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Research Paper

Mediators ofInflammation2,417-422(1993)

INCREASED plasma-andtissuelevels ofendothelin-1(ET- 1)during inflammatory diseases,havesuggestedaroleof ET-1 inthe pathophysiology ofinflammatory reactions.

The authors have studied theeffect ofET-1 on cytokine release from monocytes and monocyte-derived macro- phages. ET-1 increased secretion of TNF-, IL-1/I and

IL-6inadose- andtime-dependentmanner.Optimal ET-1 concentration ranged from 0.01 to 1nM. The maximal

response was a200 to 400%increase incytokine release.

Atime-coursestudyrevealedthatthe pattern ofcytokines induced by ET-1 wasdifferent inmonocytes andmacro- phages, althoughanearlyincrease inTNF-ffwasobserved in both monocyte and macrophage supernatants. Incon- clusion, ET-1 stimulates monocytes andmacrophages to release cytokines thereby demonstrating a potential role forET-1 inregulation ofinflammatory responses.

Keywords: Cytokines,Endothelin-1, IL-lfl,IL-6, Inflamma- tion,Macrophages, Monocytes, TNF-

Endothelin-1 stimulates human monocytes in vitro to release TNF- , IL-1/ and IL-6

E. Helset,1"cAT. Sildnes, R. Seljelid2 and

Z.S.

Konopski2

Department of Anesthesiology, Institute of Clinical Medicine, and

2Department

of Experimental Pathology, Institute of Medical Biology, University of

Troms,

N-9038

Troms,- Norway

CACorresponding Author

Introduction

Endothelin-1

(ET-1)

was originally described as a potent vasoactive peptide released from en- dothelialcells.1’2The observation that

macrophages

and mast cells both release and have receptors for ET-1, has

suggested

that ET-1 possibly

plays

a role

as a modulator of

monocyte/macrophage

func- tion.3,4

Increased

plasma-

and tissue levels of ET-1 are

reported

in patients with inflammatory diseases such as rheumatoid arthritis, Mb.Crohn and septic shock.5- Elevated

plasma

levels of ET-1 are even associated with the severity of illness in patients with septicaemia. Furthermore, in a recent

study

the authors have shown that ET-1 increases microvascular permeability in isolated rat

lungs provided leukocytes

are present in the

perfusate.

Against this

background

we

hypothesized

that ET-1 might

play

a role in the pathophysiology of inflammatory reactions. Therefore an investigation into whether ET-1 stimulation of monocytes and

macrophages

induced release of inflammatory mediators such as

TNF-, IL-I

and IL-6, was

required. Thesecytokines

play

animportant rolein the

development

of inflammatory diseases.9’

Synergistic actions of these cytokines are further

suggested

to be of crucial importance for the

outcome ofseptic shock.9-1

In the present

study

human monocytes and monocyte-derived

macrophages

were stimulated in vitro with ET-1 for specific time intervals, and the secretion of

TNF-oq IL-I

and IL-6 in the

supernatants was measured

by

bioassay.

(C)1993Rapid Communications ofOxford Ltd

Materials and Methods

Reagents: Endqthelin-1

(human)

and endothelin

antiserum were obtained from

Nova

Biochem (Liiufelingen,

Switzerland).

Human

rTNF<z

with specific activity 2.0 x

107 U/mg,

monoclonal anti- human TNF-0 antibodies, human rlL-6 with specific activity 2.0 x

108 U/mg

and monoclonal anti human IL-6 antibodies were obtained from Boehringer-Mannheim

(Mannheim, Germany).

Human

rlL-1/g

with specifiC activity 2.0 x

108 U/mg,

and goat

polyclonal

anti-human

IL-lfl

antibodies were

purchased

from British Bio-

technology (Oxon, UK).

Isolation and cultivation

of

monocytes and monocyte-derived macrophages: Highly

purified

monocytes were ob- tained using the method described

previously.

2 Briefly,

unseparated

mononuclear cells

(PBMC)

were isolated from

buffy

coat

(The

Blood Bank, University Hospital of

Tromso, Tromso, Norway) by

density centrifugation with

Lymphoprep

(Lym-

phoprep, Nycomed

Pharma

AS,

Oslo,

Norway).

