Journal

Lymphotoxin From Adherent

of Leukocyte

Biology

47:506-513

(1990)

Induces Secretion of Granule Proteins Neutrophils: Possible Role of Intracellular Free Calcium Johan

Division

of Haematology,

Department

Richter

of Medicine,

University

Hospital,

Lund,

Sweden

Lymphotoxin (LT) can activate human neutrophils. Using a hemolytic plaque assay to detect secretion of lactoferrin and myeloperoxidase (MPO) from single adherent neutrophils, we showed that LT induced secretion from both primary and secondary granules. Incubation of cells with cytochalasin B was required for MPO secretion, and it enhanced lactoferrin secretion. Pertussis toxin, which blocks a G-protein in the plasma membrane, inhibited LT-induced exocytosis of MPO, but not of lactoferrin. Incubation with LT did not induce any detectable changes of the cytoplasmic free [Ca2] in neutrophils. On the other hand, secretion of granule proteins from adherent neutrophils in response to LT was blocked by loading neutrophils with quin-2 in order to increase the intracellular calcium buffering capacity. This was achieved at a concentration of quin-2, at which the secretion induced by the phorbol ester PMA and the chemotactic peptide FMLP was unaffected. Trifluoroperazine (TFP), a dual protein kinase C and calmodulin inhibitor, significantly inhibited the LT-mediated secretion of lactoferrin from adherent granulocytes. The PMA effect was unaltered by TFP under these conditions, suggesting that the inhibitory effect was on a calcium-calmodulin dependent step. The secretion induced by TNF and GM-CSF was also blocked by buffering changes in the intracellular [Ca2l and inhibited to a similar extent by TFP. Our results suggest that calmodulin and minute changes in the cytoplasmic free [Ca2 ] may be involved in a common signal transduction pathway engaged in activation of adherent neutrophils by several cytokines. Key words:

lactoferrin,

myeloperoxidase,

pertussis

INTRODUCTION Lymphotoxin

is produced

(LT)

by stimulated

and some lymphoblastoid to the same family of cytokines (TNF),

which

phages quence

[6].

tem

LT

. To

biological also

and

homology

[ 1 3]

cause

is released [25]

shown

granulocytes, 34].

Also,

the

respiratory

in response herence was

has

directly

been

promoted

have

been published chemotaxis induced nyl-anine (FMLP), Wiley-Liss,

a 30%

macro-

amino

acid

a common

receptor

they

have

also

the function or by so-called

shown

burst was activated to a second stimulus [28].

on neutrophil migration having a chemotactic well as reports on LT

© 1990

share

extent,

to modulate

either

LT

endotoxin-treated

show

and

It belongs necrosis factor

The

to stimulate

sys-

neutrophils;

[ 10]

on the effect

or ad-

of LT

are contradictory; reports on LT influence on neutrophils [ 10] as not affecting neutrophil migration

[9, 17]. An inhibitory by N-formyl-methionyl-leucyl-pheCSa, and LTB4 has been Inc.

beit has

of neutrophil priming [16,

either directly [9]. and neutrophil data

se-

similar

TNF was originally recognised effect on tumor cells [7], but

of a cytotoxic been

TNF

a large

effects.

from

lympho-

[ 1] .

cell lines as tumor

cytes

effect observed

on

toxin,

quin-2,

trifluoroperazine

F 17] . LT has been shown to enhance the tumoricidal properties of neutrophils in an antibody-dependent cellular cytotoxicity assay [29]. LT alone did not induce granule protein secretion from neutrophils, but incubation with LT led to secretion of lysozyme and enhanced secretion of beta-glucuronidase in response to FMLP cytochalasin B-treated cells [9]. We have previously described a reverse hemolytic

plaque

assay myeloperoxidase

for detection (MPO)

phils [26]. Using this granulocyte/macrophage elicit secretion of both aim LT the

of the

present

on neutrophil mechanisms

work

of secretion from single assay,

we

of lactoferrin and adherent neutro-

found

that

colony-stimulating lactoferrin was

and

MPO

to determine

degranulation involved in the

the

TNF factor [27].

and can The

effects

July 6. 1989: accepted

of

and try to characterize signal transduction pro-

cess.

Received

in

September

8, 1989.

Reprint requests: Johan Richter, Research Department University Hospital. S-221 85 Lund. Sweden.

2, E-blocket.

Lymphotoxin

PoIy-L-Lysine

MATERIALS AND METHODS Special Reagents Recombinant

LT

was

We

supplied Vienna,

Ernst-Boehringer-Institut,

tion, assayed (by the supplier) L-929 fibroblast cells contained MPO

and

antilactoferrin

of rabbits

with

[24].

