Avian Inflammatory Macrophage Function - CiteSeerX

Journal

Avian

Inflammatory

of Leukocyte

Macrophage

Biology

Function:

Arachidonic Acid Metabolism, Respiratory Cell-Surface Phenotype During the Response K.A.

Golemboski, Institute Poultry

S. Shaw,

J. Whelan,

J.E. Kinsella,

48:495-501

(1990)

Shifts in Burst, and to Sephadex

and R.R. Dietert

for Comparative and Environmental Toxicology (K.A.G., R.R.D.), and Avian Sciences (K.A.G., 5.5., R.R.D.), and Institute of Food J.E.K.), Cornell University, Ithaca, New York

Department of Science (J.W.,

The avian inflammatory response to intraperitoneal (i.p.) Sephadex injection produces macrophages which display characteristics of an increasingly activated state over time. We examined elicited chicken peritoneal exudate cells (PEC5) with respect to superoxide anion production, arachidonic acid metabolism and cell surface Ia and transferrin receptor (TfR) expression from 4 to 96 h after i.p. stimulation. Avian PECs showed the highest level of superoxide release when harvested just 4 h after injection, and did not produce PGE2 or 6-keto PGF1,. Early (4-h) PECs produced elevated amounts of thromboxane as compared to later (42-h) macrophages. Expression of both ha and TfR Increased between 4 and 24 h after Sephadex stimulation; TfR remained elevated through 96 h, but Ia declined after 42 h. Some aspects of chicken macrophage regulation of superoxide anion, thromboxane release, and surface antigen expression are in contrast with those reported for mouse macrophages. Key words:

inflammation, chicken, ide anion, thromboxane

class

II glycoproteins,

INTRODUCTION Macrophages virtue

play

of their

gen-specific by,

and

dual

a pivotal role

(acquired) regulators

role

immunity. of,

the

to the stimulus cells, macrophages

new

capabilities,

of their cell which in turn other

These

acid antigen

ability to kill not be mutually tance of each stimulus, the

They

are

itself

by

and both

anti-

altered

response.

As

change

the

antigenic

functions,

metabolites presentation

microorganisms exclusive;

[1]. study,

secretion

of

and reactive oxygen interto lymphocytes, and the and/or however,

may vary, dependent kinetics of the resulting

species of origin In the present

including

tumor cells, may the relative imporon the nature of the response, and the

(Sephadex

macrophages injection G-50)

© 1990 Wiley-Liss,

we

extend

our

observations

[19]. In this model of a crosslinked

is used to elicit an inflammatory

Inc.

equivalent

mammalian

on

system, Dextran pop-

of the

cyte) [2,20]. responding

Recruitment macrophages

changes largely

primarily of of heterophils

macrophages, (the avian

polymorphonuclear

consistent to

superox-

leuko-

is rapid and extensive, show morphological with

initiation

by

pathway of complement have described the kinetics

progressive

Sephadex

fixation of the

and

and the func-

activation, of

the

alternate

[7]. Previous studies cellular response [ 1 1]

and the acquisition of phagocytic and microbicidal activity [8,18]. In addition, changes in antigen expression that were dependent on the eliciting agent and the time after stimulation were detected among chicken peritoneal macrophages using specific monoclonal antibodies [24]. This

study

examined

Sephadex-elicited face expression class

the inflammatory response in an avian model, the White Leghorn chicken. The chicken provides a particularly convenient animal in which to study macrophages, because of the absence of significant numbers of harvestable resident intraperitoneal

composed component

due

profile

receptor,

ulation of cells with a smaller

tional

and to the products progressively acquire

surface, and secrete a series of products may up- or down-regulate the activities of

leukocytes.

arachidonic mediates,

protection,

(innate)

inflammatory

they are exposed of surrounding functional

in host

in nonspecific

transferrin

II (Ia)

MHC

and the production ucts, arachidonic

Received Reprint Sciences,

the

time-dependent

macrophages of functionally

March requests: Cornell

antigens,

changes

in

with respect to cell relevant markers,

surthe

and

the

transferrin

receptor,

of two categories of secretory acid metabolites and superoxide

13, 1990; Rodney

accepted

R. Dietert,

University,

28, 1990.

