Stimulation of arachidonic acid mobilization by adherence of resident peritoneal macrophages to plastic substrate

Camp. Biochem. Physiol. Vol. 113C, No. 3, pp. 403-408, Copyright 0 1996 Elsevier Science Inc.

ISSN 0742s8413/96/$15.00 PI1 SO742-8413(96)00003-l

1996

ELSEVIER

Stimulation of Arachidonic Acid Mobilization by Adherence of Resident Peritoneal Macrophages to Plastic Substrate S . Llore t and _J._7.Moreno DEPARTMENT OF PHYSIOLOGICALSCIENCES, UNIT OF PHYSIOLOGY, SCHOOL OF PHARMACY, UNIVERSITY OF BARCELONA, BARCELONA 08028,

SPAIN

ABSTRACT. To interpret results of studies on arachidonic acid (AA) mobilization and metabolism in witro, it is essential that the influence of culture and conditions should be well defined. Thus, we investigated the effects

of murine

medium

resident

on arachidonic

peritoneal

macrophage

acid mobilization.

adherence

The present

and

the presence

data demonstrate

that

of foetal

calf serum

in culture

[jH] AA mobilization was triggered

simply by contact between cell and substrate. The presence of serum can modulate cell-substrate interactions but not AA mobilization. Protein kinase C, and calmodulin inhibitors failed to inhibit [‘HI AA release induced by cell adherence. stimulated

Finally,

low molecular

by cell adherence.

KEY WORDS.

weight

PLAZ inhibitors

COMP BIOCHEM PHYSIOL 113C,

were not able to inhibit

403-408,

[3H] AA mobilization

1996.

Arachidonic acid mobilization, macrophage, cell adherence, phospholipase A], protein kinase

C, calmodulin, trifluoperazine

to the plasma membrane.

INTRODUCTION Mononuclear

cells are the first line of defense against infec-

tious agents and toxic particles, and they are suitably positioned to participate in allergic and inflammatory reactions. Mononuclear number

cell activation

of secretory

such as platelet Phospholipase

including

metabolites

of arachidonic

for the mobilization

metabolized

lipid mediators,

factor (2) and cyclooxygenase

A* (PLA2, E.C. 3.1.1.4.)

enzyme responsible subsequently

products,

activating

and lipoxygenase

induces the release of a large

acid

(15).

is the rate-limiting of AA, which is

of arachidonic

acid by mono-

nuclear phagocytes requires a trigger, which can be supplied of particles such as zymosan, by im-

mune complexes, or by soluble agents, including phorbol myristate acetate, which activate PKC (1,s). This has been demonstrated

in vitro using macrophages cultured on plastic

dishes (10). Kouzan et al. (11) found that this above stimulation AA release and metabolism nalization,

serum (FCS)

during this study, the

cultured with foetal calf

increase cyclooxygenase

and lipoxygenase me-

tabolites. To interpret metabolism

ture conditions vestigated

results of studies on AA mobilization

and

in vitro, it is essential that the influence of culbe well defined. Thus, in this study, we in-

the effects of macrophage

adherence

and the

presence of foetal calf serum in culture medium on [‘HI arachidonate

mobilization.

to eicosanoids.

The release and oxidation either by phagocytosis

Moreover,

authors noted that macrophages

of

did not require particle inter-

but was dependent

on the binding of particles

Address reprint requests to: 1. J. Moreno, Department of Physiological Sciences, Unit of Physiology, School of Pharmacy, University of Barcelona, Avda. Joan XXIII s/n, 08028 Barcelona, Spain. Fax: 343-4021896. Abbreviations-AA, Arachidonic acid; BSA, bovine serum albumin; FCS, Foetal calf serum; NDGA, nordihydroguaiaretic acid; PBS, phosphate buffered saline; PLA*, Phospholipase A*; PMA, 4p phorbol-12-myristate 13 acetate. Received 1 June 1995; revised 22 November 1995; accepted 5 December 1995.

