS, carefully
layered
on top of Percoll (Sigma)
made up to a density of 1.075, and then
centrifuged
at 20,000 g for 30 minutes at 4#{176}C.
The resulting
PMNs
(>95%
with <1% platelet
contamination)
were washed
once and
suspended
in 10 mL HBSS. Viability
was assessed
by trypan blue
exclusion
and exceeded
95%.
Endothelial
cell cultures.
Human
umbilical
vein endothelial
cells (HUEC)
harvested
from umbilical
cords by collagenase
digestion as previously
described28’
exhibited
factor VIII antigen
and
Weibel-Palade
bodies. The endothelial
cells, plated either in 2-cm2,
24-well Costar
plates (Costar;
Cambridge,
MA) or 0.32 cm’ microtiter wells (Dynatech
Laboratories,
Alexandria,
VA) were grown to
confluent
monolayers
and used as primary cultures.
Neutrophil
priming
with PAF.
i-PAF
was stored at
20#{176}C
in
chloroform,
and DL-PAF
was made
to a 10 mmol/L
solution
in
phosphate-buffered
saline (PBS) and stored at -70#{176}C.For experiments, an aliquot of the stock solution of DL-PAF was prepared
in
HBSS with 0.5% human albumin
(American
Red Cross, Washington, DC) and added to the final incubation
mixture.
An aliquot of
the L-PAF dissolved
in chloroform
was evaporated
unIer nitrogen
and redissolved
in HBSS with 0.5% human albumin
to the required
concentration.
We measured
the ability of PAF to prime neutrophil
superoxide
release,
elastase
release,
aggregation,
and adhesion
by
methods
previously
described.’#{176}’3’ For example,
the priming
for
superoxide
release was assayed by adding PAF at various concentrations and for varying time intervals
to 2 x 106 PMNs in HBSS at
37#{176}C.In some
centrifuged
at I ,200 rpm for two minutes, the supernatant
removed,
and I mL HBSS added. Cytochrome
c (75 imo1/L)
was then added
to each tube and was followed by a stimulus
such as FMLP (l0
mol/L) or PMA (100 ng/mL).
Thecells
were incubated
at 37#{176}C
for
I 5 minutes
and thereafter
placed
on ice. The supernatant
was
subsequently
washed
and
as previously
described.’#{176}Priming was performed
by using
both
L-PAF
and DL-PAF.
No differences
were seen when using
either PAF preparation.
When PAF inhibitors
were used, they were
added at the given concentrations
for five minutes at 37#{176}C
before the
addition of PAF. BN52021,
L-652, and kadsurenone
were dissolved
in DMSO
and then added to HBSS to result in a final concentration
of DMSO not exceeding
0.1%. Control buffer contained
HBSS with
0.1%
DMSO.
The control
responses
to FMLP
or PMA
were
performed
by adding
HBSS
with 0.5% albumin
to the PMN
suspension,
washing as described
earlier, and then stimulating
with
FMLP or PMA. In some experiments,
cytochalasin
B (5 Mg/mL)
was added to PMNs
before stimulation
with FMLP.
A similar
priming
technique
was used to measure
the stimulated
release of
elastase
by measuring
the cleavage
of a peptide substrate,
MeOSuc-Ala-Ala-Pro-Val-nitroanilide,
as previously
described.”
We
arbitrarily
defined 100% elastase release by the amount of elastolytic
activity
in the supernatant
of PMNs
(2 x I0’/mL)
treated
with
cytochalasin
B (5 zg/mL)
and stimulated
with FMLP (l0
mol/L)
for 20 minutes
at 37#{176}C.
Neutrophil
aggregation
and adhesion
to
endothelium
were performed
as previously
described’2
upon similarly primed
PMNs.
The effect
of PAF
priming
on neutrophil
CR3
expression.
Studies
assessing
the effect of PAF priming
on neutrophil
complement
receptor
type 3 (CR3)
expression
were performed
as
described
by Nelson et al.” Briefly, PMNs (5 x 106/mL) suspended
in PBS containing
0.2% BSA were stimulated
with PAF in various
concentrations
for 15 minutes at 37#{176}C.