The cells were washed three times with HBSS

(Gibco, Glasgow, UK)

and

resuspended

inmedium consistingof RPMI-1640 with 100

IU/ml

penicillin,

100/.zg/ml

streptomycin, and

supplemented

with 25% human serum.

Peripheral

blood mononuclear cells

(PBMC)

were then seeded in 24-well culture

plates (Falcon,

Becton Dickinson Labware,

N J, USA)

in concentrationof 2 x

106 cells/well.

After incubation at 37C for 90min the cell cultures werewashed threetimes with

pre-warmed

RPMI-1640 to remove non-adherent cells. Mono-

MediatorsjofInflammation.Vol 2.1993 417

(2)

cyte cultures were either stimulated with ET-1 immediately, or further cultivated for 10

days

to obtain monocyte-derived

macrophages.

The cells

were assessed morphologically with Wright stain,

showing >

90% monocytes, andtheir viability was

>

90% as determined

by Trypan

blue exclusion.

Stimulation

of

monocytes and monocyte-derivedmacrophageswith ET- The monocyte and

macrophage

cultureswere incubated with various concentrations of ET-1 ranging from 0.005nMto 100nMormedium

only (controls)

for 1,4,8and 24 h.

To

exclude endotoxin contamination, cell cultures were incubated with ET-1 denaturated

by

boiling for 30min. Further- more,insomeexperimentsET-1 waspre-incubated with ET-1 antiserum prior to addition to the cell cultures. For positive controls, cultures were incubated with LPS 0.1

g/ml

for 24h. The supernatants were harvested at the time points indicated and stored at -20C before being

analysed

for

TNF<z, IL-lfl

and IL-6.

Supernatants

and ET-1 were tested for endotoxin using an

Endospecy

kit (Seikagaku

Kogyo

Co. Ltd,

Tokyo, Japan).

No significant amounts of endotoxin were

detected.

Detection

of

TNF-" TNF-0 activity was determined

by

its cytotoxic effect on the fibrosarcoma cellline

WEHI

164 clone 13, as described previously.13 Briefly, target cells were seeded in 96-well microplates

(Falcon,

Becton Dickinson Labware,

N

j,

USA)

with different dilutions of culture

supernatants from ET-1 stimulated monocytes and

macrophages

and incubated for 24 h at 37C.

Serial dilutions of human

rTNF- (Boehringer- Mannheim)

were included as a standard. The TNF-0 specificity ofthe assay was verified using a monoclonal antibody against rTNF-0 (Boehringer- Mannheim,

Germany),

which

completely

neutral- ized the detected activity

(data

not

shown).

Results

are

presented

as

pg/ml +_

S.D. of triplicate determinations.

Detection

ofIL-lfl: IL-1/

activity was determined

by

a two-stage bioassay. The first stage involves the mouse

thymocyte

IL-4 NOB-1 cell line, which

produces

high amounts of IL-2 in

response

to human

IL-lfl.

14 Serial dilutions of human

rlL-lfl

wereincludedasstandard. NOB-1 cellswereseeded in 96-well

microplates

with different dilutions of culture supernatants and incubated for 24 hat37C.

Then,

100/1

aliquots of the supernatants were transferredto a replicate 96-well microplatefor the

nextstage. Thisstage of theassayinvolved theIL-2

dependent

mouse T-cell line HT-2.s Aliquots

(100/1)

of HT-2 cell suspension

(1.5

x

108 cells/

well)

were added to each well of the replicate microplate and incubated for a further 24 h. The

IL-lfl

activity was neutralized

by

a

polyclonal

antibody

against

rIL-lfl (British Bio-technology) (data

not

shown).

Results are

presented

as

pg/ml

S.D. oftriplicate measurements.

