Platelet activating factor and leukotriene B4 induce hyperpolarisation of human endothelial cells but depolarization of neutrophils

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 805-810

Vo1.153, No. 2,1988 June 16,1988

PLATELET ACTIVATING FACTOR AND LEUKOTRIENE B~ I N D U C E HYPERPOLARISATION OF HUMAN ENDOTHELIAL CELLS BUT DEPOLARIZATION OF NEUTROPHILS

Richard

Lerner

M.D.,

Peter

Lindstr6m

M.D.

and

Jan

Palmblad

Section of H e m a t o l o g y , Department of M e d i c i n e II!, institute at S ~ d e r s j u k h u s e t , S-100 64 S t o c k h o l m ,

Received

April

14,

M.D.

Karolinska Sweden

1988

We studied one expression of c e l l a c t i v a t i o n in n e u t r o p h i l s (PMN) and endothelial cells (EC), membrane potential changes [assessed by the fluorescent dye, di-C-Om(3)]. Human neutrophils responded with depolarization after exposure to fMLP, LTB~ A23187, PAF and P M A . In c o n t r a s t s o n l y P A F a n d L T B 4 induced membrane potential changes in h u m a n umbilical vein EC, which responded with increased f l u o r e s c e n c e , possibly indicating membrane hyperpolarization. These discordant responses may reflect processes of s i g n i f i c a n c e for interactions between EC and PMN. ©1988AcademicPress, lnc.

Neutrophil here

to

granulocytes

endothelium

neutrophils adherence complex of

and

been

CDII/CD18

defined.

leukocyte other between

in

(i). is

(2). Among

adherence

factors these

order

endothelial

process

neutrophils

(PMN),

may

be

cells~

It

the The

activated to

emigrate

cells is

(EC)

molecular

basis

molecules

(E-LAMs)

significance electrostatic

the

active

that for

the

are (3).

for

stimuli, tissues. roles

the

on

EC

forces

Both in

this

adhesion has

not

endothelium

Nonetheless,

the

ad-

glyeoprotein

stimulated

structure

candidates

e.g.

play

likely

corresponding

various

into

very

possible

of

by

adhesive

yet

several

interaction

(4,5) ~ cell

shape

Abbreviations: EC, endothelial cells; PMN, neutrophil granulocytes; PAF, platelet activating factor (l-o-alkyl-2-acetyl-3-phosphatidylcholine); LTB4, leukotriene B.; d i - O - C n ( 3 ) , 3,3'-dipentyloxacarbocyanine; fMLP , N-formyl-methlonyl-leucyl-phenylalanine; PMA, phorbol myristate acetate; ECGS, endothelial cell growth supplement; A23187, calcium ionophore A23187; HBSS, Hanks' balanced salt solution pH 7.4; LTC~, leukotriene C.; LTD4, leukotriene D4.

805

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 153, No. 2, 1988

changes telet

(5,6),

respond

changes

cence

and

activating

PMN

Such

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

factor, to

their

a

can

variety

tion

of

function,

changes using

by

measured

a

adhesive

properties

MATERIAL

AND

the

EC

of

been

stimuli

as

change where

examined

potential reported

agents

depolarization

dyes,

hypothetically

membrane of

surface

promoting

usually

cyanine has

may

variety of

adherence

indirectly

lipophilic

cells of

endothelial

potential,

[di-O-C~(3)]

endothelial

with

be

the

pla-

(7,8).

stimulation

tyloxacarbocyanine Since

on

PAF

membrane

alterations of

expression

we

fluores-

detail

to

(10).

stimula-

assessed to

(9).

3,3'-dipen-

in

react

of

with

confer

this enhanced

(6,7, 11,*).