March Department

Ithaca, NY

prodanion.

14853.

of Poultry

and

Avian

496

Golemboski

The

same

et al.

parameters

transformed

were

chicken

also

measured

using

macrophage-like

cell

a virus-

line,

at

HD1

1.

The results provide an overview of changes in the chicken macrophage during the activation process and offer insight into possible regulatory mechanisms.

MATERIALS

AND METHODS

used

Cornell

in each

housed

K strain

experiment

White at 4-5

in thermostatically

batteries [22] and

Leghorn weeks

were

Cells

The

HD1

Birds

were

raised

wire

grow

Graf.

C ration)

and

Reagents The

following

, St.

Co. paque

were

Louis,

obtained

MO:

(Ficoll-Hypaque),

ferricytochrome Heparin,

fetal

and

bovine

serum,

Grove, (FITC) fled). from

was

PA) was labeled

Grand NY.

Exudate were

serum,

assay CA).

(SOD). Medium and

Cell

harvested

(PGE2, Seragen

(PEC) as

Co. an-

BiotechnolLabs (West

1 cell

cells

traperitoneally

(i.p.)

cific intervals PECs were with 30 ml heparin.

(dry

6-keto (Boston,

PGF1a, MA).

at 1 ml/l00

and

g body

were

phosphate-buffered

washed saline

twice (pH

7.2;

with PBS)

saline

injected

weight.

in-

At spe-

cold prior

0. 15

M

to further

analysis.

Peripheral Blood EDTA

Blood was

pooled from were separated

Monocytes

obtained

as anticoagulant.

by

brachial

Equal

each of two birds. by centrifugation

volumes

of

macrophage-like are

loosely

T.

morphology

adherent

[4]

HD 1 1

.

Cytometry

transferrin

from flasks by vigorous in PBS prior to analysis.

shaking

and

of Ia antigens

and

venipuncture of blood

with were

Mononuclear leukocytes through Ficoll-Hypaque

Analysis cell

surface

receptor,

HD1 1 cells were I :2 in PBS) to

expression

aliquots

of

1-3

exposed to normal block nonspecific

followed

tein encoded (1:200 dilution Dr.

0.

by

by

two

x

PECs

106

goat serum secondary

washed

the B-L of ascites

Vainio),

which

on the

supernatant)

postinjection, the birds were sacrificed and obtained by flushing the peritoneal cavity of cold sterile saline containing 0.5 U/mI

Cells

transformation

with avian myelocytomatosis of Dr. K. Klasing and Dr.

Flow

terminant

[18].

(0.75%)

solution

by

in PBS.

or

(diluted antibody

Cells

were

then reacted with one of three monoclonal antibodies a volume of 250 l: C-Ia, which reacts with a glycopro-

obtained kits

described

physiological

W/V)

by

removed

were removed washed twice

reacts

B-L

[32],

region fluid)

of [10], with

molecule

or JS8

the chicken MHC 21-1A6 (a gift of a monomorphic

(undiluted

(1:2

in

dilution

de-

tissue

culture

of supernatant),

a

generous gift of Dr. M.J. Hayman. JS8 specifically recognizes the chicken transfemn receptor [2 1] . After 30 mm at 0#{176}C, cells were washed 3 times with PBS and then

flow cytometry aliquots exposed

of sterile

a 3%

were

Cells were

propriate

volume

were

were grown in Dulbecco’s modified Eagle’s me(DMEM), with the addition of 8% fetal bovine and 2% chicken serum at 37#{176}C,with 5% CO,.

exposed

provide

obtained cells gift

and

Cells

cells dium serum

Sephadex G-50 super fine was weighed, then preswollen in double-distilled water overnight. After 2 washes with water, the Sephadex pellet was resuspended in the apto

cells

exhibit

cytochemistry

by trypan

95%.