Detection

of

IL-6:IL-6 activity was determinedusing

the IL-6

dependent

mouse hybridoma cell line B.13.29 clone

9.16

Briefly, serialdilutions of culture supernatants and rlL-6 as a standard, were incubatedin96-well

microplates

withcells

(5 103 cells/well),

in a total volume of

200/1,

at 37C for 72h. IL-6

bioactivity

was neutralized

by

a monoclonal antibody against rlL-6

(Boehringer-

Mannheim,

Germany) (data

not

shown).

Resultsare

presented

as

pg/ml -+-

S.D. oftriplicate determina- tions.

MTT assay: Viability of the cells in the assays for

TNF-0, IL-1/

and IL-6 were tested

by

incubation

with

MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-di-

phenyl-tetrazolium

bromide]

(Sigma,

USA)

as de-

scribed previously.5 Microplates were read at

570nm

(ODsT0)

on aNovaPathTMMiniReader

(Bio

Rad, Richmond,

CA, USA).

Results

ET-1 stimulates monocytes and macrophages to release

TNF-,

IL-lfl

and IL-6: There was a donor to

donor variability in the levels of cytokines

produced

in unstimulated control cultures, as also

reported by

others.17

However,

the

response

pattern seen after ET-1 stimulation was

generally

the same in cultures of mononuclear

phagocytes

obtainedfromdifferentdonors. Figure1 depictsthe

dose-response

of ET-l-inducedsecretionof

TNF- (a), IL-lfl (B)

and IL-6

(C)

from monocytes and

macrophages.

TheconcentrationsofTNF-0,

IL-lfl

and IL-6 measured in the supernatants showed an inverse

U-shaped dose-response

curve. The max- imal cytokine

production

was observed in a

concentration range of 0.01-1 nM ET-1. In this

range

the optimal ET-1 concentration

appeared

to be donor

dependent.

Furthermore,asshowninFig.

1, monocytes secreted

generally

more TNF-0 and

IL-lfl

than

macrophages

in

response

to

ET-I

stimulation. Denaturation

by

boiling or

pre-

incubation with ET-1 anti-serum, neutralized the effect of ET-1 on cytokine release from monocytes and

macrophages (data

not

shown).

For comparison, Table 1 shows the

response

to LPS

0.1/g/ml.

After 24h, LPS stimulation causeda strong five- toten-fold increase in release of

TNF-0, IL-lfl

and IL-6 from monocytes and

macrophages.

Kinetics

of

ET-1 inducedTNF-orelease

from

monocytes and macrophages:

As

shown in Fig. 2A, the time-course

study

revealed that ET-1 stimulationhad increased monocyte

TNF-

production after 1 h incubation.

(3)

A 400 350

300 250

200

50 100

5O

-11 -10 -9 -8 -7

ET-1 (log M)

B 350

300

250

I

200

150

lO0

5O

0

C -11 -10 -9 -8 -7

ET-1 (log M)

450 4OO 35O 300 E 250 200 0 O0 5O

C -11 -10 -9 -8 -7

ET-1 (log M)

FIG.1.Endothelin-1 (ET-1)-inducedrelease ofTNF-(A),IL-lfl(B)and

IL-6(C) fromhuman monocytes andmonocyte-derived macrophages.

Monocyte and macrophage cultures were incubated with different concentrations of ET-1 or mediumonly (C" controls). Data represent the mean

___

S.D.oftriplicate measurementsof one of three representative experiments performed in duplicate.

macrophages.

Vol2.1993 419

(4)

A 400

I

350

300

250

200

50

O0

50

C 300

4 8 24

Time (h)

250

200

150

O0

50

ND

4 8 24

Time (h)

25O

200

150

O0

5O B

4 8 24

Time (h)

FIG. 2.The time-coursestudy ofendothelin-1 (ET-1)-inducedreleaseof TNF- (A),IL-6(B),andIL-lfl(C)from monocytesandmacrophagesin responseto0.1 nM ET-1.This ET-1 concentration was in the optimal range formaximalcytokine releaseasshowninFig. 1. Themonocyte and macrophage cultures werestimulated withET-1 for 1,4, 8and24h.The results are expressedin% of controls,andgiven as the meanvalue 4-S.D.

ofthree tofourexperimentsperformedin duplicates. I,monocytes; [], macrophages.

