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

In vivo latex phagocytosis primes the Kupffer cells and hepatic neutrophils to generate superoxide anion.

Activation of liver macrophages during clearance of endotoxins, bacteria, or other particulate materials may be accompanied by the migration of polymo...
1MB Sizes 0 Downloads 0 Views