METHODS

Chemicals: PAF, fMLP, PMA, thrombin, heparin, gelatin and g]utaraldehyde were obtained from Sigma Chemical C o (St L o u i s , MO). A23187 was from Calbiochem (La Jolla, CA), di-O-C~(3) from Molecular Probes (Junction City, OR), ECG5 from Collaborative Research Inc (Bedford, MA), trypsin-EDTA solution from Flow Laboratories (Irvine, Scotland) and HBSS from Natl. Bacteriol. Lab. (Stockholm, Sweden). RPMI 1640, fetal calf serum penicillin and streptomycin were from Gibco (Paisley, Scotland). LTB~ was a generous gift from Dr.J.Rokach (Merck-Frosst~ Dorval, Canada). Endothelial cells were obtained from human umbilical veins by treatment with 0.2Z collagenase (12). Cells were resuspended in culture medium (RPMI 1640 with 20Z fetal calf serum, 90ug/ml heparin, 50~8/ml ECGS, i00 units/ml penicillin and 100~g/ml streptomycin) and grown in 8 0 a m ~ t i s s u e culture flasks precoated with 2Z g e l a t i n " EC were trypsinized when confluent, washed twice and resuspended in H B S S p H 7 . 4 . P r e t r i a l experiment showed that when a microcarrier system (13) was used to o b t a i n monolayers of EC unacceptable fluorescence was recorded, mostly dependin 8 on the carrier spherules. Likewise, when EC were grown on plastic or glass cover slips and positioned in t h e q u a r t z cuvette of t h e spectrofluorometer, too high levels of f l u o r e s c e n c e and poor accumulation of d i - O - C s ( 3 ) ensued. Thus, suspended EC were used. N eutrophil granulocytes were isolated from human blood by a one step discontinuous Pereoll gradient centrifugation (14) and resuspended in H B S S . Membrane potentials were measured in a s p e c t r o f l u o r o m e t e r (Hitachi F-3000) with excitation wavelength s e t at 4 6 0 n m a n d e m i s sion wavelength at 5 1 0 n m . D i - O - C s ( 3 ) in H B S S (final conc. 33 n M ) was placed in t h e s p e c t r o f l u o r o m e t e r to w a r m . EC or PMN were added (final conc. 2.5 x 10S/ml) and allowed to t a k e u p t h e d y e f o r 5 to I0 m i n u t e s until a stable level of f l u o r e s c e n c e was obtained. S u b s e q u e n t l y , a stimulus (PAF, LTB4, PMA, thromhin~ f M L P or A23187) was added at i n d i c a t e d concentrations. Addition of P A F

*

Lindstr6m,

P.,

Lerner,

R

and

Palmblad,

806

J. : u n p u b l i s h e d

data.

Vol. 153, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

conferred fluorescence in cell free as well as in EC-containing systems. Thus~ cell free controls were run to allow assessment and deduction of this part of the response. Shape chan~es: ' Cells, treated with indicated stimuli (or HBSS as the control) for 5 min at 37°C were fixed with glutaraldehyde (5%) and assessed by interference contrast microscopy.

RESULTS PMNs and

responded PMA

tics and

(7.5~M)

of

the

fMLP

tion,

-

to

with

conferred in

with ly

45

a

membrane seconds

After

-

no

at

10-1000nM),

depolarization

and

PMA

response

after

repolarization

for

caused

a

have

with a was

lag

by

slow been

PAF

I),

each

followed

curves

stimulation

A23187

(Fig

specific

depolarization

depolarization and

(both

highly

A23187

Dose

(9, 15, 16).

were

rapid

contrast~

LTB4

membrane

responses

depolarization. where

fMLP,

agent:

and

LTB4

continous

reported

period

kine-

repolariza-

(I-10~M)

observed

The

(2~M)

of (Fig

PMNs

elsereacted

approximateI).

addition of stimulus--% --~ ~ 1 rain

• lpM

/

/

-1~~._

A23 187

A.

B.

C.

7.5~M

PMN

F i g I. C h a n g e s of d i - O - C ~ ( 3 ) fluorescence in P M N s s t i m u l a t e d as indicated. Typical curves from one representative experiment are presented, Micrograph A shows unstimulated P M N s ~ B: P M N s s t i m u l a t e d w i t h L T B ~ ( 0 . 1 ~ M ) , C: P M N s s t i m u l a t e d w i t h P A F ( I ~ M ) .

807

Vol. 153, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

S lm

~

PAl= ,,lpM

addition of sumulus/

~

/

..

,

s,,

u,os

PMA A23187

J A.

B.

C.

fMLP

Eq F~ 2 . C h a n g e s of d i - 0 - C S ( 3 ) fluorescence in EC s t i m u l a t e d as indicated. Typical curves from one representative experiment are presented. Micrograph A shows unstimulated EC, B: EC s t i m u l a t e d w i t h LTB 4 ( I ~ M ) , C: EC s t i m u l a t e d w i t h PAF ( 1 0 ~ M ) .

EC

accumulated

did

not

PMA

(I-10uM)

ed

to

exhibit

the

duced

of

after

system.

the

LTB4

with

1-10~M

to

be

(after

per

PAF cent

remained

(where

microscopy

readily

of

reacted

in

PAF

and

the 2).