prior to analysis. Monocytes at 37#{176}C,5% CO2. for 2 h,

line,

These

binding,

Collection

previously

as determined exceeded

nonadherent

bone marrow was a generous

To quantitate

chicken

Island Biological C-Ia, monoclonal

reagents were Radioimmunoassay

Viability, always

(PMA),

the source for fluorescein-isothiocyanate goat antimouse IgG/IgM (affinity-puri-

for arachidonic acid metabolites and TXB,) were purchased from

PECs

acetate

from Southern AL. Jackson

Dye-binding protein Bio-Rad (Richmond,

Peritoneal

Histo-

dismutase Eagle’s

goat

purchased Birmingham,

Chemical

EDTA,

myristic

superoxide Modified

serum were obtained from (GIBCO), Grand Island, tichicken Ia, ogy Associates,

Sigma

G-50-50,

phorbol

C, Dulbecco’s

(DMEM),

from

Sephadex

mm.

time

HD11

of age.

day length. Feed (Cornell provided ad libitum.

20

exclusion,

after which washing.

chicken virus,

controlled

with a 15-hr water were

chickens

for

dye

washed 3 times in PBS were isolated by adherence

Birds Female

lOOg

blue

to the

secondary

antibody,

antimouse IgG/IgM (diluted utes on ice. Cells were washed

antibody.

analysis. Controls only to goat serum

Samples

son Fluorescent (Becton Dickinson, a 488

nm

FITC-labeled

1:50 in PBS), 3 times with

argon-ion

were

analyzed

Activated Mountain laser

were and

with

Cell View, (Spectra

goat

for 30 mmPBS prior to duplicate cell the secondary

a Becton

sorter, CA)

Dickin-

Model equipped

440 with

and

a four

Physics)

decade logarithmic amplifier. Data were collected on i0 cells per sample. Discriminators for background fluorescence levels were based on values obtained from control samples. Cell diameters were determined by comparison of time of flight, a measure of cell diameter, with that of standard-size fluorescent eas were calculated [23]. resent ranging

two

or three

from

9 to

latex beads and cell surface Results for each timepoint

separate 16 (HD11,

experiments n

=

with 5).

arrep-

N numbers

Avian Superoxide

Anion

Production ing

procedure

anion

release

with since

that from harvestable

anion

was

of Kitagawa

from

elicited

analyzed

et al.

peripheral resident

Superoxide

was

Briefly,

PEC

samples,

U/ml).

Samples

were

tested

80 (I) .J -J Li

60

in triplicate.

C reduced

verted

by superoxide

to nanomoles

cient 2.1 determined solubilized with the

using

the

tissue to rebuffer C (80 (133

After

anion, molar

1 h of

and

in distilled water Bradford dye-binding

was

extinction

x i04 M’cm. Protein using duplicate (untreated) cell

con-

PECs

Acid

or HD1

by 4-5 freeze-thaw (Coomassie blue)

Metabolite

1 cells

were

1 h at 37#{176}C,5%

moved

by

washing.

C02,

Two

HOll

96

INJECTiON

Statistical

Analysis

Data

were

(ANOVA),

analyzed

cycles, assay,

[6] . Results experiments of 3-6 per

cells ysis

by a one-way

followed

were (data

by

t-test, Results

arc-sine presented

with a probability significant.

analysis

of variance

least-significant

if significance expressed as

difference

was indicated percentage of

by total

transformed prior to statistical analare not transformed). Comparisons value

of < Li

C

0

z

:

>

UU

2.0

C-) m

>

0

1nn

1.0

24

4 HOURS

Fig. 2.

Changes

In intensity

of fluorescence

AFTER

HF

200

42

96

SEPHADEX

HD11

INJECTION

due to ha and TfR

on PECs or HDI I (as determined by flow cytometry) and changes in cell size (as determined by time of flight). MCF index was determined by dividing mean channel fluorescence for positIve samples by that of control samples. Results are cx-

c:: + PMA pressed as mean ± SEM and represent the mean of at least two experiments at each time point. Results of statistical analysis are described under Results. Time-of-flight data were obtained from a single experiment, with n =6 for 4- and 42-hr macrophages, n = 2 for HD1 1 (1O cells/sample).