Alternatively,
in sequential
agonist experiments
10_12 mol/L
PAF (or HBSS as a control)
with
0.5% human
albumin
was added to the PMNs
for five minutes
followed
by stimulation
with FMLP
(I0
mol/L)
or buffer.
At the
end
of the
incubation
on ice, adding
the
reaction
was
stopped
by placing
the
PMNs
anti-CR3
antibody
(5 L OKM-l),
and mixing, and
incubation
at 4#{176}C
was continued
for 30 minutes.
After incubation,
the cells were washed
twice with cold PBS, treated
with goat
antimouse fluorescein-conjugated
secondary
antibody,
incubated
for
30 minutes at 4#{176}C,
again washed and treated with I % paraformaldehyde, and stored
in the dark at 4#{176}C
until analyzed
by a Cytofluorograf 50H and 2150 computer
(Ortho Diagnostic
Systems).
The flow
cytometer
was calibrated
as previously
described,33
and control
studies
were done to establish
that staining
was specific for the
primary antibody
used.
Assay of PMN intracellular
Ca2.
We measured
intracellular
calcium levels by using Fura-2 as a fluorescent indicator of cytosolic
free calcium.
PMNs
(1 x l08/mL)
were loaded with Fura-2
(5
imol/L),
washed,
and resuspended
in HBSS (1 x 107/mL).
The
change
in emission
fluorescence
(5 12 nm) over time after stimulation with FMLP and/or PAF was measured
in a Spex Fluorolog
Spectrofluorometer
(Spex,
Edison,
NJ). The intracellular
calcium
concentration
was calculated
by measuring
the ratio of the fluorescence intensity
at excitation
wavelengths
of 340 nm and 380 nm as
described
by Grynkiewicz
et al.’ The resting neutrophil
intracellular calcium
concentration
was 40 ± 10 nmol/L.
PMN-mediated
endothelial
cell detachment
and killing.
The
ability
of PMNs
to cause endothelial
cell lysis and detachment
was
measured by using 5’Cr-labeled
HUECs
grown
to confluence
in
24-well 2-cm2 dishes as previously
described.3’
Statistics.
Results
are expressed
as means
±
SEM. Statistical
analysis used Student’s
t test.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
VERCELLOTTI
1 102
ET
AL
20
30
a.
z
a,.,
#{149}
(I)
z
0
ia.
a
Wa,
a. 0
a.
<
a.
0
:
0
PAF+FMLP
CE
A
:#{149}#{149}?
0
0
-11
PAFALONE
-9
-10
PAF
(log
-‘8
#{149}‘
U)
20
10
40
30
TiME
40
60
50
(MINUTES)
IX
Fig 2.
Time course
for PAF priming.
PAF (10
mol/i)
was
added
to PMNs for varying
time intervals.
after which
the cells
were stimulated
with FMLP (iO
mol/i)
and superoxide
production measured
over
1 5 minutes.
Results
are expressed
as the
mean increment
(nmol superoxide/10
PMNs/1
5 mm) above that
of buffer-pretreated.
FMiP-stimulated
PMNs (5 nmol/ 1 0 cells/ 15
mm).
30
20
I
10
0
_
a,
B
mm
j
-11
a
I
,
,
.
0
-10
9
PAFALONE
-8
PAF (log U)
five
Fig 1 .
(A) Effect of PAF upon FMiP-stimulated
PMN superoxide production.
PMNs (2 x 1 0) were pretreated
for five minutes
with
varying
concentrations
of PAF and stimulated
with
FMLP
(iO
mol/i)
or buffer
for 15 minutes
at 37’C
and superoxide
measured
as in Methods.
Results
are expressed
as means
± SEM
nmol
superoxide/
1 0 PMNI 1 5 mm released.
(B) In analogous
incubations,
elastase
release
after 20 minutes
was measured
as in
Methods.
One hundred
percent
elastase
release
equaled
the
amount
released
by PMNs
(2 x 10/mL)
pretreated
for five mmutes with cytochalasin
B (5 isg/mL)
and stimulated
with FMLP
(1O
mol/i).
Results
are expressed
as the mean percentage
±
SEM of elastase
released.
PAF
primes
PMN
respiratory
addition
of
concentrations
superoxide
mol/L),
FMLP.
up
to
also
then stimulated
superoxide
are
of
synergistically
were
PMN/15
priming,
primed
mm
±
was
(I0_8
I nmol/l06
.01).