EC,

a

This

but

not

resting

the

after with

shape

time

0. I u M

of

stimulation ruffling

at

least

5

with

and

808

elongation

in-

30

observ-

calculated

for per

10uM cent

PAF for

fluorescence

min.

cells

and

was

13

The

endothelial LTB4

add-

rapidly

was

potential

occured).

,

approximately

and

and

EC

hyperpolarizaoccurred

of

LTB4

were

dependently

response

lag

PMNs.

(I-10uM)

units/ml)

indicating

membrane

for

fMLP

dose

autofluorescence)

stable

than

hyperpolarization

autofluorescence and

(10

LTB4

This

exhibited

slower when

thrombin

PAF

(Fig

I~M

the

of no

of

hut

and

increased

change

or

stimulation.

subtraction

LTB4

No

membrane

slightly

fluorescence

contrast,

addition~

ed

of

fluorescence

PAF

only

(l-10~M)

In

cell

after

24

change

, A23!87

seconds

by

any

increased

Lion

IuM

fluorescence

could PAF, (Fig

be

detected

whereas I

&

2).

PMNs

Vol. 153, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION Di-O-Cm(3) are

fluorescence

believed

to

fluorescence rescence that

EC

in

reflect (17).

a It

a

is of

polarity

(but

less

centration changes latter that

in

(6).

Membrane of

release as

the

vation.

exist. at

and

PMN

other. and

PMN

Yet,

discrete

Although

interaction~

the

are So

is

of

changes

adherence

(13),

and

adherence, the

fluorescence

differences

discordant

membrane

to

significance

endothelium

to

c19).

may

been

did

with

for

e.g.

PMNs conto The

adherent react

or

as

PAF.

an

early

Since

EC

can

(18)

as

well

to

were cells

result

reported

not

LTB4

PMNs.

al-

(17).

of

mV

upon

contribute

has

system

potential

between

809

it

the

-50 for

alterations

changes

as

relationship

may

similarities

hypothesis

to

critical

radicals

between a

a

interpreted

oxygen

certain

as

the

above

in

res-

inside

structures

been

in

di-O-Cs(3) changes

since

coupling

attractive

unknown

also

have

an

estimated

responsible

dye

stimulation

response

contents

augmented

to

pointing

on

shape

are

dye

our

is

approximately

changes

in

assume

cells

di-O-Cn(3)

cytoskeleton

their

-

the

here,

cells

stimulus

granule

in

EC

fluorescence

endothelial

potential

exhibit

appear

of

and

of

for

shape

addressed

changes

However,

changing

step

was

or

to

fluo-

accHmulated

fluorescence

of

decreased

increased

previously

lipophilic

solvent

pumping

and

dye

potential

that

-

reasonable

the

in

neutrophils

endothelial

PMNs,

the

the

charges

possibility

in

aggregation

Ion

induces

of

of

likely)

surface

as

proposed

changes

(I0).

PAF

with

was

in

mechanisms

fluorescence

is

since

membrane

what

It

of

changes

membrane~

changes

manner

negative

It

the

phenomena

similar

unclear

stimulation.

EC

similar

activation

potential

(9, 1 5 ) .

fluorescence

net

terations

from

of

hyperpolarization

of

during

membrane

depolarization

expression

or

reflect

di-O-Cm(3)

ting

changes

PMNs

opposite~ during

cell

to

acticell

responses

in

EC

adherence

to

each

surface

charge

changes

Vol. 153, No. 2, 1988

Rapid LTD4

adherence

can

(7,8).

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

be

PMA

responses

mediated and

LTB~

by

of

increased

also

PAF

expression

(8,20).

and

but

not

thrombin

fluorescence and

PAF

act

~ctiva~ors

:~n

PMA

endothelial

rapidly of

or

and

endothe~

EC We

cells.

found

This but

by

thrombin,

expression

adhesivness

induced

directly,

ia!

induced surface

increase

inducing LTB4

EC

by

in

study

increase

other

of

of

indicate routes

and

PAF

promptly

our

could

LTC4

without

that

PAF

di-O-C~(3) that than

LTB~ PMA

as

ce!]s.

ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council (19P-7095, ]9X-5991), the funds of Karo]inska Institute, Swedish Medical Society, S6dersjukhuset, P&A lledlund, Gamla Tj~nnarinnor, F6renade Liv. The ski] fu] technical assistance of Mrs L Riede], Mr B Coital and S Jones is g r a t e f u l l y acknowledged. We are also grateful to the staff of the obstetric department, S6dersjukhuset, for their kind cooperation in obtaining umbJ lical cords.

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