Changes in subcellular localization and surface expression of L ...

Changes L-selectin,

in subcellular localization and surface expression alkaline phosphatase, and Mac-i in human

neutrophils

during

stimulation

with inflammatory

Niels Borregaard Lars Kjeldsen Henrik Timothy A. Springer,’ H. Clarke Anderson,’ UI

The

Granulocyte

Denmark;

The

Research

The

Center

University

of

Department

Laboratory,

for

Kansas

Department

Blood

Research,

Medical

Center,

of Immunology,

of

Harvard

Ridgefield,

Abstract: The localization of the adhesion L-selectin in human neutrophils was determined cellular fractionation and immunoelectron and compared with the localization of Mac-i

Medical

School

of Medicine,

and

tDepartment

C7

protein by submicroscopy (amfl2) and

Words:

tory

fMLP

vesicles

.

GM-CSF

.

IL-8

.

LTB4

.

PAF

.

Hospital,

Boston,

Kansas

MA,

City;

and

Mac-l

on

the

tips

and

seemingly

ideal

neutrophils

face lated

cells an

[231.

of intact of secretory

plasma

membrane

in

matory

mediators

[29,

by

their

membrane

on the Because

30].

activation

mobilized

organelle

important

as

of

and which [31],

microvilli

of

secretory

vesiand

appearance of can be accounted membrane into the with

are

inflamidentified

phosphatase

most

sur-

the

alkaline

present ignored

present

in the matrix. in studies of

rapidly

[32],

mobilizable

the nonstimu-

monocytes,

vesicles

albumin is largely

is the

whereas

over

stimulation

ofneutrophils

a rapidly

cells,

that

to

[24] contact

randomly

Secretory

content

neutrophil

rolling

neutrophils vesicle

response

luminal surface this organelle

initial

that

and

prob-

of L-selectin

[25-28],

Mac-I

is

L-selectin

tail

demonstrated

contain

neutrophils

that

endothelial

from

Inc.,

a localization

the

neutrophils

on the surface incorporation

of

23],

mediating

have of

[29],

[2,

on

Oncology,

Francisco

surface

cytoplasmic

excluded

We

San

demonstrated

is distributed

organelle

and

Pharmaceuticals

the

P-selectin

but

eosinophils

Mac-i for by

for

present,

membrane

des,

the

with

when

on

been

ofmicrovilli

through

Mac-i,

of Pathology

of Ca4fornia,

It has

mediated

Copenhagen,

Ingelheim

(a0j32)

ably

of

‘Department

University

understood.

well

F. Bainton11

Rigshospitalet,

SBoehringer

of Pathology,

is present

secre-

.

School,

less

J-

Key

University

selectin

alkaline phosphatase, the marker for secretory vesicles. L-selectin was found to be localized exclusively on the plasma membrane of unstimulated cells and also of stimulated cells, although markedly diminished. This was in contrast to Mac-i, which was also localized in secretory vesicbes and in specific/gelatinase granules as shown previously [Sengelv, H., et al.J. Clin. Invest. (1993) 92, 1467-1476]. Stimulation of neutrophibs with inflammatory mediators such as tumor necrosis factor (TNF), platelet-activating factor (PAF), or f-Met-Leu-Phe (fMLP), induced parallel up-regulation of the surface membrane content of alkaline phosphatase and Mac-i and down-regulation of L-selectin, as evidenced by flow cytometry. Preimbedding immunoelectron microscopy confirmed that L-selectin was present mainly on tips of microvilli in unstimulated cells and showed that alkaline phosphatase and Mac-i were randomly distributed on the surface membrane of fMLP-stimulated cells. These studies indicate that the transition of neutrophils from L-selectin-presenting cells to Mac-i-presenting cells induced by inflammatory mediators is mediated by incorporation of secretory vesicle membrane, rich in Mac-i and devoid of L-selectin, into the plasma membrane. Leukoc Biol. 56: 80-87; 1994.

mediators

Sengel#{243}v Michael S. Diamond,’ Takashi K. Kishimoto,S and Dorothy Hematology,

of

and

most

easily

it is potentially

source

of

very

neutrophil

sur-

face proteins. We therefore decided to address the question whether secretory vesicles contain L-selectin in addition to Mac-i and thus might play a role in continued rolling contact of neutrophils with endothelium by replenishing Lselectin

lost

whether

secretory

case

a highly

tion

of

to

from

the

selective

by

are

devoid

of

secretory

role

neutrophils

adherent,

surface

vesicles from

rolling,

Mac-l-presenting

shedding

[33,

34],

of L-selectin,

or

in which

vesicles

in

the

transi-

L-selectin-presenting cells

must

cells

be

suspected.