Other
PAF,
that
is,
mol/L)
also
induced
by PMA (34%
with PAF plus PMA,
P
PAF
dependent
enhances
a maximal
priming
PMA
enhanced
±
<
2%
increment
limited
mm
release
with
This
the
of PMNs
to
before
with
20
2
±
pretreated
addition
ofFMLP
still
stimulation
washed
away
provokes
1 nmol/l06
±
sion
(l0
(line
mol/L)-a
2)-significantly
of FMLP-stimulated
Similarly,
become
non,
PMN
PAF
(lO_8
induce
first
with
primed
more
5 and
(lines
aggregation,
mol/L)
only
to
the
with
themselves
in contrast,
and
then
greater
degree
Stimulated
PMN
Adhesion
PMNs
stimulated
(Fig 3), and
to HUEC
PMN
PMN Adhesion
Treatment
PMN
17.91
±
17.54
±
2.73
±
4.93
PMN
+ PAF + FMLP
69.74
±
2.61
release
PMN
+ PMA
37.20
±
3.921
2%
PMN+PAF+PMA
were
minutes
and
PAF
with
ng/mL)
and
Results
e
after
experiments
(%)
P
2.81
32.71
is time
by
to a much
Table 1 . PAF Primes
treated
phenomeenhanced:
mol/L)
minutes
4).
with
cells
synergistically
(l0
for five
aggregate
than
aggregation;
PMN
PAF
3 and
stimulated
adhesive
+ FMLP
noted
hyper-
not itself
the adhe-
lines
and
adherent
is also
modest
treated
FMLP
PAF
6). Another
FMLP
or
that does
enhances
+ PAF
release
the
mm).
(compare
with
significantly
alone
dose
PMNs
neutrophils
PMA
also
±
to
from
identical
before
PMNs/l5
PMN
v 52%
need
PMN
mm-virtually
and then
washed
PMN
in Fig 2. Pretreating
PMNs
with
to subsequently
exhibited
FMLP,
production
(19
PMNs
with PAF
increase
adhesion
PMNs
in superoxide
addition,
effect
PAF
FMLP
exposed
with
elastase
elastase)
PAF,
PMNs/l5
with
PAF
nmol/l06
PMNs
PAF
.01).
(and
response;
priming
Moreover,
subsequent
FMLP
the
60 minutes.
responsiveness.
For example,
PMNs
treated
with PAF (I0
mol/L)
for five minutes
and then
stimulated
with
FMLP
(with the PAF remaining
in the reaction
solution)
produced
when
than cells
Preincubation
PMA
PMNs
PMA
first
functions
are
FMLP-stimulated
PMN
an enhanced
1 nmol/106
±
during
present
obtain
with
large
production
(38
PMN/15
neutrophil
PAF-primed,
of superoxide
as shown
the response
at
no
are
as
pretreatment
neutrophils
release
much
more
elastase
either to PAF or FMLP
alone (Fig 1 B).
PAF
PAF
alone
virtually
not
superoxide
with
P <
by
various
by the
mm).
If PMNs
PAF-as
low
production
(l0
mol/L)
stimulated
v 46
elastase
with
FMLP,
however,
produced
(solid
line).
enhanced
later
and
in Fig IA,
mol/L
induces
PMNs/15
amounts
of
for superoxide
FMLP;
PAF
PMNs
l0
burst
or primed
with
minutes
followed
(dotted
line); likewise,
FMLP
(i0
alone to neutrophils,
provokes
minimal
superoxide
(5 nmol/l06
primed
with
minute
priming
agonist
seen
As
production
when added
mol/L-and
quantities
incubated
for five
be
not
thereafter
the next
over
PAF primes
PMN
adhesive
responses.
Preincubation
of
PMNs
with PAF also enhances
their
adhesion
to endothehum. Data
shown
in Table
I demonstrate
that priming
of
RESULTS
release.
PMNs
were
concentrations
of PAF
of preincubation;
minutes
deteriorates
1
J
<.001
71.95±3.07]
pretreated
stimulated
PMN
with
with
adhesion
expressed
( 10
FMLP
‘
(i0
to endothelium
as the
done in triplicate.