MATERIALS

AND

METHODS

Cell culture medium RPM1 1640, foetal calf serum and antibiotics were obtained from GIBCO (Grand Island, NY) and heat inactivated (56”C, 30 min) before used. RPM1 1640 medium was also purchased from GIBCO. myristate 13-acetate acid, cycloheximide, perazine, toluidine

blue and staurosporine

from Sigma Chemical donic acid (180-240

Co. (St. Louis, MO). Ci/mmol)

England Nuclear (Boston, analytical

4P_phorbol-12-

(PMA), actinomycin D, aristolochic p-bromophenacyl bromide, trifluowere obtained [‘HI Arachi-

was purchased from New

MA). All other reagents were of

grade.

Animals Male CD-1 mice (20-25 g) (Charles Spain) were used in these experiments.

River, Barcelona, Mice were main-

404

S. Lloret and J. J. Moreno

tained under standard conditions ad libitum.

and given food and water

sure consistency of the observerations. Significance of differences between data points and control was determined using a 2-tailed Student’s t-test.

Cellular Adherence To determine the cellular adherence, macrophages were allowed to adhere to the substrate for a variable period, after which the supematant was removed and nonadhered mononuclear cells were determined using a differential microscopic count with Toluidine blue (0.025%).

Lubelkd Macrophages Male CD-1 mice were killed by carbon dioxide asphyxiation, and the peritoneal cavity was lavaged with Hank’s balanced salt solution containing 1% bovine serum albumin (BSA), 20 units of heparin/ml, 100 units of penicillin/ml and 100 &ml of streptomycin to collect peritoneal macrophages. Lavage fluids were pooled and centrifuged at 400 g for 10 min at 4°C. Then, macrophages were resuspended at 106 cells per ml in RPM1 1640 supplemented with the antibiotics and containing 2.5 @ fat-free bovine serum alblumin plus 2 /G/ml [‘HI arachidonic acid and were incubated with shaking for 60 min at 37°C. The cells were then washed three times in phosphate buffered saline (PBS) containing 0.5% BSA to remove unincorporated [‘HI arachidonate. The macrophages labellcd in these experimental conditions incorporated 52 it 3% of the [‘HI AA. Cell Culture The labelled cells were cultured in plastic tissue culture dishes 25 mm in diameter ( lo6 cells per dish) (Costar, Cambridge, MA), in RPM1 1640 supplemented with antibiotics, in the presence or absence of 10% heat inactivated foetal calf serum. Cells were allowed to adhere to the plastic substrate at 37°C in 5% CO:, after which the medium was removed and centrifuged at 400 g for 10 min to pellet nonadherent cells, and the supematant was analyzed for [‘HI AA mobilization. Then, adhered cells were washed three times in PBS and overlaid with RPM1 1640. After a determined period, the medium was removed for analysis of radiolabelled compound. At the end of each experiment, the cell monolayer was overlaid with 0.1% Triton X-100, and the cells were scraped off the dishes. [‘H] AA content of the cell lysate was determined by scintillation counting, using a Packard Tri-carb 1500 counter. The amount of [‘HI AA released to the medium was dctermined and expressed as percentage of cell-incorporated [‘HI AA, which was determined in solubilized ceils. Background release from unstimulated cells (about 7 2 2% of [‘HI AA incorporated) was subtracted from all data. Statiscical

Analysis

Data are expressed as the mean ? standard error (S.E.M.). All experiments were performed at least three times to cn-