TNF MATERIALS

AND

METHODS

INTRODUCTION Human The key

dynamic event

invading

the

process in

ofneutrophil-endothelial

inflammation

in

microorganisms

in

demonstration

selectins

in

trophils

with

both mediating

from surface

and 21J

80

mediators

intracellular

stores

Journal

the

in

[1-7]

vivo

[22],

of Leukocyte

but

and

of

/32

CSF.

role of

dynamic

[16]

Volume

to

E-selectin

56,

freshly

drawn blood of red cells was Uppsala, Sweden), was centrifuged on Residual erythno-

[35].

1994

fl’s’l LP,

the

TNF,

L-

phosphate; tumor

Reprint tory.

V-lorisiylrnethionyl-leucyl.phenylalanine;

granulocyte-macrophage

Ringer

[20, of

July

were

described

Abbreviations:

synthesis

presentation

neutrophils

previously

and

P-selectin

subsequent

as

neu-

adhesion cells of

of

by

the

integrins

bodies) and

Biology

to

against

irreversible of endothelial

membrane the

of

transbocation

[17-19]

of

a

clarified

contact

(Weibel-Palade plasma

defense been

rolling

subsequent response

cells

insertion into and ICAM-l

in has

and

involves

ofendothelial

and

initial

cells

ICAMs in mediating the [5, 8-15]. The immediate inflammatory

vitro

the

endothelial

general particular,

in

isolated from Sedimentation induced by dextran T-500 (Pharmacia, and the leukocyte-nich supernatant Lymphoprep (Nygaard, Oslo, Norway).

interaction,

Received

Niels of

Copenhagen

January

B4;

ZAS,

zymosan-activated

Borregaard,

Hematology, 0.

factor;

leukotriene

factor;

requests:

Department

DK-2100

CUB4,

necrosis

GM-

colony-stimulating

The

PAF,

platelet-activating

Granulocyte

Rigshospitalet

accepted

March

P.

Krebs-

flictor;

serum. Research L-4042,

Denmark.

7, 1994;

KR

21,

1994.

9

Labora-

Blegdamsvej,

cytes

were

lysed

resuspended

by

in

carried

out

at

of

fractionation, sorter

mM

i0

for

analysis,

was

terminated

ice-cold

buffer

and

pelleted

cells

were

samples

were

dilution at

200g

in

FAGS

2

with for

ice-cold

analysis

mm.

for

gradient This

scribed

fractionation

[35].

In

containing

short,

S

Chemical

Milwaukee,

containing

and

bottom

in

3 mM

15

NaCl,

1

ml

mM

and

200g of

previously

resuspended

kept

for

10

disruption

Na2ATP,

A

on

in

KRP

Marker

mm. were

Azurophil

cells

(100

mM

KC1,

using

as

mM

body

(Dakopatts

for

10

vesicles,

cytosol.

Gradients

the

pump ml

in

band

intermediate and

gelatinase

plasma

clear

were

that

bottom

containing the

described

regions the

granules

and

Percoll as

separate

granules,

a peristaltic 1.4

two-layer

gradient:

(y-band)

MgCl2,

0.5

granules

measured

B

attached

identified

by

(S2) from

to

fraction.

mem-

supernatant

aspirated

each

by

were

enzyme-linked

the

a fraction

col-

catching

antibody A

myeloperoxidase

immunosorbent rabbit

398,

assay

(ELISA)

antimyeloperoxidase

Glostrup,

anti-

Denmark)

after

affinity

D

C

Geiatinase,

Lactofertin, pg/mi

CD11b, U/mi

L-Seiectin, U/mi

28-ml

specific

at super-

proteins

the

ice

mm

mM

the

centrifugation postnuclear

centrifuged

offour in

secretory

deliver

a

and

generation

band

the

at

Nuclei

(Aldrich

buffer 3.5

top

to

of

azurophil

through set

top

of

7.2)

pH

cavitation.

