by phospholipase. D determines phosphat- idate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. J. Bwl. Chem. 264. 17069-17077.
Transforming
growth
oxidant
factor-1
stimulates
release
by adherent
human
J. Balazovich, J. Suchard,
Rosemarie and Laurence
Kenneth Suzanne Department
Abstract:
The
of Pediatrics,
signal
University
transduction
by cytokines and growth their receptors on human neutrophils understood. When PMN in suspension of these primed
agonists they for subsequent that
ported
when
stimulated
tide
are
PMN
with
of Michigan
pathways
activated
cytokines
plated or with
that
but and
forming
onto librinogen the chemotactic
by releasing hydrogen granule component factor43i
growth
cells
including
PMN.
It has
stimulates chemotaxis oxide production by cells sized when
that UF-31 they were
mediated determined
by
would adherent
Ii
activate to
by PMN
adherent
In
‘RF-
this study, H202 and
to fibrinogen.
IXW-
and
rin
release.
lactoferrin
release
phospholipase
D pathway.
In
3H]phosphatidylcholine,
we
treatment caused Propranolol (150 phosphohydrolase, rin
release,
phatidic
that
suggesting
acid
to
PMN
labeled
observed
the
cliradylglycerol
with that
by
lyso-
1XF-31
Riot.
60:
mechanisms.
one
specific
members
generate
proteins,
This
heat,
latent
growth,
In
opposing
a reviewj,
apparent
paradoxes
TGF43i kocyte
are or released
adhesion,
infiltration,
synthesis
as
fibronectin
13i
administered systemically has the expression of endothelial
iting
and
decreasing
tion
[4].
blocks
In
vitro
PMN
[1-
ester PMN
expression
adhesion
Thus
PMN
step
these results activation of
conditions.
activation
lipase
D
772
Journal
.
peroxide
.
a common pathway and cytokine ac1996.
772-777;
phosphatidic
kocyte;
H202,
KRPG,
Krebs-Ringer
tidic
acid;
TNF-a,
acid
.
Biology
Volume
60,
leu-
regulating
for integrins other hand,
such TGF.
opposite effect, inhibcell adhesion proteins
in response
to phorbol
selectively
promote upon the
December
transforming
role
growth PMN,
hydrogen
peroxide;
KRP,
phosphate
lyso-[3HPC, tumor
or
inhibit
specific of
en-
TGF-1
in
buffer
factor-1i;
fMLP,
polymorphonuclear
Krebs-Ringer
phosphate
with
I3HIPA,
glucose;
N-
leubuffer;
phospha-
1O3HJoctadecyl-sn-glycero-3-phosphochohne;
necrosis
factor
a;
1996
Laurence
of Pediatrics, Dr.,
Received
phosp/io. 30,
Leukocyte
by
ELISA,
enzyme-linked
immunosorbent
assay.
secretion
of
in vivo
to promote
to sites of inflammaPMN chemotaxis but
Presently,
TGF431,
Abbreviations:
Center
hydrogen
2
is unclear.
Correspondence:
Words:
ref.
example,
depending
formyl-methionyl.leucyl-phenylalanine;
ment
Key
may
[see
these
activation
to fibninogen
responses
vironmental
For
recruitment promotes
TGF431
functional
cells
of receptors On the
leukocyte TGF-31
matrix
promotes
underlying
is said
the
by
reportedly
TGF-
[31.
laminin
other
activated
TGF-f3
same
known.
the
and
be
to
two
extracellular
the
and
a large
dimenized
with
active
locally
and
expressed as
and
mechanisms
not
applied and
in
the
are
can
cases,
is
related
synthesized
Active
activities
although
understood
and
some
membranes (TGF43i)
complex
treatment.
These
factor-31
complexes
differentiation,
synthesis.
PMN
altered
that
cells
1J.
of poorly
of which is
TGF43i
or enzyme
seemingly
three
TGF41
peptide
many
of structurally
is proteolytically
a 25-kDa
protein
growth
tissues. that
on
a variety
of a group
similar
by (PMN)
receptors
through
mammalian
released
are
leukocytes
Transforming
of five
for
factors
of phos-
important
Overall, for fMLP
PMN and suggest that in both chemoattractant
J. Leukoc.
conversion is an
PMN activation by 1XF-31. are similar to those reported
tivation.
detected
an increase in I3Hlphosphatidate. .tM), an inhibitor of phosphatidate blocked both H202 and lactofer-
in
adherent is involved
were
‘ontinued for at least 180 min. Adhesion of PMN paralleled H202 and lactoferEthanol (200 mM) blocked both H202 and release, suggesting the involvement of the
to them
regulates
Both
lactoferrin
bind activate
acid,
H202 and lactoferrin release from adin a concentration-dependent manner, seen in the range of O.i to 100 pg/mL.