1500

175

1250

z

EzJ

150

-

PMA

Li I-

0

1000

0’

750

50 A

250

C

25

I PBM

4

24

42

early production

HD11

4

I.P. SEPHADEX

P. SEPHADEX

macrophages

of thromboxane

also

A7 (TXA2;

exhibit

measured

as

production is in contrast to mammalian macrophages [5]. The predominance of TXA2 production may be a characteristic of the response to i.p. Sephadex, since Sephadex-elicited macrophages can produce PGE2 under in vitro culture conditions (K. Kline and B.G. Sanders,

sponsible

for

prostaglandin

It has been reported that macrophages may be largely synthesis,

while

newly

resremi-

HD11

Fig. 4. Thromboxane A2 production (measured as TXB2) by Sephadex-elicited PECs or HD1 I , with and without PMA. ResuIts are expressed as mean ± SEM and represent the mean of two separate experiments, each sample assayed in quadruplicate. Asterisk indicates significant difference from cells not cxposed to PMA.

grated macrophages produce [26]; the lack of prostaglandins

to PMA, without detectable produc(POE2 or PGI, measured as 6absence of detectable prostaglandin

personal communication). ident murine peritoneal

42

HOURS AFTER

inflammatory

TXB2) in response tion of prostaglandins keto-PGF1). The

S

HOURS AFTER

Fig. 3. Superoxide anion production, determined by the superoxide dismutase-inhibitable reduction of ferricytochrome C, by peripheral blood monocytes, Sephadex-ehicited PECs, or HD1 I. Results are expressed as mean ± SEM and represent the mean of two or more separate experiments at each timepoint, each sample assayed in triplicate. Columns with different letters are significantly different.

The

100

E 75

500

peak

125

a-

the

absence

of

resident

proportionately in our system

peritoneal

more may

macrophages

TXA2 reflect in the

chicken. In the murine system, TXA, is produced at constant levels throughout the inflammatory response [25], while thromboxane production during the avian response to Sephadex decreases after 4 h postinjection. TXA2 is a vasoconstrictor [9], and could play a role in the rapid conclusion of the recruitment period after Sephadex injection by reducing blood flow to the pentoneal region. The regulation of macrophage plex and arachidonic

involves acid

secretory events

multistep pathways. may be mobilized

is com-

In macrophages, from cellular phos-

500

Golemboski

pholipids pendent

in response phospholipase

phospholipase pacity for

to surface A, or

by

factors: an NADPH

Ca2

-dependent

cell

membrane.

therefore

of

increase oxidase

translocation These

both

influx

two

Ca2

ions.

at an

Sephadex. produce tamed

early

Early a Ca2 to

point

promote

shown

via

sive

pathways

that

acid

in our

system,

in the response in PECs that

4 h after

production

as

polymorphonuclear macrophages of superoxide

well

as

production,

may

chicken

and

In-

.

accom-

4 and 24 have been

macrophages

production in part

TfR

expression

PECs

between PGI

by murine

account through

96

by Sepha-

for

the

rapid

in-

remained h after

ele-

Sephadex

the percentage of cells expressing Ia de42 and 96 h. Time-dependent changes in

of Ia expression, 24 and

96 h. This

in contrast,

42 h) and

may

declined

coincide

of

increased

later

to earlier

with

tation activity [31]. The functional profile

which

[3]

were

amount of cell surface Ia and TfR expression may reflect their relative importance at different points in the response-TfR intensity increased between 4 and 24 h, possibly indicating an increased requirement for iron. tween

leukocytes [28]. display a progres-

anion

PECs

Amount

which

dramatically Both PGE2

of prostaglandin

injection, while dined between

i.p.

activation

Ia expression

absence

on

in Ia expression

decreases in TfR expression [14]. described here, however, expression

in Ia expression.

vated

may sus-

to that

the

rise during

antigens increased i.p. Sephadex.