PAF
either
mean
±
mol/L)
or
mol/L)
measured
SEM
PMN
for
buffer
or PMA
as
in
adhesion
five
(100
Methods.
of four
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
PAF
PRIMED
PMNs
AUGMENT
ENDOTHELIAL
DAMAGE
1 103
C
PAF+FMLp
300
a,
U
U,
a,
0
200
a,
C
C
100
C
-12
-11
.10
-9
PAF
(log
-8
-7
-6
PA)
Fig 4.
Effect of PAF on PMN CR3 expression.
PMNs (5 x 10)
were stimulated
with varying
concentrations
of PAF. and the CR3
expression
was measured
as in Methods.
Results are expressed
as
the mean ( ± SEM) channel
fluorescence.
FMLP
PAF
HBSS
1MlN-
-4
l0”
mol/L)
no change
occurs;
however,
if PMNs
in
are
intracellular
first primed
calcium
with these
levels
same
tiny concentrations
a dose-dependent
and then stimulated
in intracellular
with
calcium
FMLP
levels
of PAF
increase
above
evident.
that
The
priming.
order of addition
of agonists
is critical
As shown in Fig 6, the addition
of PAF
five
minutes
tion
of FMLP
mediated
by
followed
over,
Fig 3.
Effect
of PAF priming
on PMN
aggregation.
PMNs
(5 x 10) were pretreated
with PAF (10’
mol/L)
or buffer for five
minutes
at 37’C
before
stimulating
with
FMLP
(iO
mol/L).
Curves
depict
the change
in light transmission
(ST)
over time.
which reflects
cell aggregation.
was verified
this
and
microscopically
not simply
enhanced
Mechanism
of
Both
expression
of adherent
monoclonal
fashion
small
tude
less
light
adhesion
aggregation.
such
4),
doses
than
and
(lO_12
that
increasing
FMLP
Because
FMLP-induced
lyso-PAF
at position
superoxide
with FMLP
responses
due
aggregation
glycoproteins
1 ) that
as 0KM-
PAF
in
to
the
(assayable
by
also
reverse
is slowed
by cold
because
by
6).
Moreover,
in ancillary
was found
provoked
function
increases
as
the
bar).
experiments
the mean channel
fluorescence
(l0
mol/L)
alone.
receptor-ligand
PAF
2)
incubation
PAF
priming
subsequently
others
have
signal
transduction
intracellular
in Fig 5, at very
calcium
low concentrations
must
be
metabolism
(4#{176}C).This
is
in PMNs
of PAF
also seems
prevented
at
so
cold
8
a,
U,
-13
PAF
in
by
is
As
(l0”
bound
by
of such
PAF
compared
accumulation.
stimulated
shown
that
100
#{149}1
4
(lO_12
increase
FMLP
by 50%
More-
g
a dose-dependent
to amplify
the
by l0
mol/L
In marked
is seen if the
(second
order
addi19 nmol
requires
an intact
PAF
molecule
has a hydroxyl
group
instead
of an
or methoxy-PAF
does
not prime
PAF
(which
production
in PMNs
(bars
3 and 4). Finally,
priming
for maximal
to PMNs
for
the subsequent
bar,
Fig
production
is
PAF
involve
alone
alone
(first
superoxide
in the
alone
production
PAF,
to exert
its other
known
actions,
specific
membrane
receptors’2’36-the
for
thought
to be mediated
by changes
in intracellular
calcium
levels and phosphatidylinositol
turnover,
we assayed with the
calcium-sensitive
probe
Fura-2
the effect
of priming
upon
shown
by
and
superoxide
mol/L)
significant
amplification
occurs
with
mol/L),
some three orders
of magnineeded
to produce
perceptible
PMN
mol/L)
preincubation
CR3 receptor
expression
with
and
surface
antibodies
because
acetate
PAF
aggregation
added
priming
transmission.35
PMN
for iC3b (CR3 receptors).
of these
CR3
receptors
(Fig
very
to be enhanced
enhanced
priming.
receptors
expression
are
agonists
01
StIMULUS
(I0
by washing
increases
over either
agonist
contrast,
no enhanced
ADDITION
FMLP
to
-I 2
-11
(log U)
Fig 5.
