In vivo latex phagocytosis hepatic
neutrophils Abraham Department
Abstract: ance of materials
to generate
P. Bautista, of Physiology,
Activation of liver endotoxins, bacteria, may be accompanied
primes
Agnes Louisiana
Schuler, State
superoxide
Zoltan
University
Key mutase
Kupffer to 1992.
Words: perfused
cells. hepatic
The
macrophages during clearor other particulate by the migration of poly-
toxic injury.
oxygen-derived radicals liver, receptor-mediated
oxygen
J.
radicals Leukoc.
ibuprofen phagocytosis
.
may
Biol.
superoxide rat
Spolarics,
Medical
morphonuclear neutrophils (PMNs) into the liver and priming of the hepatic phagocytes to release toxic oxygen metabolites. In the present study we investigated the effect of in vivo administration of latex particles on the hepatic sequestration of PMNs and the release of superoxide anion (Of) by the in situ perfused rat liver and isolated hepatic phagocytes. One hour after an intravenous injection of latex beads, a significant amount of Of (0.7 nmol/min/g) was produced by the in situ perfused liver. Administration of latex particles into the perfused liver also elicited Of production. Hepatic phagocytes from latex-treated rats generated large amounts of 02 (2-14 nmol/60 min/106 cells) when these cells were stimulated in vitro with opsonized zymosan or phorbol myristate acetate (PMA), whereas phagocytes from saline-treated rats released less than 0.8 nmol 02. Intravenous infusion of superoxide dismutase or ibuprofen did not prevent the immigration of PMNs to the liver. However, ibuprofen inhibited the production of 02 by the perfused liver. Also, after addition of ibuprofen in vitro to isolated cells, there was more than 50% inhibition of 02 generation by Kupffer cells and hepatic PMNs treated with either zymosan or PMA. These observations suggest that arachidonic acid metabolites play a role in 02 release under these conditions. Thus, activation of the reticuloendothelial system by latex phagocytosis induces the migration of PMNs into the liver and enhances the production of toxic oxygen-derived radicals by these cells and the resident contribute 39-45;
the Kupffer
also 51:
is
due
to
anion
and
Center,
cells and
New
John
Orleans,
these
cells.
J. Spitzer Louisiana
In
the
course
of
these
events,
Kupffer
cells become activated and cytotoxic by virtue of their enhanced capability to generate toxic oxygen-derived radicals [1, 3, 14] and cytolytic proteases [i21. Resident macrophages are normally quiescent cells but, upon activation by bacterial products such as endotoxin or lipopolysaccharide (LPS), secrete a variety of cytokines [19], superoxide anion [3, 14], cytolylic proteases [i2], and chemotactic factors [22]. This complicated array of metabolic and functional events is rather difficult to dissect; therefore, it wasdeemed useful to employ a model that elicits an endotoxin-like response in some respects but does not involve quite as complicated immunomodulatory responses. We have previously shown that in vivo latex phagocytosis elicits an endotoxin-like response with respect to increased glucose utilization by Kupffer cells, endothelial cells, and Sequestered hepatic neutrophils [i8]. Receptor-mediated phagocytosis is an energy-requiring function of macrophages that is accompanied by the generation of superoxide anion [9]. Oxygen-derived radicals generated by phagocytic cells are implicated as one of the mediators of hepatic injury in endotoxemia. Superoxide anion is also suspected to induce the release of chemotactic peptides that leads to the migration of neutrophils into different tissues [21]. Certain agents sensitize the hepatic phagocytes to the effect of an appropriate secondary stimulation. Following such priming, these cells produce increased quantities of 02 when they are later challenged with soluble (phorbol ester) or particulate (zymosan) stimuli. Therefore, it was the aim of this investigation to test the hypothesis that in vivo stimulation of the hepatic phagocytic system by administration of latex evokes sequestration of blood neutrophils into the liver and primes these cells and the resident Kupffer cells for the generation of toxic oxygen metabolites that could contribute to subsequent hepatic injury.
dis-
MATERIALS INTRODUCTION The liver is an important organ for the maintenance of metabolic homeostasis [17], clearing of bacteria and particulate antigens, and detoxification of endotoxin [10, 12, 16], in addition to performing many other functions. The parenchymal cells play a major role in maintaining metabolic homeostasis, and the sinusoidal cells are responsible for some of the other functions. Thus, the Kupffer cells clear the circulation of bacteria translocated from the gut and detoxify circulating endotoxin in gram-negative sepsis. Although the Kupifer cells represent less than 10% oftotal cellular components of the liver [7], they account for a significant portion of the hepatic glucose uptake during fasting [ii]. Also, the major portion of the increased glucose uptake by the liver during endotoxemia [ii] and latex phagocytosis in vivo [18]
AND METHODS
Experimental
Animals
Male Sprague-Dawley mington, MA)
were
HC1.
was
rats
A catheter
another one was placed tic surgical technique.