de-

fluorophosphate WI)

at

as

were

diisopropyl

centrifugation

resuspended

performed

neutrophils

mM

Co.,

After

was

in

PIPES,

by

ml

g/ml)

containing

the

lector,

Subcellular

on

containing

and

10

identified

(/3-band)

branes

fractionation.

loaded

mM

nitrogen

sedimented

and

(1.05/1.12

visually

granules,

subcellular

was

by

were

(P1),

resulted

be

The

buffer

and

(S1)

10

disrupted

cells

mm

natant

(a-band)

of

and

15

density

band

volumes

10

for

could

period

fluoride,

cells/ml

unbroken

F35].

(KRP):

0.95 mM pH 7.4.

by

i0

400g

subcellular

MgSO4, NaH2PO4/Na2HPO4, a 5-mm preincubation

resuspended for

subsequent

x

and cell

fluorescence-activated

centrifugation

taken

cells.

37#{176}C at a

at

3

1.27 mM

mm

15

red

phenylmethanesulfonyl were

Krebs-Ringer-phosphate

after

after

for

subsequent

in

initiated

of

performed

cells/mi

i07/ml

neutrophils

procedures

sedimentation

NaCl, S mM KC1, mM glucose, 10 mM

Stimulation

the

All

was

x

at

(FAGS)

CaCl2, and

3

or

and

indicated.

ofneutrophils

concentration

130

shock

as

4#{176}Cexcept

Stimulation

cell

hypotonic

buffer

pg/mi

2.000

1.500

1,000

I

5

10

15

Fraction

20

25

No

E

10

15

Fraction

No

20

251

25

Fraction

F

10

20

25

Fraction No

No

G Phos, mU/mi

200

E

E 150

:D C

.0

a 0 0 Cl)

0 0

Frtion

No

Fraction

No

15

11

20 Fraction

Fig. other

1.

Subcellular

localization

stimulated

with

is shown.)

(A)

of

membranes);

plasma

branes) and

are Figure

l0

L-selectin;

shown 2 in

left

L-selectin

fMLP

(B) (F)

by the the

of M

Mac-l; latent

following y axis

and

at 37#{176}C(Fig. (C) alkaline

symbols:

lactoferrin

Mac-I. 2).

(a

phosphatase

Mac-l

Isolated

Subcellular marker (a

(*),

marker

L-selectin

neutrophils

were

fractionation of specific

was

divided then

granules);

of secretory

(#{149}), latent

(D)

vesicles); alkaline

in

two

performed gelatinase (G)

is an

phosphatase

equal

parts,

in parallel. (a

marker

insert (U),

one

kept

the

HLA

at

4#{176}C(this

representative

of geiatinase

where and

was

(One

25

No

granules);

results (Y).

for Note

figure)

experiment fractions difference

(E)

HLA 11-20

between

of G.

Borregaard

et al.

Human

neutrophil

adhesion

and

out

receptors

(a (light this

the

of three marker memfigure

purification

on

antibody (Dakopatts as

a

was A

purified sured

by

isolated

azurophil

granules

were

identified

measured

by only

(latent

alkaline

1271.

human

leukocyte

ELISA

(MELISA)

Mac-i

antigen

the

an as

IgGl

by

a

IgG2a

catching

mouse

of

L-selectin

sg/ml)

tions

as

ref.

8 of

against

detecting

antibody.

by

in

with

batch

all

experiments

I domain

using

DREG

biotinylated of

55

buffer

pH

7.2,

1 h. was

diluted

in

After

three

and

200

2-fold

serial

(55

arb.

dilu-

in

B

buffer

(except

for

A.

once

citrate,

1 h, in

pH

mM Color

were

CDiib

A,

[30]).