crease

metabolism

the

by concomitant avian system

dex-elicited

require

to Sephadex is sufficiently

preceded

to suppress

[27];

can

are regulated indepenand do not usually reach but both responses

superoxide

deactivation

C to the

processes

arachidonic acid mobilization, analogous results from N-formyl-methionyl-leucyl-phenylalanine binding to human Later Sephadex-elicited

panied In the

of protein

kinase

days

in Ia expression

of both h after

Arachidonic

events influx

3-5

creases

in the affinity of a memfor its substrate, and the metabolic

be up-regulated

of

a Ca2 involving

[ 1 3] . The cadepends on at

lipases anion

and superoxide anion production dently in the murine system [29] maximum levels concurrently, peaked

stimuli via a pathway

C and diacylglycerol secretion of superoxide

least two brane-bound

an

et al.

levels

levels

(be-

before

of antigen

presen-

Sephadex-elicited

macro-

may result from changes in the affinity of the membrane oxidase for its substrate [30] , or from the resolution of early events that lead to divalent ion flux, or both. Sim-

phages varies considerably during the course of the inflammatory response. In addition to the changes reported here for Ia, TfR, SO, and TxB, parameters, phagocytic

ilan factors may also be responsible for the thromboxane release later in the response. The regulation of macrophage cell surface

ability phages;

molecules

is also

a complex

interaction factors.

of the Changes

process,

macrophage in antigen

decrease

in

receptors/

dependent

upon

with many expression

also varies among both FcR-mediated

erythrocytes the

extracellular may reflect

(SRBCs)

4 and 42 uptake

and

the

opsonized SRBCs nod [8 , 1 1 1. FcR

increase induction

and

by essentially

is expressed

h Sephadex of opsonized

ability

macrosheep

to phagocytize

un-

dramatically during this begins by 6 h post-Sephadex all macrophages

pe-

at 24 h;

changes in protein synthesis, membrane flow, or turnover rates; quantitative or qualitative alteration of surface phenotype may have both metabolic and functional im-

by contrast, phagocytosis of unopsonized SRBCs is negligible prior to and at 24 h and peaks late in the response. Sephadex-elicited macrophages could ingest opsonized

plications

particles, such as bacteria, while SO production is high, before the optimum time for antigen presentation. Later in the inflammatory response, macrophages have a higher amount of Ia antigens for increased efficiency of

for the cell.

In the present

the induction of two functionally molecules: class II (Ia) glycoproteins receptor The cell

(TfR). transferrmn

to bind

is present

receptor

to and

on proliferating

gens is directly ing cell-cell Clearly, pressing

related contact,

nonimmunologically glycollate) did

the

capacity

[ 15]

to functional especially

is dependent elicited percentage

of the

glycoprotein-bound cells

the nature In murine

tigen expression ulus; macrophages increases in the

cell surface the transferrin

, and

iron; may

processes that involve reaction [2]. Expression

the percentage of these two antigens

could influence at a given time.

we examined

modulates

internalize

an effect on metabolic as the Haber-Weiss

study, relevant and

also

it

have

iron, such of Ia anti-

parameters involvantigen presentation.

penitoneal and their

exudate relative

of the inflammatory systems, regulation on the by Listeria of cells

cells exdensities response of Ia an-

nature of the stimshow substantial expressing Ia, but

mediated responses not enhance Ia expression.

(e.g., A latent

thioperiod

functions

requiring

cell-cell

communication.

It remains murine and

to be seen whether the differences between avian regulation of macrophage function and

cell

antigen

surface

elicited

expression

macrophages,

or

are confined if they

apply

flammatory response as a whole. chicken represents an excellent model examine macrophage metabolism inflammatory response.

to Sephadexto the

In either system

during

avian

in-

case, in which

the to

the course

of an

ACKNOWLEDGMENTS We gratefully acknowledge Dr. 0. Vainio of the 21-1 A6 monoclonal antibody-producing

for

and Drs. K. Klasing and T. Graf The excellent technical assistance

1 cell line. Sanders,

for

the HD1 of Forrest

the gift cell line

Avian Joseph Puzzi, tance of Diane was supported gram York

and Belur Lokesh, and the editorial Colf are greatly appreciated. This in part by a Cornell Biotechnology

grant 157-8507, Sea Grant Institute

Hatch grant

157-442,

grant 7917.

assiswork Pro-

and

16.

New

transduction.

Lokesh, lipids

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