H202
agonists and
growth
polymorphonuclear
proteins.
we lac-
Arbor
and
including
precursor
stimulated herent PMN with effects 60 min ant and spreading
Cytokines
in
that
Ann
INTRODUCTION
functionally
PMN to release H202 fibrinogen, a response
132 integrm receptors. whether ‘JXF-31 stimulated
release
toferrin
reported
Hinkovska-Galcheva,
of Medicine,
(H202) Trans..
but not exocytosis or superin suspension. We hypothe-
and
neutrophils
Vania
(fMLP)
by many
is released
been
are reand pep-
peroxide lactoferrin.
(1F431)
1i
are
rather others
N-formyl-methionyl-leucyl-phenylalanine
they respond and the specific
School
factors binding to (PMN) are poorly encounter many
not activated, activation. We
are
Fernandez, A. Boxer
degranulation
1996.
Room April
Section L-2110, 2,
1996;
A. Boxer,
University
of Michigan,
of Hematology/Oncology, Ann revised
Arbor,
MI
August
1500
DepartB.
Medical
48109-0238. 29,
1996;
accepted
August
Human
peripheral
blood
fense lenges
against bacterial primarily by
such
as
superoxide
and
ent
to plastic,
strates
and
It
is
For
PMN
versus as
the
that
lag period
suspended
PMN
agonists
that
suspension
is
monophasic
the
second
corresponding
nived
to
from
the
TGF-Ii
of
promotes
PMN
and
lactofernin
also
when
release
were
we
able
and
to activate
oxidants
if the
to release
[9, i6J. investigated
stimulate
lactoferrin
and
were
have
TGF-1-dependent to involve phosphatidate
duced
by the
activation
In
a
to extracelof
by
PMN
activation and
(3
nm
activated PMN release of H2O2 manner. FurPMN pro-
manufacturer acrylamide choline Amersham.
and
human
found
platelets.
to be
gel electrophoresis.
factor. used
assessed
was
by sodium
dodecyl
by the
sutfate-poly-
l-O-(3Hjoctadecyl-sn.glycero-3-phospho-
(lyso-platelet-activating Reagents
> 95%
Its purity
for
lipid
161
Ci/mmol)
analysis
were
was
obtained
from
by
myeloperoxiin a final
were
warmed
to 37#{176}C.Plates to
The and
by
H2O2
an
emission
for
substituted
remove
cells
in relative was
measured wavelength
spectrometer.
specific
of scopoletin
con-
indicated
reduction
fluorescence the
con-
peroxidase,
centrifuged
nm
glucose
for
generating
Omis-
decrease
in fluores-
H2O2.
Control
agonists
and
samples inhibitors.
a standard
curve
using
of H2O2.
evaluated
for
the
been
samples
diluted
before
optical
densities
also
calculated
from to
the
the
examined
for release
developed
elsewhere
within
for
Lactoferrin
ELISA
addition
fell
were
reported
was their
(ELISA5)
granules.
sensitive
have
in
release
of specific
highly
of which
assays
H2O2
a component using
Adhesion
by (8J.
Birgens
The
lacto-
a standard
assay
linear
in
curve.
order
portion
to
of the
enstan-
assay
Adhesion
assays
were
Measurement plated onto The
conducted
as
previously
of phosphatidate fibrinogen
measurements
of phosphatidic
161.
described
PMN
factor,
lyso-[3HPC)
trifuged
at 300
at
x
1
KRPG.
PMN
mM
acid
10
Veldoven
described
in PMN
were
out
carried
were
wells
and
response.
(18,
p1
Balazovich
the
KRPG,
with
200
mM
at 37#{176}Cbefore during
the
cells
were
stimuli
M
for
For
to
this
i08
were
medium
were
et al.