(225-280
anesthetized
implanted
g;
with
in the
Charles
River,
Wil-
xylazine-ketamine-
right
in the left carotid Food was removed
jugular vein and artery, using asep24 h prior to the
Abbreviations: ANOVA, analysis of variance; GM-CSF, granulocytemacrophage colony-stimulating factor; IL-l, interleukin 1; LPS, lipopolysaccharide; PMA, phorbol myristate acetate; PMN, polymorphonuclear neutrophil. Reprint requests: Abraham P. Bautista, Department of Physiology, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, LA 70112. Received March 12, 1991; accepted May 6, 1991.
Journal
of Leukocyte
Biology
Volume
51, January
1992
39
experiment,
while
The use approved Medical
Administration Latex a dose (iv.)
allowing
the
of experimental by the IACUC Center.
animals
free
in these Louisiana
access studies State
to water. has been University
were
for
total
white
Co., St. injected intervals cell
Louis, MO) at intravenously 100-j.d blood and
differential
counts.
of Superoxide
Dismutase
A single bolus of 3000 U of Mn-superoxide bovine liver (Sigma Chemical Co.) or 1 mg dium ibuprofenate; Upjohn Co., Kalamazoo, jected iv. followed j.tg/min, respectively, start of infusion.
Liver
Perfusion
or Ibuprofen dismutase from of ibuprofen (soMI) was in-
by continuous infusion of 90 U/mm or 40 for 2 h. Latex was injected 1 h after the Control rats received sterile saline.
and
Cell
Isolation
After anesthesia with ketamine-xylazine (30 mg/kg, 3 mg/kg intraperitoneally), the liver was perfused in situ with 0.025% collagenase (Sigma Chemical Co.) and 1 mM CaC12 in Hanks’ balanced salt solution for 5 mm at 37#{176}C. Subsequently, it was cut into small pieces and further digested for 40 mm at 37#{176}Cwith 0.2% pronase (Sigma Chemical Co.) in Gey’s balanced salt solution at a final density of 1 g wet tissue/25 ml in the presence of 0.001% deoxyribonuclease (Sigma Chemical Co.) [2, 3]. The rest ofthe isolation procedure was performed at room temperature. Nonparenchymal cells were separated from parenchymal cells by centrifugation at 50g for 2 mm. Nonparenchymal cells-endothelial cells, Kupffer cells, and polymorphonuclear neutrophils (PMNs)were further separated by centrifugal elutriation Q2-2i centrifuge, JE-6B elutriator rotor, Beckman Inc., Palo Alto, CA) at constant speed (850g) and different flow rates as described previously [5, 6]. Endothelial cells (90-99% pure) were obtained at a flow rate of 23 ml/min. Small Kupffer cells (60-70%, 10 jsm) arbitrarily designated as KC1 and some endothelial cells (30-40%) coeluted at 29 mi/mm; large Kupffer cells, referred to as KC2 (90-99% pure, 12 jim), were collected at 45 ml/min. The largest of the Kupffer cells (>95%, 15 m) were eluted at 45 ml/min at 1g. In latextreated rats (but not in controls), PMNs were found in all postelutriation fractions. It was therefore necessary to separate the PMNs from mononuclear cells by FicollHypaque, using a two-step gradient of 1.077 and 1.119 g/ml [4]. With these procedures, cell purity was above 90%, as assessed by peroxidase and Wright’s stain. Viability, checked by trypan blue exclusion, was above 96%.
Superoxide
Anion
Assay
Using
the in Situ Perfused
Liver
The liver was perfused with 200-400 ml oxygenated Hanks’ balanced salt solution until the organ was thoroughly cleared of blood. Then buffer containing 50 M ferricytochrome C (Sigma Chemical Co.), 0.8% bovine serum albumin, and 5 mM glucose was perfused through the liver at 25-30 mi/mm at 37#{176}Cfor 10 mm. Aliquots of the perfusates (0.8-1.5 ml) were collected every 5 s and centrifuged to remove contaminating debris. The change in absorbance was measured in a Beckman spectrophotometer at 550 nm. Superoxide anion release was expressed in nmol/min/g liver wet weight. For negative controls, superoxide dismutase at 7500 U/liver was
40
Journal
in
Isolated a six-well
Sigma Chemical body weight were appropriate time
collected
Administration
introduced described
into the substrate more detail in
Superoxide
of Latex
beads (1 pm; of 0.3 g/kg into rats. At
samples
animals of the
of Leukocyte
Biology
Volume
51,
January
1992
the
Anion
Assay
supernatant
described
was
was
sence
of
14,
used
15].
