ace- tate-stimulated. SO generation. Endotoxin infusion significantly up-regulated ...... Gallery,. M.P.,. Mangino,. M.J.,. Kamei,. T., Fly, MW. (1990). Interleukin-6.
Functional
characterization
recruited
neutrophils
J.A.
Spitzer,
Department
t?/
P. Zhang, Physiology,
of peripheral
in endotoxic and Louisiana
A.M.S. State
neutrophibs functional (PMNs), we
can also cause injury alterations in liver studied the functional
to tissues. To recruited neucharacteris-
cytic activity than blood PMNs or Kupifer cells. The percent distribution of prostaglandins D2 and E2 of total i4C.. eicosanoids was significantly higher and that of thromboxane B2 and 5-, 12-, and 15-HETEs was significantly lower in liver than in blood PMNs. Our study demonstrates several functional differences between liverrecruited and circulating PMNs in an acute endotoxic model. The implications of altered neutrophib function may extend to mechanisms of host defense and hepatotoxicity associated with sepsis and endotoxemia. 3. Leukoc. Biol. 56: 166-173; 1994. Words:
/3
integrins
. phagocytosis
. nitric
oxide
production
INTRODUCTION Polymorphonuclear leukocytes (PM Ns, neutrophils) constitute 70% of circulating leukocytes and are considered to be terminally differentiated phagocytic cells. They circulate passively in the blood, ready to be recruited to sites of infection and inflammation, where they protect the host against invading microorganisms. The neutrophils’ host defense mechanism includes phagocytosis and the killing of microbes through the generation of reactive oxygen metabolites, nitric oxide, and the secretion of several enzymes, e.g., myeloperoxidase and lysozyme, which are located in granules [1, 2]. Activated neutrophils can also cause immunologically induced inflammation and tissue damage. Within minutes of stimulation by a variety ofactivators and chemotactic factors acting on the neutrophils in the local microvessels, circulating neutrophils attach to the endothelial wall of the blood
, 166
Journal
of Leukocyte
Biology
Vcume
56,
rats
Aledical
tics of circulating blood and liver sequestered PMNs in terms of host defense mechanisms, such as nitric oxide (NO) and superoxide (SO) generation, 132 integrin expression, phagocytosis, and eicosanoid profile. Cells were isobated from rats infused with a nonlethal dose (320 .tg/kg) of E. coli endotoxin (ET) or pyrogen-free saline for 90 mm. Liver PMNs produced significantly more NO both in the absence and in the presence of an in vitro endotoxin challenge than did blood PMNs. No significant difference was observed in phorbol myristate acetate-stimulated SO generation. Endotoxin infusion significantly up-regulated the expression of CD11b/c in circulating and even more so in liver PMNs. Phagocytosis was significantly enhanced by in vivo ET treatment in blood PMNs, and liver PMNs showed even greater phago-
Key
and liver
Mayer University
Abstract: Neutrophil accumulation in tissues is a hallmark of inflammation and is associated with a variety of pathological conditions. In bacterial infection neutrophils are selectively attracted in large numbers to phagocytose and kill invading microorganisms. However, activated investigate trophibs
circulating
August
1994
Center,
vessel
of
New
the
at
the
Orleans
site
releasing
defensive
Neutrophils
the
tissue
system
perform
this
activation
for
sites
be
of
trophils
to
spreading,
have of
in
the
liver
phagocytosis by
3-h
and selectively
and [4,
significant and
even
their
5]. by
accumula0.5-
infusion of a nonlethal dose of endotoxin into Within the same time frame we also characterized in [1-14G]arachidonic acid metabolism by Kupffer PMNs and respiratory burst activity in 45 mb/mm fractions, which characteristically contain 50-55 as well as in endothebial cells [5]. We have presented cell-PMN sequent
order
vascular endothelium adThe CD11/GD18 integrins role in adhesion of neu-
demonstrated
the
In localization
endothelium and
previously
PMNs
recruitable
Neutrophils
cytokine-stimulated diapedesis,
We tion
their
regulated.
adhere to and migrate through jacent to a site of inflammation. of neutrophils have a significant
major
inflammation.
appropriately,
tightly
out readily,
[3].
the
acute
migrate
degranubate
products
represent
function
must
then
and
antimicrobial
neutrophils
defense
to
infection. into
Circulating host
of
vasculature
and
1.5-h
rats
[6, 7]. changes cells and elutriated % PMNs, previously
evidence showing the “priming” of mixed fractions from endotoxin-infused animals in vitro PMAor opsonized zymosan-stimulated
Kupifer for subsu-
peroxide release [8]. Furthermore, circulating PMNs from endotoxic rats in numbers corresponding to their presence in the 45 mI/mm elutriated fraction (containing Kupifer cells and PMNs) of endotoxic animals, when coincubated with Kupifer cells of saline-treated rats, produced less superoxide upon PMA stimulation than did the mixed fractions of endotoxic rats [7]. These results suggested that the functional states of peripheral circulating and liver-recruited PMNs are different.