100

l

of

appropriately

1 h followed l

by of

buffer

washing

developed

10O-jl

standard

washing

peroxidaseB

was

three

added times

Na2HPO4/NaH2PO4,

C Ltoferrin,

U/mi

of

a

standards

buffer

l

albu-

A,

applied,

in

by

serum

of

100

P347) followed

100 5.0.

for Then

(Dakopatts, for

A and

incubated

buffer

Addi200

Na2HPO4/

dilutions

quantitation in

mM

buffer

and

was

X-lOO).

bovine in

The KC1,

with

8

1%

serial

washes

B and in

KC1,

samples

antibody

avidin

sodium

All

Triton

times

with

h.

additional

incubated

buffer

U/ml).

1

of catch-

3 mM

incubating

X-iOO,

along

buffer

by

three

l

NaCl,

7.2, 1%

Triton

100

temperature.

mM

3 mM

washing for

times

and

1%

After applied

conjugated

are

serum

NaCl,

catching

three

(500

blocked

[36}

Immunoplates

Na2HCO3/NaH2CO3,

at room

A

were

mM

incubated

B CD11b,

U/mi

L-S.lectin,

B (500

diluted

as 200

human

sample

A

55

DREG

normal in

diluted

of

buffer

pH

sites

biotinylated

DREG

One

an

overnight in

previous

lactoferrin

used. with

mM

in-

in

and

not

coated

50

Na2HPO4/KH2PO4,

and

of

incubated washed

binding

sample

chain

LM2/l,

the

ELISA and

a 20-fold

a

and

KH2PO4, mm) for

using

against

the

mM

were in

procedure

specified

[39] was

Denmark)

general as

MELISA

diluted

were

tional

a mixed

antibody of

9.6,

wells

by

[301

biotinylated

antibody. 40.

standard

starting

by

described

antibody

g/ml)

detecting

in

used

as

and

as

(2.5

as

characterized

mea-

identified

I assayed

Roskilde,

same

except

interaction

antibody,

pH

the

antibodies

regarding

(Nunc,

be

albumin

region

quantitated

antibody

(0.63 was

Mac-i,

was

catching

could

using

the

avidin-biotin

ing

by

monoclonal

antibody

monoclonal

chain

by vesicles X-lOO

were

ELISA

extracellular

mouse

the

where

Triton

and

class

publications

mea-

identified that

performed of

stan-

(v/v)

membranes (HLA)

quantitated

COOH-terminal

Mac-i

26]

as

[39].

was

CBRM1/23,

0.2%

[25, Plasma

used

[381

of

was

dependent

Secretory

phosphatase

presence

ELISA

used

lactoferrin were

[37].

ELISA

and

was by

granules

alkaline

ELISA

Myeloperoxidase,

granules,

phosphatase)

by ELISA

sured

1 mg/mI

identified

by

the

in

at

Gelatinase

measured

The

antimyeloperoxidase

1:10,000.

were

[36].

[36],

column.

Rabbit

biotinylated diluted

ELISA

gelatinase

was

antibody from

Specific

1:100.

398)

detecting

dard.

myeloperoxidase-Sepharose

diluted

in

100 during

mM

a 30-mm

in-

D gig/mi

Geiatinase,

pg/mi

2.500

2.000

1.500

1,000

500

I

5

10

15

20

25

No

Fraction

Fraction

E

No

Fraction

F

HLA, U/mi

No

Fraction

No

G

Latent Alk. Phos.,

mU/mi

40

20

20

E

E

C

.0

a 45

10

0

1,000

0

Fraction

No

Frtion

-

No

15

11

20

25

Fraction No Fig. lated shown.)

82

2.

Subcellular

with

108 See

localization M

legend

Journal

fMLP of

of L-seiectin at

37#{176}C(this

Figure

of Leukocyte

1 for

and figure).

explanation

Biology

Mac-l.

Isolated

Subcellular of

neutrophils

fractionation

A-G.

Volume

56,

July

were was

1994

then

divided performed

in two

equal in

parallel.

parts,

one (One

kept

at 4#{176}C(Fig.

representative

1) and

experiment

the

other out

of

stimuthree

is

M.F.l.

M.F.l.

cubation

1,800 1,600 1,400 1,200

50

40

by

sodium

10

addition

nm

Fig. 3.

EL-Selectin

Immunofluorescence.