(5)

Table1. LPS-induced secretion of TNF-z, IL-lfland IL-6 by

monocytes and monocyte-derived macrophages

Cytokine Cells Controls LPS,0.1 #g/ml

(pg/ml) for24h (pg/ml) TNF- Monocytes 290_+13 2008_+81

Macrophages 109_+10 585_+70 IL- fl Monocytes 109_+15 3876_+79

Macrophages 225_+50 814_+13 IL-6 Monocytes 202+_40 1226+_78 Macrophages 163_+30 1246_+34 The monocyte andmacrophagecultureswere incubated with LPS0.1 #g/mlor medium alone(controls)for24h.The culture supernatants were analysed for TNF-z. L-lfl and L-6 as

describedunder Materials and Methods.

TNF-0productioncontinuedtoincrease

by

4 h and reached a maximum, with three-fold increase,

by

8h.

In the

macrophage

culture supernatants, ET-1 had increased TNF-0productionthree-fold

by

1 h;

whereas

by

4h and 8 h the

TNF-

level had returned to baseline.

By

24 h the

TNF-

level had increased

by

250%.

Kinetics

of

ET-l-induced IL-6 release

from

monocytes and macrophages:

As

shown in Fig. 2B, ET-1 increased monocyte IL-6

production

slightly after 4h stimulation, and levels were still elevated at24 h.

In the

macrophage

cultures ET-1 had induced IL-6

production by

1 h, reaching a two-fold

peak

concentration in the supernatants within 4h, and

declining,

but still increased as

compared

with controls,

by

24 h.

Kinetics

of

ET-l-induced IL-1 release

from

monocytes and macrophages:

As

shown in Fig. 2C, ET-1 increased monocyte

IL-lfl

production, as measured in the supernatants, after 8h, reaching more than a two-fold increase

by

24 h. In

macrophage super-

natantsincreased

IL-lfl

levelswere detected

by

4h,

reachingamaximaltwo-foldincreaseafter 24 h. No release of

IL-1/

was detected in monocyte or

macrophage

supernatants after1 hincubation time.

Discussion

The present results show that

ET-I

stimulates human monocytes and

macrophages

to release TNF-0,

IL-lfl

and IL-6 in a dose- and time-

dependent

manner. So far, thepotential of ET-1 to

aect

immune and inflammatory cells is

largely unexplored.

The

only

previouslyencountered

study

was done

by

Millull etal., who demonstrated that ET-1 stimulationofalveolar

macrophages

increased arachidonic acid

(AA)

release

by

amaximal 300%.18 In the present

study,

the same concentrationrange ofET-1, 0.1 to 1

nM,

induced an optimal

response

with a 200 to 400% increase in cytokine release.

The monocyte cultures secreted

generally

more

TNF-z

and

IL-lfl

than did the

macrophage

cultures,in

response

toET-1.Thesamepatternwas observedafterLPSstimulation inthe present

study.

These

findings

are in accordance with previous studies,

concluding

that the capacity of mono- nuclear

phagocytes

to secreteTNF-0 and

IL-1/

may

vary

with their state of dit:ferentiation.19’2

Stimulation with0.1

#g/ml

LPScaused afive- to ten-fold increase in cytokine release from mono- cytes and

macrophages.

The

generally

lower release ofcytokinesin

response

to

ET-1,

indicates thatLPS and ET-1 activate dierent mechanisms for cytokine release in mononuclear

phagocytes.

Denaturating ET-1

by boiling

did not increase cytokine production, whichexcludes the

possibility

that the observed

eect

of ET-1 was caused

by

contamination with endotoxin.

The time-course

study

revealed a sequential release of

TNF-, IL-lfl

and IL-6 from both monocytes and

macrophages.

TNF-0 was released prior to

IL-lfl

and IL-6.

A

similar pattern was

reported

previously after LPS stimulation of mononuclear

phagocytes

in vitro, in septicaemia in experimentalanimalsandinpatientswithmeningo- coccal septicshock.9’11’20Previousreports have also documented that TNF-0,

IL-lfl

and IL-6 might

interact on the

production

and release of each other.1’11’17’21 IL-6 has been

reported

to

suppress

TNF-0 production in mononuclear

phagocytes.