In order to characterize the functional status of circulating and liver-infiltrated PMNs in an early endotoxic insult (90-mm infusion) model, the aims of the current investigations were to explore nitric oxide and superoxide generation, to study the expression of f32-integrins and phagocytosis by flow cytometry, and to assess arachidonic acid metabolism in these two cell populations.
Abbreviations:
dotoxin; IL-I,
DMEM,
FITh, interleukin-l;
arginine;
NOS,
University
Received A.M.S. western
requests:
Medical January Mayer’s University,
E2;
SO,
B4;
synthase;
PMA,
TNF,
Department
Center, 1901 Perdido 20, 1994; accepted current Downers
address: Grove,
acetate; tumor
saline; PMN,
necrosis
Street, New April 4, 1994. 60515.
en-
G2a;
N#{176}-monomethyl-L-
of Physiology,
Department IL
ET,
phosphate-buffered
myristate
superoxide;
medium;
immunoglobulin
NMMA,
PBS,
phorbol
Spitzer,
Eagle’s
IgG2a,
leukotriene oxide
J.A.
modified
isothiocyanate;
LTB4, nitric
PGE2, prostaglandin phonuclear leukocyte; Reprint
Dulbecco’s
fluorescein
of
polymor’ factor.
Louisiana
Orleans, Pharmacology,
LA
State
70112. Mid-
MATERIALS
Measurement
AND METHODS
Superoxide
Materials Endotoxin (Escherichia coil, 026:B6 prepared by the Boivin method) was purchased from Difco Laboratories (Detroit, MI). Tissue culture reagents and all other chemicals were from Sigma Chemical Co. (St. Louis, MO). Fluorescein isothiocyanate
(FITG)-conjugated
microspheres
(1.5
m
in
polystyrene
diameter)
were
latex
obtained
from
Poly-
were
purchased
[1i4G]Arachidonic purchased from
from
Pharmingen
(San
Diego,
CA).
acid (specific activity 58 mCi/mmol) Amersham Corp. (Arlington Heights,
was IL).
Animals Male
Sprague-Dawley
rats
(320-350
g, Charles
River,
Wil-
mington, MA), maintained on a standard laboratory diet and housed in a controlled environment with a 12-h light/dark cycle, were used. On the day preceding each experiment catheters were placed in the jugular vein using aseptic surgical techniques. The rats were then fasted overnight with free access to water. Next morning the animals were infused intravenously with endotoxin or sterile isotonic saline (Baxter Corp., Toronto, Canada) for 1.5 h. The total amount of endotoxin in a total volume
infused of 2.5 ml
lLg of
this
was
infused
administered
at
a rate
rapidly
of
1 ml/h
as
over the of sterile at
the
1.5-h saline. start,
previously
period One and
was 112 milliliter
the
reported
rest
[8].
was
This
nonlethal conformed
dose was equivalent to 320 jig/kg. The use of animals to National Institutes of Health guidelines.
Isolation
of PMNs
Liver-infiltrated elutriation Hypaque Viability trypan
fractions [9] density ofthe purified blue exclusion.
were
obtained
from
45
culture
The
isolated
PMNs
Leukocytes
from
1 x
(circulating
or recruited)
were
Ficoll[5]. by
plated
on
of nitrite
The accumulation of nitrite, a stable inactive nitric oxide production, in the medium was ing the Griess reagent as described previously
end product determined [10].
obtained
106
by
fixed
with
0.5
ml
latex
whole
blood 0.9 and
37#{176}C for
60
was
incubation, lysed
and
0.1%
gelatin
5%
fetal To
blood by
hypotonic
cold
PBS
and
finally
100
l
of
1 x
liver-
106
cells
serum
in a metabolic
of
shock.
an
aliquot
At
the
blood
The
containing
of
cells
of
0.5
108
end were
glucose
ml
of
were
leukocytes
5 mM in
x
adhesion
cells, red
resuspended
were and
1.25
samples,
with
cells (PBS)
of
at 4#{176}C simultaneously.
whole
or iso4#{176}Cin the
nonspecific
surface
of
containing
measure
the
g
paraformaldehyde.
or
bovine
of
l
I
the saline
containing
of DMEM
incubated
removed twice
tl)
ml
on
for
washed
at
measurement (100
mm.
microspheres
mixtures
antibody
or
100
and
incubated
suspension
with
at
latex
serum
1%
U/ml)
cells
in
monoclonal were
for the
microspheres
shaker of
bovine
(10
blood
suspended
of PBS-buffered
neutrophil
incubated
as
of CD11b/c
At the end of incubation, cold phosphate-buffered
Sample preparation phagocytosis
was
fetal
mixtures
with
sequestered
were
anti-CD11b/c
mm.