TGF-31
and
onto time
course iden-
At designated
times.
x
the
modification
of the
the added
with and
neutrophil
medium to each
a Teflon
combined
of
in an (8
was
or
fibrinogen.
determination
removed pelleted
cen-
ethanol
(0.64%)
entire
were
resuspended
each
procedure,
methanol
PMN
incubated
or inhibitors.
according
PMN
as
with
and
plating
present
of ice-cold
Adherent with
with
combined
191.
labeling,
twice
treatment)
without were
extracted
Bell
250
mm
(no
in buffer eight
at 37#{176}C.After
washed
were
Control
essentially
incubated
(lyso-platelet-activating
mm
5
I#{149}
(6.
formation
cells/mL
preincubated for
propranolol
and and
30
mm,
were
propranolol or
removed
for g for 6
cells/sample)
the
cells/well)
curve.
moved
high-performance
10
PMN
warmed
NJ) PMN
inhibitors
plate,
was
abolished
by
ig/mL).
H2O2
of scopoletin
was
(50
air at 37#{176}C.At the
LS-50B
their
from
from
were
of 365
were
manner
isolated
to the
wells
accomplished
concentration
cells
was
of
and/or
to cuvettes.
oxidation
KRPG
were
details
that
the
Falcon
5 mM
x
(1
agonists
immunosorbent
determined the
in
specificity
but
release,
tical MA).
the
that
Reagents (Bedford,
as
Park,
of horseradish
in humidified
peroxidase
was
experiment.
Products
viable
described
containing
5 tg
plates
wavelength
concentrations
Ethanol
Biomedical
greater
98%
Lincoln
wells
addition
and
to the
PMN
buffer
destruction
their
in a Perkin-Elmer
the Collaborative
were
resid-
polystyrene
1-O-(3Hloctadecyl-sn-glycero-3.phosphocholine
D.
of
as previously
Dickinson,
indicated
transferred
indicating
dard
on
sedi-
lysis
than
of fibrinogen
prevent the
solution
excitation
150
from
from
of dextran
Preparations greater
assayed
(Becton
incubated
ofhorseradish
sure
in
obtained
were
due
an
was
use
flat-bottomed
scopoletin,
temperature the
M ETHODS
TGF-1,
(to
supernatant
Samples
to
plated
of adherent diradylglycerol
of phospholipase
the
ferrin
adherent ability
intervals,
and
that
exocytosis
the
time
(17J.
reported
release
PMN
sup-
to undergo
fibninogen. We demonstrated that TGF-1 adherent to fibninogen, stimulating the and lactofernin in a dose- and time-dependent thermore, appeared
[15].
tM
Before
taming
was
phosphate
24
of 1 mL.
lactoferrin
to fibrinogen,
PMN
to fibrinogen-coated
and
Samples
H2O2
by
hypotonic
f6J. and
concentrations
Enzyme-linked lactoferrin
hypothesized
receptors PMN
cells
lular matrix proteins In this study we H2O2
we
blood
in 24-well, plates
azide
to room
known
promote
[141,
others
obtained
otherwise.
exclusion.
adherent
catalase),
contained
TGF-31
to
blue
and
slowly
of 473
or induce
PMN
volume
using
de-
staining
added
Q uantitation
since
ability
adherent
.32-integrin
hypothesis,
was only
activate
they
by
burst
its
were
stated
and
described
conducted
sodium and
cence,
and
whole
as previously
in Krebs-Ringer were KRPG
sion
from
culture
fluorescence
formation
However,
through
and
D [6J.
respiratory
to fibronectin
would
mediated
TNF-a and
chemotaxis i
of our
the
suspension.
binding
the
port
in
reagents
unless
centrifugation,
saturating
suspended
1 mM
fibninogen are is biphasic,
of phospholipase
with
dase
[10-131.
release
coated
taming
of
D
diradyiglycerol
lactoferrin
other
MO)
by differential
by were
tissue
(KRPG)
For excells in
C
plated on formation
of
and
to activate PMN
integrin
that
response
phase H2O2
activation
fails
exocytosis PMN
when PMN diradylglycerol
91.
isolated
trypan
released Assays
Primaria
activation
phospholipase
hand, fMLP,
All
Louis,
assay
H2O2
factor
from
PMN by
H202
in suspension,
phospholipase
98%
[6J.