1. j; axis),

bar,

l.y

by

FACS mm

(light

alkaline

phosphatase antibodies

were

either

with

each

of

TNF,

doses

regulation dependent

surface dotted

monoclonal

Cells U/mI

These

and

using

CD11b

The

L-selectin

axis),

tensity.

37#{176}C as

formed

by incubating

monoclonal

0.2

were

kept the

pg/mI

found

IL-8, to be

ofMac-l and experiments.

at

membrane bar, (solid

42],

alkaline

as

ZAS,

M

LTB4,

l08

M

maximally

phosphatase.

axis) mean

was

for

fMLP,

at U/mi

and

effective

Results

are

4.

with

the

goid-5 label

is

on

inonocional

00

the

surface

antibody

finally

the

ELISA

reader

for

DREG

1,

37#{176}C or

cells

was

i07/ml

200,

per-

with

lBi2,

osteosarcoma

SO l

an

alkaline

Dakopatts

IgGi

phos-

M736)

or

Mountain twice, SO jl

of

rabbit anti-mouse antibody (Dakothe cells were incubated for 30 mm at washes, the cells were resuspended (PBS)

a FACScan

intensity for fluorescence

preimmune

of

(Becton-Dickinson, 4#{176}C.After washing

saline in

4#{176}C or

at 1 x

class

at

at

containing

1%

formalde-

(Becton-Dickinson).

specific by

Mean

antibodies subtracting

was mean

corrected fluores-

IgG1.

37#{176}C for

Immunoelectron

microscopy

GM-CSF, l0

M

Because

PAF. up-

ofthree

of fixation

monoclonal

in-

sensitivity

antibodies

(alkaline

of the

antigens

CBRM1/29

phosphatase),

detection

[43] ofthese

recognized (Mac-i)

two

by

and

antigens

iBi2 on

the

my

L-Seiectin was

mm

analyzed

kept

human IgGl

30

my

Fig.

was

was

in-

in causing

means

and

cence

determined

fluorescence

stimulated 100

dotted

automatic

ofcells

anti-HLA

preimmune

fluorescence for nonspecific

(open

(heavy

and

Absonbance

Labeling

(LM2/i,

against

[41,

CA)

either

indicated.

antibody

antibody

phosphate-buffered

hyde

of HLA

Mac-l

2.)

37#{176}Cor

l0 5%

bar, given

4#{176}Cor

individually

content

1. y axis),

and

following:

in

#{149}AIk.Phos.

H2SO4.

1 M

Plus

100 l

fluorescein-conjugated patts) was added, and 4#{176}C.After two additional

0HLA

of

containing H202

Finland).

were

at

View,

I

l

in a Multiscan

neutrophils

phatase munine

$()%:bc;tP4

100

buffer

0.03%

analysis

murine 0

of

stimulated of

acid and

Helsinki,

Isolated

.1

phosphate-citric

o-phenylenediamine

at 492

FACS

2”

0

500

stopped

read

30

800 600 400 200

15

(w/v)

(Labsystems,

1,000

bar,

in

0.04%

periormed. tips

of

microvilii

of neutrophils. DREG-200 Transmission (m).

Unstitsiulated against electron

L-seiectin. micrograph

neutrophils A bridge of

were of

a portion

fixed

rabbit

in

2%

anti-mouse of

a neutrophii

formaldehyde. antibody is

0.05% was

given.

As

glutaraldehyde,

applied, can

be

and seen,

washed,

and

immunolabelini. the

majority

with of

the

incubated protein

A

immunogold

x 45,000.

Borregaard

et al.

Human

neutnophil

adhesion

receptors

83

plasma

membrane

was

performed

by

incubating

unfixed

PBS,

cells at 4#{176}Cfor 45 mm with either CBRM1/29 (1 mg/mi) diluted 1:100 or lBi2 (1 mg/ml) diluted 1:20 followed by fixation in 2% paraformaldehyde, 0.05% glutaraldehyde for 30 mm at room temperature. Rabbit anti-mouse antibody (Zymed) diluted 1:200 was then added, followed by protein A-gold-S (1:50 dilution). Immunogold labeling of L-selectin was

performed

before

fixation

as

described

above

or

could

(fMLP)

colony-stimulating

in ethanol.

factor

(GM-CSF;

NJ),

10 U/ml

Granulocyte-macrophage Sandoz

Pharmaceuticals,

ig/ml

ethanol.