Heparinized
PMNs 5%
The
dark for 30 washed twice
c
of heparinized of red
p1
destruction
liver-sequestered
antibody.
finally
100
hypotonic
DMEM containing Flit-conjugated type
superoxide
[8].
for measurement
blood
by
ferricytochrome
Sample preparation expression
phagocytic
PMNs
23-mm plastic 12-well tissue culture plates (Gostar, Cambridge, MA) in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum in the absence and presence ofendotoxin (8.5 jzg/well). Each well contained 0.85 ml of medium. Gell densities were 1 x 106 cells/well. Gells were maintained for 20 h in primary culture.
Measurement
described
measured of
of
and
the
same
Flow cytometry
Girculating PMNs were isolated by exsanguinating the rats through either a previously placed carotid arterial catheter or the abdominal aorta. Exsanguination was maximized by using Krebs-Ringer-HEPES (20 mM) buffer with 5 mM glucose (approximately 20 ml) through the venous catheter. The blood was then processed for PMN isolation using a discontinuous Ficoll-Hypaque density gradient as described for elicited PMNs. Cell viability was >95%.
Cell
previously
was
reduction
solution.
mb/mm
by using a discontinuous gradient, as previously described PMNs was >95% as determined
anion
generation
inhibitable
The
PMNs
anion
dismutase
whole
sciences (Paul Valley Industrial Park, Warrington, PA). FITG-conjugated monocbonal antibody against rat CD11b/c (mouse immunogbobulin G2a, IgG2a, clone OX-42) and FITC-conjugated isotype-matched control antibody (mouse IgG2a)
of superoxide
of us-
ing
a
488-nm
(using
as
the
gated
the cells
and
CDlib/c
or
mean
argon
Light
scatter
angle
light
scatter)
laser.
sample
analyzed.
engaged
in
Green
5000
Results
channel
us-
were
cells
were
wi-
expressed
phagocytosis
fluorescence
gates
(% in
of
phagocyto-
expression.
Preparation Previously
were
of
percentage
an
nm.
In each
on
cytometer
525
forward
PMNs.
expression
flow
from
at vs.
regions
phagocytosis) sis
scatter
CDiib/c
ELITE
line
monitored
to identify
and
a Coulter
excitation
90#{176}light
thin
on
was
selected
of PMNs
assessed
fluorescence
of [1-14C]arachidonic published
acid metabolites
procedures
[6]
were
used.
Specifically,
cell suspensions were incubated (1 jCi/ml, specific activity 58.3
with [1-’4Cjarachidonic mCi/mmol, final
tion
17
the
were
washed
tM)
for
with
30
mm.
buffer
After
labeling
containing
1%
acid concentra-
period
bovine
the
cells
serum
albu-
mm (fatty acid free, w/v) and resuspended buffer at a concentration of 5 x 106 cclls/ml. of arachidonate metabolites released by ( 0.45 ml) were transferred to polystyrene cubated in the presence or absence ofGa2 (5 tM) for 15 mm. The reaction was stopped of cold buffer. Unlabeled standards of (PGE2), PGD2, PGB2, 6-keto PGF1a, 5-HETE, and leukotriene B4 (LTB4) (5 tg
For the analysis the cells, aliquots tubes and inionophore A23!87 by adding 4 ml prostaglandin E2 HHT, 12HETE, each) were added
as
lO,000g
markers.
at 0#{176}C.The acid
Spzt:er
activity
were
ci al.
(88%,
Functional
Samples
were
supernatant w/v),
diluted
differences
centrifuged
was to
20
at
in
acidified
to pH
ml
water
in blood
with
and
liver
the
incubation
for
3.4
10
with
and
PMNs
applied
mm
formic to
167
Sep-Pak 20
ml
G18 cartridges
prewashed
distilled
as
of
tridges
were
ml),
and
water eluted
ethyl
appropriate
with
acetate
water to
solvent
20 ml ofethanol described
(10
a small
for
chromatographic acid metabolites.
with
previously ml),
vial
subsequent
(HPLG)
The
petroleum
and
and
[4].
ether
of
I
in
systems
products,
of
liquid
for [1-14C]arachidonic
cycbooxygenase
reverse-phase
HPLC
jected into a column ofWhatman cm) and eluted at a flow rate increasing
acetonitribe
[ii].
The
235
for
mono-HETE
tion
of
radioactive
lected
in
ultraviolet
for
profile,
LTB4,
metabolites.
( 1 00
presented samples was done by
variance,
10 ODS (0.46 x 25 with a gradient of
at
acid
192
used
nm
for
Fractions
for
the
(0.5
(pH
2)
ml)
PG
acid
(1
NMMA
(30
zM)
,‘ O/(.
current study we assessed and state of peripheral circulating neutrophils in terms of NO Peripheral
press
constitutive
while
inflammatory,
iNOS-like
were
and
oxide
[12].
synthase
As
can
(n
be
of
to
an
in
a
seen
ci)
activity,
an
in
inducible, 1, liver-
Figure rats
NO
both
under
vitro
ET
challenge
produce
basal than
cir-
conditions. Withe in vitro ET ofNO producIn one experi-
a p