Effect of PAF priming
on PMN intracellular
Ca2. PMNs
(107/mL)
were
primed
for five minutes
with varying
concentrations of PAF and then stimulated
with FMLP (10’
mol/L)
or buffer,
and the intracellular
calcium
concentration
([Ca2Ji)
was measured after 20 seconds
as in Methods.
The resting
PMN [Ca2]i
was 40 ± 10 nmol/L,
and FMLP (10’
mol/L)
increased
[Ca2Ji
within
20 seconds
to 142 ± 1 1 nmol/L.
PAF (10h1
to 1O’
mol/L)
itself did not increase
[Ca2]i
over resting
levels
(dotted
line).
Solid-line
results are expressed
as the percent
increase
of [Ca2]i
over that in cells treated
with FMLP alone. The depicted
data are
representative
of those from five separate
experiments.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
1104
VERCELLOTTI
surenone,
and
subsequent
L652
all inhibit
agonists
and
of
enhancement
compounds
mediated
PAF
LYSOPAF
AFTER
METH010
PAP
PULP
that is, the addition
at 4#{176}C
followed
by washing
PMNs
of PAF (lO_8
and subsequent
with
superoxide
FMLP
at 37#{176}Cdoes
not result
production
over either agonist alone
tion
Not
of superoxide
trophil
inhibit
PAF priming
detachment.
enhances
PMN-mediated
We previously
showed
mediated
endothelial
PMNs
to endothelial
tion
and
adhesion
release,
and
we
release.43’
aggregation
that
suspected
Because
as well
such
(line
PMN
damage,
(line 5);
this
even during
notwithstanding,
PMA-mediated
Inhibition
recently
the
damage
of
PAF
described
(line
priming
PAF
receptor
Table
2.
shown
further
antagonists
PAF Priming
PAF
activation
neutrophil
Enhances
by enhanced
reported
We
now
that
superoxide
release,
release,
observations
shown
were
For
to be completely
PAF
antagonists
suggest
that
mechanisms
instance,
to diverse
We
Although
Lysis and Detachment
neutrophil
of Endothelial
1.90
±
+ FMLP
1.29
± 0.521
2.68
±
4. PMN
+ PAF
5.04
±
1.26]
6.26
±
5. PMN
+ PMA
8.63
±
2.901
9.80
6. PMN
+ PAF
±
3.65]
as means
2%.
±
SEM
One hundred
of three experiments
percent
endothelial
done in triplicate.
cell lysis was
measured
28.01
Spontaneous
Iysis with buffer
by the addition
a critical
endothelial
chemotaxin
role
cell
and
(%)
± 0.55
3. PMN
detachment,
play
powerful
2.27
and are expressed
spontaneous
its
Monolayers
0.48
.01
exposed
modulate
in sepsis-associated
pulmonary
1.28 ± 1.07
‘#{176}<
insights
inflammation.
may
may
(C5a)
is a
recently
is probably
that
that,
to cause
C5a
.05
et
kadsure-
new
during
mediators
suggested
and
provide
may
Detachment
‘#{176}<
Gay
PMN
dam-
by the
L-652,
damage
complement
neutrophils
inhibited
results
circulating
previously
activated
in activating
damage.2
these
inflammatory
function.
by
as well as
confirm
PAF-primed
endothelial
BN52021,
of vascular
the
as
as measured
et a123 and
1.16
“<
.02
±
0.51 1
1.26]
2.90 1
±
3.64]
‘#{176}<
.01
PMNs (5 x 106) were primed with PAF (i0
mol/L) or buffer for five minutes
at 37#{176}C
and then stimulated
with FMLP (i0
ng/mL)
and the specific
endothelial
cell lysis and detachment
after three hours measured
as in Methods.
Values are percentages
release
to subsePMA.
The
burst
as
degranulation
of Ingraham
+ PAF
27.08
quantities
for the first time
that
these
are also excessively
damaging
to
2. PMN
+ PMA
neu-
priming
minuscule
and adhesion
Lysis (%)
+ FMLP
by PAF
the agonists
FMLP
and
responses
include
respiratory
age
into
2
±
endothelial
responses
endothelial
cells.
Moreover,
and subsequent
PMN-mediated
none.