Tumor

necrosis UK),

Amersham,

with

the

main

peak

of

the

phosphatase localizing close to marker HLA and L-selectin, while vesicles are found in a broad region

factor (TNF; iO4U/ml in

region

to

the

region

marker

the the from

latent

alkaline

plasma membrane rest of the secretory the peak in the light

containing

specific

granules.

The separation of HLA and L-selectin from latent alkaline phosphatase and from Mac-i is evident when focus is made on the light membrane region of the gradient as seen in Figure 1G. The localization of Mac-i in secretory vesicles and L-selectin in the plasma membrane is further illustrated by the translocation in response to stimulation with fMLP (Fig. 2). Stimulation of intact cells with fMLP results in nearly selective and complete exocytosis of secretory vesicles. This is evidenced by the disappearance of latent alkaline

(Sigma),

East

in H20. Recombinant interleukin-8 from Dr. K. Thestrup-Pedersen, Department Marselisborg Hospital, Aarhus, in PBS. Leukotriene B4 (LTB4; Sigma), Platelet-activating factor (PAF; Sigma),

ous gift matology, 10

gradient,

membrane

1 mM

(Sigma).

The distribution of L-selectin in subcellular fractions of unstimulated neutrophils (Fig. 1) indicates that L-selectin is present exclusively in the plasma membrane, whereas Mac-i is localized with a main peak in specific/gelatinase granules and a second peak colocalizing with secretory vesicles. The distribution of secretory vesicles is bimodal in this two-layer

be

of stimuli

N-Formylmethionyl-leucyl-phenylalanine

BSA

International,

RESULTS AND DISCUSSION

paper.

Source

(w/v)

H20.

performed after fixation by adding mAb DREG 200 diluted 1:200 (stock 2 mg/ml). The cells were subsequently refixed in 1.5% glutaraldehyde for 45 mm at 22#{176}C,washed in sodium cacodylate buffer, postfixed in 1% 0s04 in veronal-acetate buffer, stained with aqueous 1% uranyl acetate, dehydrated in ethyl alcohol, and embedded in Epon. Detailed morphometric analyses of these localizations are the subject of a separate

0.5%

Amersham

Hanover,

(a generof DerDenmark), 1 mM in 1 mM in

F, I,

Fig. 5.

Alkaline monoclonal

with rabbit OIl

the

84

anti-mouse

the

resting

l)re5efl(e

Journal

phosphatase antibody

on lBl2

antibody neutrophil of alkaline

the surface of neutrophils. against alkaline phosphatase.

followed (Fig.

4),

phosphatase

of Leukocyte

by the

protein

plasma on

Biology

A gold-5. membrane

the

plasma

Volume

Neutrophils The cells and has

processed become

membrane

56,

July

for very

in

were were

transmission irregular

a random

1994

stimulated then fixed

with l0 M fMLP in 2% formaldehyde, electron

with distribution.

large

microscopy. extensions

x30,000.

for

10 mm at 37#{176}C, chilled 0.05% glutaraldehyde,

Note or

ruffles.

that The

instead immunogold

ofthe

to 4#{176}C,and immunolabeled small label

microvilli (arrows)

labeled with found indicates

phosphatase

(alkaline

secretory

vesicles

phosphatase [26]).

Now,

present

on

complete

the

inside

colocalization

of of

Mac-i and L-selectin in the plasma membrane is observed as a result of translocation of Mac-i from secretory vesicles to the plasma membrane, whereas the main pant of Mac-i present in specific/gelatinase granules is unchanged. The level of L-selectin is reduced as a result of stimulationinduced shedding, the mechanism of which is still unclear [33]. This demonstrates that secretory vesicles, although endocytic in origin [27], are not simply inside-out plasma membrane vesicles but are organelles organized with a selective membrane composition, highly enriched in Mac-i and cytochrome b558 relative to the plasma membrane [29, 30] and devoid of HLA and L-selectin as demonstrated here, but sharing alkaline phosphatase with the plasma membrane. Thus, the incorporation of secretory vesicle membrane into the plasma membrane can be expected to affect profoundly the profile of proteins present in the plasma membrane rather than just adding more membrane with the same protein composition. Such changes in the protein profile of the plasma membrane can be observed and quantified by immunofluorescence on single cells.