These

react

with

iments

other

rified

proteins,

fura-2,

Pertussis Campbell,

quin-2,

and

TFP

Company, St. Calbiochem-Behring

Louis,

MO. Comp.

toxin was CA.

surplus

from

List

pu-

13-acetate were

from

Cytochala, La Jolla,

Biological

Laborato-

protein

ries),

Blood 2%

was

obtained

dextran

from

(Pharmacia,

healthy

volunteers.

Sweden)

in 0.15

One

part

M NaCI

was

granulocytes the erythrocytes

and

granulocytes once in Hanks

were washed once in 0. 15 M NaCl balanced salt solution (HBSS, Flow

oratories, Irvine, (FBS) (Gibco,

erythrocytes. with sterile

containing

Scotland) with Paisley, Scotland).

was

used

for

was

used

for checking

granulocyte bility was

staining. 30 mm hemolytic

cell

counting.

cells

in HBSS plaque

lysis of sec. the and Lab-

1% fetal bovine serum A Coulter Counter

Initially.

the

purity, in excess The

After hypotonic water for 30-45

Coulter

a Burker Counter

chamber

readings

and

which was from 96% to 98%. Viaof 97% as judged by trypan-blue were

allowed

to equlibrate

with 1% FBS assay.

before

for at least

being

used

in the

Protein red

of Protein

A to Sheep

A (Pharmacia,

Sweden)

blood

cells

(SRBC,

kindly

Red Blood Cells was

coupled

supplied

by

partment of Microbiology, as described previously

University of Lund, [12]. In short, SRBC,

Alsevers

1-3 One

times mixed

in 0.15

solution,

aged

in 0. 15 M NaCl. with

M NaC1) in 0. 15 M NaCI. for 1 hr. washed HBSS, and kept week.

one

part

buffer plaques

protein

A

(0.5

mg/ml

and ten parts 2.5 X l0 M CrC13 The mixture was incubated at 30#{176} C once in 0. 15 M NaCl and twice in at 4#{176}C. The cells were used within 1

medium KC1,

used

off,

ml

MO, were

followed of protein at 4#{176} C, followed

by

or anti-MPO ( 1/ Flow Laborato-

a stimulatory

for

agent

dissolved

in

and

fura-2

contained I . 1 mM

MgSO4,

NaHPO4,

5 mM

of

of

quin-2

analysis

1 mM

1 mM

loading

and

138 mM NaC1, 6 CaCl-,, 0. 1 mM

NaHCO3,

5.5

mM

glu-

cose, and 20 mM Hepes (pH 7.4). For spectrophotometer experiments, cells were suspended in the above-described medium at a concentration of 5 X l0 cells/ml and allowed fura-2/AM to a final

to equilibrate

of the

1 x

cells/ml

l0

at 37#{176} C for 5 mm,

(from a stock solution concentration of 2 jiM.

addition

probe, with

the

cell

suspension

the

same

type

tration.

Cells

Fluorescence

kin-Elmer Cells

were

kept

at room

measurements fluorimeter (LS used

at a concentration concentration during Loading

of I .0 x was 1 .0 jiM, 1 hr without under

these

was

temperature

plaque cells/ml.

106

and any

diluted

until

assay The

were

loaded

quin-2/AM

the loading was dilutions during

conditions

this

used.

with a Perdescribed

was

would

lead

perthat

previously

found to result in an intracellular quin-2 content ± 0.08 nmole/l06 cells [27]. Ifthe average cell

assumed to be 4.2 um [30], cellular quin-2 concentration

to

supple-

loading procedure at this concen-

were performed 3B) as previously

in the hemolytic

which

of medium

with 0.5% (w/v) albumin. The completed after 50 mm incubation

was

after

in DMSO) was added Ten minutes after the

mented

time.

three was

poured

per

and stored at 4#{176} C until the number were counted under a microscope.

fluorometric

mM

the

weeks, were washed part of packed SRBC

and

free

The

formed

De-

jig

St Louis. The dishes

in HBSS,

( 1/2,560) (1/100,

and Manipulation

to sheep Sweden) stored in

were

Ca2

[2].

Coupling

A-SRBCs

Determination

Norway)

in a pellet

(25 .

once

Cytosolic

EGTA,

resulting

and

of antilactoferrin pig complement

the

Approximately 330 granulocytes were added to each dish. The dishes were incubated for 2 hr at 37#{176} C and then fixed with a 5% solution of glutaraldehyde in

during

centrifuged,

solution

I ml of a I % suspension After 15 mm of incubation

granulocytes.

added to one part of heparinized blood. After red cell sedimentation, the leucocyte containing supernatant was layered on top of Lymfoprep (Nyegaard and Co. , Oslo, and

of

[15]. Plastic dishes, 60 Oxnard, CA, USA).