RESULTS The culture of murine resident peritoneal macrophages used in these studies have been shown by both morphological and functional criteria to contain more than 95% mononuclear phagocytes. These cells were strongly adherent. Thus, when we incubated macrophages at 37”C, 62% of cells were adhered to plastic after 30 min and 80% after 1 h incubation. We next measured the possibility that FCS modify the macrophage attach to substrate. The presence of different concentrations of FCS (O-20%) during the experiment did not cause a significant increase in adherence (data not shown). Macrophage adhesion to the plastic substrate in RPM1 medium without FCS induced a marked stimulation of AA mobilization. Fig. l(A) shows the time-course of [‘HI AA release. These significant increases in AA release occurred within 45 min and remained high until a plateau that was reached at 4 h. As shown in Fig. l(B) the time-course for adhesion-stimulated [‘HI AA release was linear for at least 90 min. The maximum velocity and acceleration of the process of AA mobilization were reached at 56 min and 24 min, respectively. This suggests that the mechanism of AA release induced by the cellular adherence is a rapid process that probably does not involve protein synthesis. To examine whether or not the effect of cellular adhesion on [‘HI AA mobilization is mediated by protein synthesis de nouo we tested the effect of protein and RNA synthesis inhibitors on [‘HI AA mobilization. A combination of cycloheximide (10 k/ml) and actinomycin D (1 &ml) did not inhibit adhesion-induced secretion of [‘H] AA (data not shown). In a subsequent experiment we determined that macrophage adherence to the plastic surface covered by gelatin did not modify the [‘HI AA mobilization (Fig. 2A). Furthermore, the presence of FCS during the adherence period did not cause an increased release of arachidonate (Fig. 2B). Early studies reported that AA mobilization by PLAI is stimulated in vitro by Ca+2; nevertheless, it has been commonly assumed that Cat’ is the major regulator of this enzymatic activity in Y&. In our experimental conditions, the role of extracellular Ca” concentration in I’H] AA mobilization was studied using Ca+!-free medium. Our results indicate that adherence of macrophages releases similar amount of [‘H] AA independent of the presence ofCa+’ in the medium (data not shown). Recently, some reports suggest that cytosolic PLA: may he partially regulated by phosphorylation. To investigate the role of kinases in AA mobilization induced by macrophage adherence, parallel studies were conducted using different inhibitors. Staurosporine is a microbial product, first described by Tamaoki et al. (20), which is a highly potent inhibitor with broad specificity of protein kinases (18). Tri-

Stimulation of Arachidonic Acid Mobilization

100

Q

F

40

-

t-3

K

FIG. 1. Time-course [3H] arachidonic acid release stimulated by cell adhesion of resi. dent peritoneal macrophages on plastic surface (A). Timecourse of the velocity (--) and acceleration (--) of the [W] AA mobilization induced by macrophage adherence (B). The culture medium employed was RPM1 1640 without FCS. The data represent the means + S.E.M. of four

20 -

Accstsroti6n

was

pooilive

far

56

minutes

T

duplicate experiments.

01 0

’

’

1

’

2

’

3

’

4

5

’

6

TIME (HOURS)

fluoperazine is a known inhibitor significantly

inhibits

of calmodulin

calmodulin-dependent

(16) that

kinases.

Nei-

[jH] AA mobilization nificant

induced by cell adherence was as sig-

as the effect induced by these agonists.

Further-

ther staurosporine nor trifluoperazine affected [3H] AA mo-

more, we observed that the effects of stimuli such as PMA,

bilization up to concentrations

zymosan or ionophore

nases (Table

that significantly

l), thus ruling out a significant

inhibit ki-

contribution

of these pathways to mediate [3H] AA release in adherent

to indicate

macrophages. The next experiments

had a disruptive action.

mine whether the mobilization

were designed to deter-

A23187

were raised by cell attachment

on [3H] AA mobilization (Table 2). However, we have

that cell adherence

effect was not added and

of AA in mouse peritoneal

macrophages prelabelled with [3H] AA and stimulated by the cell adhesion could be modified by inhibitors of the low

DISCUSSION

molecular weight PLAz activity. In this study, we used aris-

Cell adherence to plastic is a common method used to sepa-

tolochic acid (100 PM), p-bromophenacyl bromide (25 PM) and NDGA (20 and 100 ,uM). Incubation of macro-

rate macrophages from other cell types (4). This initial step

phages with the drugs for the period of adhesion

rophage metabolites

showed only a weak, nonsignificant mobilization. Maximal inhibition reached by p-bromophenacyl

studied

inhibitory effect on AA was about 7% and was

bromide at 25 ,uM (data not

shown). Finally, we compared the effect of cell adherence on [3H]

of adherence is carried out in a variety of studies where macwere assayed (21). The present study

was undertaken to investigate the effect of cell adherence and cell culture with FCS on arachidonic acid mobilization. The data presented here support the hypothesis that contact between macrophage cell membranes and a solid plastic substrate

stimulates

arachidonic

acid mobilization.