We
of
cell
0.72
also
enhanced
induced
responses
described
, kad-
1. PMN
L652
the PAF antagonists
did not
PMN-mediated
endothelial
neutrophil
demonstrate
neutrophils
PAF-primed
PMN-Mediated
by
by elastase
cultured
PMN Treatment
only
endothelial
production
PAF-enhanced
demonstrate
enhance
measured
The
BN52021
7A),
(Fig 7C).
superoxide
and
by inhibiting
the increment
studies
markedly
recently
and then
signifioccurs
antagonists.
and
probably
but only
present
ARDS,
PAF
kadsurenone,
adhesion,
8B),
evidenced
6).
by
not shown).
al.22
in Table
aggravates
These
(Fig
adherence
of neutrophils
to human
endothelium
PMN
aggregation.
These
findings
essentially
PMN
a more powerful
stimulant
PMN-mediated
endothelial
brief incubation
PAF priming
itself,
(data
quent
primed
wrought
by activated
2, we used concentra-
If PMNs
are first primed
with PAF
minutes
later with FMLP,
however,
endothelial
cell lysis and detachment
4). PMA
(100 ng/mL),
responses,
does
cause
and
(Fig
injury
of
(l0
mol/L)
of FMLP
and PAF
that
when
added
to PMNs
cause
little endothelial
lysis or detachment
(lines 2 and 3).
stimulated
five
cantly
enhanced
adhesion
neutrophil-mediated
by inhibiting
enhanced
elastase
release. Again,
the agonist-stimulated,
The
as superoxide
and elastase
priming
might
aggravate
endothelial
cell lysis and detachment
PMNs.
In support,
as shown
in Table
tions
alone
enhances
injury.
inhibit
PAFPMN
functions,
DISCUSSION
in amplified
(last bar, Fig
endothelial
lysis
that
neutrophil-
PAF
BN52021,
mol/L)
to
stimula-
damage
depends
on close adhesion
of
monolayers
as well as oxidant
produc-
protease
endothelial
PAF
release,
or adhesion
by themselves,
but
PAF priming
response
(data
not shown).
surprisingly,
detachment
to
abrogate
neutrophil
AL
of neutrophils
(Fig 7B), and elastase
release
did not inhibit
FMLP-induced
inhibit
PAF-aggravated,
lysis (Fig 8A), probably
6).
and
PAF-primed
release, elastase
the incremental
Fig 6.
Requirements
for PAF enhancement
of PMN superoxide production.
PMNs
(2 x 10)
were
primed
with
PAF (1O
mol/L)
for five minutes
at 37’C and stimulated
with FMLP (iO
mol/L)
and superoxide
(nmol/1O’
PMNs/15
mm) measured
as in
Methods.
In some experiments.
FMLP (1O
mol/L)
was first added
for five minutes.
the cells washed.
and then PAF (1O
mol/L)
added
(bar 2). Alternatively.
lyso-PAF
(iO
mol/L.
bar 3) or
methoxy-PAF
(iO
mol/L.
bar 4) were
used instead
of PAF. or
PAF (10’
mol/L)
was added
for five minutes
at 4UC
before
stimulation
with FMLP at 37’C (iO
mol/L,
bar 5). Results
are
expressed
as the mean ± SEM increment
in superoxide
produced
(nmol/1O’
PMNs/15
mm) above stimulation
with FMLP alone.
temperatures,
PMN-mediated
superoxide
release
These
antagonists
PAP UPTAKU
SLOcKADE
(4C)
priming
concomitantly
in micromolar
concentrations
incremental
responses
in several
including
PAF
PAF
ET
alone was
of H202 (0.3%)
to control
mol/L) or PMA (100
of maximal
lysis or
approximately
wells.
5% and
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
PAF
PRIMED
PMNs
AUGMENT
ENDOTHELIAL
DAMAGE
1 105
30
50
40
20
-a
fi
30
a,
20
10
Al
_10
A
,
.
0
SN
KAD
L852
A
0
SN
KAD
L652
0
BN
KAD
L652
20#{149}
30
zo.
u0
ZI
<0
a.
20
01
0
oz
I
-I-
B
.-
,
‘
0
BN
1652
KAD
10
I-.
U
B
Fig 8.
Effect
of PAF antagonists
on PAF-enhanced,
PMNmediated
endothelial
cell lysis and detachment.
BN52021
, kadsurenone.