[8,
were
determined
are activators of adactivated by fMLP are
results
phosphatidylinositol-specific
only
than
of H2O2
adherent PMN. in fMLP-treated
and
(St.
Ficoll/Hypaque
erythrocytes
ual
PMN
colony-stimulating
a (TNF-a), pathways
grade. Co.
PMN
mentation,
in suspen-
fMLP
PMN
phosphatidylcholine-specific other with
with
prime
Chemical
Human
sub-
adherent
detection
activated
only
chromatography
Sigma
Cells
adherother
PMN
before
also different in suspended versus ample, diradylglycerol formation
with
and
than
liquid
cytoplas-
become
proteins,
granulocyte-macrophage
On the treated
have
differently
and tumor necrosis factor herent cells. The signaling
both
of de-
is significantly longer, and the total amounts lactoferrin released are greater in adherent
Furthermore, such
line
to host chalmetabolites
secreting
documented
quite
example,
and lactoferrin of H2O2 and
by
they
matrix
well
to agonists
sion.
and
after
extracellular
[6-9].
respond
a first
respond oxygen
that contain proteolytic enzymes. to release these compounds when
in suspension
are
provide PMN toxic
H2O2,
mic granule contents PMN can be stimulated they
PMN
infection. releasing
was well
scraper. with
activation
l0 Van
the
re-
to stop PMN meth-
773
anol
extract
from
chloroform (1:2:0.8,
v/v),
were to
corresponding
and
water vortexed,
centrifuged
a clean
water
the
and
and
centrifuged
and
washed
centrifugation,
an
the
under acyl
a stream
raphy
plates
group
in a solvent
by
treatment
with
corresponding lipid
The
areas
and
assayed
by
Statistical
dried
0 Ii
20#{176}Cto mm-
-
chromatogand
(12:2:3:
D fl9j.
with
primulin
were
identified
and
0
separated
0
of ethyl
10,
v/v).
10
1
1000
100
LOG [TGFJI,]
Unla-
from
Thin-layer areas
under
acid
scintillation
at
After
and
0
pg/mi
dioleoylphosphatidylcholine
has
of the
a UV
chro-
Fig.
2.
plate
plated were
on fibrinogen-coated added to wells (10
with
fibrinogen
lamp.
a slightly
In
faster
mo-
dioleoylphosphatidylcholine.
to phosphatidate
liquid
removed
(9:1).
of a mixture
acid:water
produced
was
to thin-layer
phase
l-O-alkyl-phosphatidic
corresponding
were
scraped
from
the
plates
counting.
Dose-response
bated
for
H2O2
release.
curve
for
stimulation
plastic cells each)
containing
varying
Data
are
samples
means
release
±
PMN
three
also
Fig.
PMN coated
43
of
withdrawn
from
SE
by
with TGF431. 24-well plate
concentrations
mm at 37#{176}C, and
120
of H2O2
and stimulated of a Primaria
and
incu-
.
assayed
experiments
for
done
in
triplicate.
analysis
Statistical
significance
was
between
means
differences are
reported
RESU
upper
final
vortexed
tube
0
phases, the
were
phase
stored
w
that
as a standard,
from
sprayed
standards
lower
were
-J
two
so
Samples
applied
-J
supernatant
To obtain
I phospholipase
type
were
system.
than
Data
were
to phosphatidate
solvent
bility
Samples
generated
cabbage
plates
1 h. Samples
the
to a clean
phosphatidate
was
and
spin.
The
of
transferred
supernatant
transferred
of the
for
5
with ratio
1 M NaCl:methanol
pentane:acetic
matography this
was
consisting
phosphatidate
first
mm.
Lipids
unlabeled
mixed
chloroform:methanol:
10:10:9.
of
of nitrogen.
system
the
10
phase
acetate:2,2,4-trimethyl
beled
above.
volume
migration.
with
described to the
g for
lower
was
supernatant
with
was
equal
the
re-extracted
added
at 2,000 with
sample
temperature
g and
from
were
of chloroform:methanol:water
imize
as
supernatant
water
ratio
down
was
centrifuged
with
chloroform
at room
at 2.000
pellet
Each
a chloroform:methanol:water
kept
10 mm
The
(1:2:0.8),
wells.
obtain
and
for
tube.
combined
to
determined
by
were
as the
mean
use
of
considered values
the
Student’s
significant
±
SE
unless
1-test;
if P