Thus,

AA release with the effect induced by certain recognized

previous studies by Grinnel

(6) showed that cell spreading

stimulatory agents of the AA cascade such as zymosan, calcium ionophore A23187 or PMA. In Fig. 3, we can see that

on a surface and phagocytes are similar cell responses to different-sized substrates. Our results support this idea since

S. Lloret and J. J. Moreno

406

100

I:A) IJ

80

Plastic

q Gelatin

TABLE 1. Effect of staurosporine and tiuoperazine arachidonic acid released from mouse peritoneal phages induced by cellular adherence

on [‘H] macro.

% [‘HI AA released Control

PMA

60

PMA

+ Staurosporme

Adhesion

4h

2h TIME (hours)

(B)

4.1 25.6 9.7 25.3

-c -t 2 2

0.2 1.4 0.9+ 1.3

z 1.5 + 1.6

Aclhcsion Adheslon

+ Staurosporlnc + Trifluopcrazine

26.3 27.1

Adhesion

+ Staurosp.

18.5 + 2.1

+ Trifluop.

Macrophages were Isolated and belled with [‘HI arachldonic acid. Control cells were m.>inralned wth shaking to prevent cellular adherence. The macn~pha~es were mcuhated m RPM1 1640 for 1 h, then nonadhered cells were wshed. After 2 h, aliqucxs of the medium were removed and counted. The cells :mxhed fc, plastic were lysed wth Triton X-100 (0.1%) and rhc r,ldloactlvq was determined I)uring the cxpenment the medium was xlded with staurospormc (100 nM) and rrifluo~razinr (100 PM). Macrophagcs prelahcllcd were mamtaincd wirh shaking and rhcy were also acwirared wth I’MA (IO PM) m presence or absence of staurospormc (100 nM). Resulrs arc means + 5.. F_.M. of at least three expenments. *P i 0.01, .qruficantly different from cells snmulare with PMA.

c] Medium

q Medium

80

+ FCS

10%

was a sufficient ticle sponsc.

60

since tween

stimulus

internalization The

present

data

AA mobilization the plastic

to trigger the AA cascade,

was not was

substrate

required

further triggered

to initiate

illustrate simply

this

and parthis

hy contact

and the cell membrane.

rc-

concept, beThus,

40

20

0

Ln

4rl

TIME (hours) FIG. 2. [‘H] AA mobilization of macrophages by adherence on plastic tissue culture dishes and on plastic dishes coated with gelatine (A). Effect of FCS on [‘H] AA release induced by adherence (B). The data are the means + S.E.M. of 45 determinations.

the macrophages cytizing

the plastic

face contact

studied

here can ;11so bc viewed

substrate.

appeared

The stimulatory

to be independent

as phago-

effect of sur-

of the nature

of

that surface, at least with respect to the surfaces that WC have tested. Thus, gelatin and uncoated tissue-culture plastic, both conditions induced a similar stimulation. These data indicate that contact alone is sufficient to induce the behavioral

change

in the cells. The variety

L

of surfaces

that

are able to induce this phenomenon also suggested that it was not due to binding of a specific ligand by macrophages membranes. Furthermore, Kouzan et al., (10,ll) have demonstrated that during phagocytosis of iron beads, initial contact of the particles with the macrophage plasma membrane

Adherence

Zymoran

A23107

I

i PMA

I i23187 + PMA

FIG. 3. Effect of cefl adherence, zymosan ( 150 lug/ml), ionophore A23187 (10 &I), PMA (10 ,uM), and A23187 (10 PM) + PMA ( 10 PM) on [‘H] AA mobilization. Macrophages prelabelled with [‘H] AA were left to adhere for 2 h. Macrophages culture and labelled with (‘H] AA were incubated with shaking with the agonist for 2 h at 37°C. The data represent the means YZ S.E.M. of 4-5 determinations.