1652, or control
buffer
was added
to PMNs
as in Fig 7
before
priming
with PAF (10
moIhL).
PMA
(100
ngbmL)stimulated
specific
endothelial
cell lysis (A) and detachment
(B)
were
measured
as in Methods.
Results
are expressed
as the
mean ± SEM
increment
that PAF priming
induces
above
PMA
stimulation
alone.
00.
C
BN
‘
KAD
PMNs
Fig 7.
Effect of PAF antagonists
on PAF-enhanced
PMN adhesion, superoxide
release.
and elastase
release.
BN52021
(100
pmol/L,
BN). kadsurenone
(lOOpmoI/L.
KAD). 1652 (100 smol/L).
or control
buffer
(HBSS
with 0.1 % DMSO.
0) were added to PMNs
for five minutes
at 37’C before
priming
with PAF (10’
moth).
FMLP
(10
molbL)-stimulated
PMN
adhesion
(A). superoxide
release
(B). and elastase
release
(C) were measured
as previously
described.
that PAF
promotes
stimulus
Results
are expressed
priming
induces above
as the mean
FMLP stimulation
catastrophic
ARDS.
endotoxin,
although
a sluggish
In fact,
others
neutrophil
responses
to other
PMN
soluble
thus
it is a weak
This sug-
agonists
promote
have
might
be
full-blown
demonstrated
agonist
that
itself,
stimuli.’9
primes
of toxic
“stickiness”
and
mediated
vascular
of PMN
damage
is by its capacity
surface-adhesive
to increase
glycoproteins
(Fig
the
4);
this would
presumably
enhance
the margination
of endothehal-damaging
PMNs.
Second,
because Dahinden
et al3t have
demonstrated
that cell-cell
contact
augments
production
by
thrombin-exposed
are layered
atop
hyperresponsive
PMNs
become
agonists
such
per
enhanced
se
further
Finally,
we
mechanism
by
in inflamma-
endothelium
and
thrombin
that
may,
attending
of PAF
activated
FMLP,
mediation
becomes
an
blocked
occurs
endothelium
they
ineffectual
doses
of
PMA.
this
PAF
PMNs.
These
paracrine
amplification
of
for instance,
accompany
damage
findings
intravascular
provide
that
by
an
damage
coagulation
the small amounts
in the vicinity
of
trivial
activators
then become
is correct,
syndromes
rebe
suggests
stimulated
endothelial
sepsis.
That is, we propose that
derived
from
endothelial
cells
coagulation
potentiate
otherwise
vascular
inhibitors.
amplified
elastase
damage-can
by PAF
inhibitors;
between
endothelial
marginated
These
production,
of endothelial
of marginated
PMNs;
these cells
same endothelium.
If this construct
mediated
by PAF
or
superoxide
actual
and
thrombin-treated
to otherwise
as C5a,
responses-including
interesting
and Etienne.’8’24
One
involved
in endotoxin-
that
might
adhesive
substrate
for neutrophils2627-perhaps
of this production
(Table
I ). Stimulated
by these
observations,
we recently
preliminarily
reported39
that
if
endothelium.37
indeed
play
has been recently
reviewed
by Braquet
way in which PAF might
be critically
likely
cells
excessively
by virtue
specifically
“cross
talk”
evidence
that PAF does
role in endotoxic
states
it seems
,
cells,
endotoxin
induces
from
macrophages
the release
of the
monokine
I L- I , which
can alone augment
neutrophil
adhesion to endothelium30
and also can induce
PAF synthesis
in
In this latter
regard,
an important
etiologic
species,
PAF-primed
tory
states:
activation
by inflammation
of intravascular
coagulation
with
production
of thrombin.
Thrombin,
like
IL- I has been shown to induce PAF production
in endothe-
lease,
In addition,
02
of
aggravate
PMN
aggression
toward
endothelium.
have recently
provided
evidence
for a third
which PAF probably
worsens
vascular
damage
hal
alone.
neutrophil
adhesion
and aggregation,
the respiratory
burst
and exocytosis.
inflammatory
PMNs
and
increment
SEM
±
of
gests
that
additional
required
to fully provoke
expression
0
should
lethal
to that
inflammationbe ameliorated
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
VERCELLOTTI
1106
ET AL
REFERENCES
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