The

incorporation

plasma

membrane

against

alkaline

stimulation

may

be

of secretory

with

phosphatase.

of

with

in

results

inflammatory

controlling

in

membrane

into

a monoclonal

the

incorporation

the

antibody

(Fig.

3) that

mediators

that

It is demonstrated

neutrophils

operative

interaction

vesicle

is monitored

neutrophil-endothelial

of

secretory

vesicle

mem-

brane into the plasma membrane, as evidenced by the parallel increase in the amounts of alkaline phosphatase and Mac-i in the plasma membrane. It is observed that the amount of L-selectin is reduced following stimulation. This has previously been shown to be due to stimulation-induced shedding with

of L-selectin

exocytosis

[33].

Although

of secretory

this occurs in parallel these two phenomena do

vesicles,

not seem to be causally related because stimuli that mobilize secretory vesicles efficiently may differ in their capacity to induce shedding of L-selectin (TNF versus PAF) (Fig. 3). It has previously been shown by enzyme cytochemistry that alkaline phosphatase, the membrane marker of secretory vesicles [25-27], is localized in intracellular vesicles in unstimulated neutrophils [44] and that stimulation causes these to fuse to form intracellular tubular structures that are in communication with the cell surface [45]. Our results demonstrate

that

after

mobilization

of secretory

vesicles,

the

i...

0-

,

.

1.,

.

:

: :

.

,

‘

0. :

.

45 ,

.

,.

:

,

:: ,1

. .-i-.+

;

-

. -. .

‘t

‘

,

.

S

.

,.-.

:

e

., -. :.

“

.

.

- . Cl

._‘;

--

on the surface

antibody CBR Ml/29 with rabbit anti-mouse is found

randomly

ofneutrophils.

against the a chain antibody, followed distributed

on

both

Neutrophils of Mac-I by protein ruffles

and

were stimulated with l0 M fMLP for 10 mm at 37#{176}C,chilled (CD11b). The cells were then fixed in 2% formaldehyde, 0.05% A gold’5, and processed for transmission electron microscopy.

other

parts

of

the

plasma

membrane.

Borregaard

-

‘

4’ ‘

. _t.

Fig. 6. Mac-l

,

;,4%

\,*1.

--- I .

‘

.-.

.

.

-

-

,‘...:_,,,,_.

to 4#{176}C, and labeled with monoclonal glutaraldehyde and immunolabeled The immunogold label (arrowheads)

x 60,000.

et a!.

Human

neutrophil

adhesion

receptors

85

associated

alkaline

phosphatase

and

Mac-i

become

-independent

accessi-

ble to macromolecules such as antibodies and hence are incorporated into the plasma membrane, where they may interact with the surroundings of the cell. To further visualize this, intact cells were labeled with monoclonal antibody against L-selectin, alkaline phosphatase, or Mac-i, and the membrane topology of these proteins was determined by immunoelectron microscopy. Figure 4 confirms the localization of L-selectin on the tips of microvilli by preimbedding immunoelectron microscopy of unstimulated cells as previously demonstrated both in frozen thin sections [2] and by scanning electron microscopy [23]. Figures 5 and 6 show that both alkaline phosphatase and Mac-i are randomly distributed on the surface of fMLP-stimulated cells. We found no lar

indication structures

that alkaline as reported

phosphatase previously

5. von

J.D.,

The expert technical assistance of Charlotte Horn, Pia L. Olsen, and Yvonne Jacques is gratefully appreciated. This work was supported by the Danish Cancer Society, the Danish Medical Research Council, NIH-CA 31799 (T.A.S.), NIH-DK10486 (D.F.B.), the Novo Fund, the Lundbeck Fund, Anders Hasselbalch’s Fund, Amalie Jcrgensen’s Fund, and Ane Kathrine Plesner’s Fund. NB. is the recipient of a Neye research professorship.

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