Stimulation

of Arachidonic

Acid

407

Mobilization

TABLE 2. Effect of cell attachment on [3H] AA mobilization

sidered. Moreover,

induced by PMA, zymosan or ionophore A23187

from this study is the lack of inhibitory effect of low molecu-

% [3H] AA released

another

lar weight PLAz inhibitors

interesting

observation

on AA mobilization

arising

by adher-

ence. These results agree with a previous paper, where we Control Adhesion Adhesion Adhesion Adhesion

4.0 25.5 45.2 56.2 46.3

+ PMA + Zymosan + A23187

2 2 ” -t +

0.2 l.O* 3.5* 4.8* 3.7*

demonstrated

tivity was determined. During these experiments cell were also activated with PMA (10 ,uM) zymosan (150 pgplml) or ionophore A23187 (10 PM).

*p <

0.01, sig

ings of Wijkandler

and Sundler (22), who observed that a

high molecular mass PLA2 was likely to be responsible for stimulus-induced

AA

mobilization

in mouse

peritoneal

we have shown that attachment

of macro-

macrophages. In conclusion,

phages to a plastic substrate induces the production of AA mobilization

in a similar form to other stimulus such as zy-

mosan, ionophore

A23187

or PMA. Besides, this dramati-

cally alters the [3H] AA mobilization stimulation. cell adherence

might disturb [3H] AA incorporation

phospholipids

and might alter the release of AA induced

by subsequent stimulation. 6 h after initiation

linked with

mobilization in murine resident peritoneal macrophages (12). Th ese observations were in line with the find-

were isolated and labelled with [‘HI AA. Control

Results are means ? S.E.M. of at least three experiments. nificantly different from control.

relatedness between se-

PLAz activity

AA

cells were maintained with shaking to prevent cellular adherence. The macrophages were incubated in RPM1 1640 for 1 h, then nonadhered cells were washed. After 2 h, aliquots of the medium were removed and counted. The cells attached to plastic were lysed with Triton X-100 (0.1%) and the radioac-

Macrophages

the immunochemical

cretory PLAz and intracellular

to cell

This effect was appreciable until

substrate

The

interactions

induced by subsequent

of serum can modulate

but not arachidonic

cell-

acid mobiliza-

tion. The knowledge of AA release consequent

to cell ad-

herence is essential when interpreting from experiments ing macrophages

of cell attachment.

presence

us-

in culture.

Since serum is frequently used for macrophage culture in many laboratories,

and since macrophages could be exposed

to serum or plasma in inflammatory component,

states with exudate

are very grateful

to Mr.

Robin

Rycroft

for his valuabk

in the prepmation of the manuscript.

it seemed appropriate to study the effect of se-

rum on the AA mobilization

in macrophages.

Ravinovitch

and De Stefano ( 17) reported that the adherence of macrophages to a solid substrate is an energy-dependent

process

that is enhanced by high serum concentration. However, we did not observe a significant increase in cellular attachment in the presence of FCS. Furthermore,

neither FCS nor

extracellular

effect on subse-

calcium

had any significant

quent [‘HI AA mobilization. ments indicate

Thus, the results of our experi-

that [3H] arachidonic

by cell adherence

acid release induced

is not a consequence

of stimulation

calcium influx across the plasma membrane,

of

in agreement

with our previous results on influence of calcium on arachidonic acid mobilization

by murine resident peritoneal

mac-

rophages stimulated by zymosan (14). Recently,

The authors assistance

several authors have proposed that a protein

kinase C (5) could be involved in the activation of phospholipase A*, the main enzyme involved in arachidonic acid mobilization.

In this context,

to determine

the importance

of these kinases in the [‘HI AA mobilization cell adherence,

the effects

of protein

kinase

induced by inhibitors,

staurosporine and trifluoperazine, were examined. It is noteworthy that these inhibitors AA mobilization,

wholly failed to inhibit

[‘HI

suggesting that these kinases did not trig-

ger AA release in our experimental conditions as we also observed in murine peritoneal macrophages stimulated with zymosan ( 14). PLAz is a growing class of enzymes. With the recent discoveries of novel forms of PLAz (3,9,19), new schemes for the roles of these enzymes in lipid metabolism must be con-

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