opsonized
Hepatic buffer
and
for
the
as a substrate
zymosan
(1.98
mg/106
Phagocytes placed After
(Sigma
in the
is
in 1 h, with fresh as previously
ofO2 c
method
were MA).
replaced
assay
Ferricytochrome
at 50 tM
This
Cambridge,
removed
buffer
[3,
Co.)
on Isolated
cells in HEPES-bicarbonate plate (Costar 3406,
HEPES-bicarbonate
mm.
at 7 1.
ref.
Chemical
presence cells)
or
or abphorbol
myristate acetate (PMA, 106 M) (Sigma Chemical Co.). Superoxide dismutase (700 U/ml, Sigma Chemical Co.) was also added to the negative controls. The amount of reduced ferricytochrome c was measured using a molecular extinction coefficient of 21.1 mM cm from the change in absorbance at 550 nm against a cell-free blank [6]. Superoxide formation was expressed as nmol/106 cells or per mg protein/60 mm. Earlier experiments demonstrated that there was a linear relationship between the amount of O2 released and cell concentration.
Histology The livers were fixed in 10% formalin saline. The tissues were dehydrated paraffin, and thin sections (5-7 tm) with hematoxylin and eosin.
Estimation
of PMNs
Approximately scanned and
the
in phosphate-buffered and embedded in were cut and stained
per Microscopic
Field (40 x ) of the livers were per area was counted.
50 high-power fields number of PMNs
Statistics Data presented in this to eight independent Statistical significance (ANOVA)
and
paper represent experiments was assessed
nonparametric
means ± unless stated by analysis
statistical
SEM of five otherwise. of variance
analysis.
RESULTS Accumulation Administration
of PMNs of Latex
Neutrophils were detected histological sections of the tion of latex. The estimated power field (40 x ) in these control animals, the number was less than 2 ± 0.8 (P Although the number of power field (40 x ) after
in the Liver
Following
in liver
significant numbers in the 60 mm after the administranumber of PMNs per highlivers was 130 ± 18. In the saline of PMNs per microscopic field < .001 versus latex treatment). granulocytes observed per highlatex plus superoxide dismutase
(95 ± 10) or ibuprofen (86 ± 24) was lower, these differences did not reach statistical significance compared to latextreatment alone as determined by nonparametric statistical analysis (P > .05). Isolation of the nonparenchymal cells and separation of the different cell types further demonstrated that latex treatment was associated with migration of PMNs into the liver. Table 1 shows that a significant number of PMNs was isolated from the livers following latex administration. Their presence accounted for almost 25% of the total nonparenchymal cells that were collected. More than 60% of the nonparenchymal cells were endothelial cells. Administration of
TABLE
1.
PMN
Effect
of
Superoxide
Infiltration
into
Dismutase
the of
Liver
or
!buprofen
Following
Latex
the
in
Vivo
on
120
Administration
Particles’
100-
4.,
Group
PMN
s/liverb
(x
Latex
Latex
+
SOD
Latex
+
IBU
Control
C
106)
0
60
±
12
___%
30
±
16
_C
28
±
8
1.5
±
0.8
80 0
1g g
60
U V
.
‘n
=
nificant tate;
5-8. P < as assessed
IBU, hPMNs
ibuprofen. were obtained
,
digestion
.001 all values versus by the nonparametric from
centrifugal
described
in
the
and
liver
,
elutriation
Materials
control; method. after
and
all
other SOD,
values Superoxide
collagenase
Ficoll-
are
perfusion,
Hypaque
zo
not sigdismu-
‘
40 0 0
pronase
centrifugation
cE
as
either superoxide dismutase or ibuprofen did not alter the distribution of the nonparenchymal cells (data not shown). KC3s were most phagocytic among the liver cells, containing more than 50 latex particles per cell. Ninety-three per-
0J
50%
(Table
Fig. P > tex
Figure of latex
2 shows particles,
TABLE
2.
that the
Percent
1 h after reduction
of Hepatic Latex
by the the
Nonparenchymal
Cells
type
Latex
Endothelial
Kupifer Kupifer Kupifer
cells
cell cell cell
I 2 3
Granulocytes “Means after
“P ‘P (‘P
iv.
±
SEM;
5-7
administration