17’22

This might explain the reduction of TNF-0 observed in the

macrophage

supernatants in

parallel

withthe

peak

concentration of IL-6. Inthe monocyte

cultures,

ET-1 caused

only

a

slight

increase inIL-6 secretion, andno

parallel

reduction of TNF-0 was observed. This also demonstrates that ET-1 induces a differential modulation of TNF-0 and IL-6 secretion from monocytes and

macrophages.

In experimentalanimals

TNF-

is showntocause a

leukocyte dependent lung

injury, and to increase microvascular

permeability

in the

pulmonary

circulation.23 Thus the ET-1 induced TNF-0 secretion from monocytes and

macrophages

as shown in the present

study,

might explain the earlier observation of ET-1 induced

leukocyte dependent lung

injury in isolated rat

lungs.

8

In patients with septic shock,

plasma

levels of ET-1 have been

reported

to be 20

pg/ml.

This coincides with the lower

range

of the ET-1 concentrations causing cytokine release from monocytes and

macrophages

in the present

study.

However,

many workers favour the belief that ET-1

primarily

functions in an autocrine manner, and that circulating levels of ET-1

only

reflect an overflow to the circulation,

indicating

that local

concentrations ofET-1 are substantially higher.2 The present observations extend the under- standing of ET-1 as a vasoactive

peptide,

and

2.1993 421

(6)

Vascular smooth muscle

Activated tissue macrophage

TNF-=

IL-6 IL-1

Monocyte TNF.=

"

IL.6

IL.1 ELAM-1

Vascular endothelium

CAM

RBC ET-1

Thrombin Shearstress

TNF.=

Ischaemia

ET-1

)

Activated tissue macrophage

FIG.3.Endothelin-1 (ET-1)isreportedtobe released fromendothelialcellsinresponseto thrombin, endotoxin,TNF-and injury caused byischaemia and shearstress.2’24-27Inthepresentstudyit isdocumented thatET-1stimulates monocytesand macrophagestorelease TNF-0,IL-1/tandIL-6. These resultssuggesta new possibility forinteractionsbetween mononuclear phagocytes and endothelial cells.

demonstrate that ET-1 is

capable

of activating mononuclear

phagocytes, thereby

causingrelease of

TNF-0, IL-lfl

and IL-6. The present results also suggest a new possibility for the interactions between endothelial cells and

macrophages,

as illustrated in Fig. 3.

References

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12. Boyum A.Isolationof mononuclear cells andgranulocytes from human blood. ScandjClinLabInvest1968; 97: 77-89.

13. EspevikT,Nissen-MeyerJ.A highlysensitivecell line,WEHI 164 clone

13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes.JImmunol Methods 1986; 95: 99-105.

14. GearingAJH,BirdCR,BristowA,PooleS, Thorpe R. A simplesensitive bioassayfor interleukin-1 whichisunresponsiveto103 U/mlofinterleukin-2.

JImmunolMethods1987;99:7-11.

15. MosmannTR,CherwinskiH,BodnMW,GiedlinMA,Coffman RL.Two types ofmurinehelper Tcell clone. I.Definitionaccordingtoprofilesof lymphokineactivitiesandsecretedproteins.JImmuno11986;136: 2348-2357.

16. Aarden LA, De Groot ER, Schaap OL, Lansdorp PM. Production of hybridoma growthfactorbymonocytes.EurJImmuno11987;17 1411-1416.

17. SchindlerR,Mancilla J, EndresS,GhorbaniR, ClarkSC,DinarelloCA.

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ACKNOWLEDGEMENTS.Thiswork carriedoutwith grant support from NordicInsulin Fond. The authors thank O. H. Olsen at the Blood Bank, University HospitalofTromso,for technicalassistancewithbuffycoats,andW.

Johnsenand KristinKjaerfortechnical assistance withcytokine analysis.

Received 9

J

u ly 1993"

accepted in revised form 9 September 1993

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