HBSS.

hemolytic

of Granulocytes

in 0. 15 M NaC1

the addition 320), guinea

507

a modification

assay Plastics,

a poly-L-lysine

addition of in HBSS.

phosphate

Isolation

with

once

was

not

described

plaque (Falcon

M NaCI, Sigma Chemical Co. for 15 mm at room temperature.

by exper-

did

Lactoferrin 1 2-myristate

mm were treated

Degranulation

Assay

previously

monolayer in diameter

0. 15 USA)

Neutrophil

Plaque

have

by the A-SRBC

and

as judged

original

rinsed

described

specific

[4] . Phorbol

FMLP,

activity on U/ml. Anti-

by immunization

immunodiffusion.

Sigma Chemical sin B was from

G.R. Adolf, The prepara-

as previously

highly

neutrophil

as described

(PMA),

CA. ries,

were

radial

obtained

proteins

antisera

with

by Dr. Austria.

by its specific 1 .5 x l0

were

purified

Induces

of 0.54 radius is

to an

intra-

of approximately I .7 mM. The reason for using this different loading procedure was that it made it possible to minimize the DMSO concentration. The cells were used in the plaque assay within 30 mm

after

chelation under

completion of the loading was not found to affect the circumstances described.

procedure. neutrophil

Calcium adherence

508

Richter TABLE

1.

Maximal Secretory

GM-CSF, and PMA, and Concentrations Secretory Response

Response

Obtained

by LT, TNF,

as Percentage of Plaque-Forming Necessary to Obtain Half-Maximal (EC

Cells

Cl,

Secretion

ci

100

Maximal response plaque forming cells)

0

Stimulus

0.

(e/(

LT 0 a-

50

E

610 150’

46C4

GM-CSF

37/J

PMA

82e%

Data

z

ECS( pM

36C%

TNF

C) .0

of lactoferrin

is from

Richter

et al.

1271.

1989

.

0

0

LT

i02

LT

(U/mI)

200 Cl)

Fig. 1 . Effect of rLT on formation of lactoferrin (A-A) and MPO plaques (Li-U). Cytochalasin B was required for MPO plaque formation. Data represent the mean of at least five separate experiments.

0 Ce

0.

C

100

RESULTS Secretion

0 0 Cl)

of Lactoferrin

-J

Using the hemolytic plaque induce secretion of lactoferrin ncr (Fig.

1 ). Secretion

U/mI

reached

and

centration was to

be

started

a maximum

necessary

assay,

at 10,000

for half-maximal

lactoferrin and the

LT resulted

2). Cytochalasin

Secretion

of MPO

The

[27]. Addition in an increased B by itself was

of cytochalasin B number of plaques also found to elicit

formation.

when

of cytochalasin

cytochalasin

B within

B was

0.3-0.5

this

concentration

jig/ml. range

Even was

added,

only a small fraction of the neutrophils released enough MPO to create detectable plaques. MPO plaque formation started at 300 U/ml, but it did not reach a plateau within the concentration range investigated.

The

tactic

receptor

Toxin

on the granulocyte

peptide FMLP is coupled located in the plasma membrane. the G protein can be inhibited

with

pertussis

toxin,

which

causes

ADP

ribolysation

of

of the G protein [1 1]. In the present neutrophils for 2 hr with pertussis

work, toxin

(300 ng/ml) and monitored release of MPO and lactoferrin in response to LT using the hemolytic plaque assay (Figs. 3, 4). FMLP and PMA were used as positive and negative controls, rin plaques was

by pertussis was

reduced

the

plaque

37] the

respectively. slightly, but

toxin, almost assay

whereas to zero. when

and

The number not significantly,

the number Cytochalasin

MPO

of MPO B was

secretion

Buffering

of lactoferreduced

was

plaques added to

monitored.

of Cytosolic

Ca2

We

for the chemo-

to a GTP binding protein Signal transduction via if granulocytes are treated

10U/ml

symbol.

Free surface

io

Fig. 2. Effect of cytochalasin B on lactoferrin plaque formation induced by LT: 0 tg/ml (s-U), 0.5 tg/ml (A-A), or 5 g/ml (A-A) was added to the assay of degranulation. Each point represents the mean ± SEM of three different determinations. SEM not marked in the figure falls within the corresponding

Determination

Effect of Pertussis

io2

0

the alpha unit we preincubated

Exocytosis of MPO after LT stimulation was observed only in the presence of cytochalasin B. The optimal concentration

0

con-

of TNF, have been

along (Fig.

plaque

U/mI.

to

manof 100

secretion with the effects phorbol ester PMA, which

investigated

lactoferrin

found

(EC5O) and found the effect

previously with

was

exocytosis curve 1 compares

calculated from the dose-response 1,500 U/ml (610 pM). Table

of LT on GM-CSF,

LT

in a dose-dependent at a concentration

and

others

nor GM-CSF intracellular

suspension

when

have

shown

[27, Ca2

32] induce concentration

measured

that

neither any

as fluorescence

TNF

[27,

35,

rapid changes in neutrophils from

quin-2

of in

Lymphotoxin

Induces

Neutrophil

PMA

LT

Degranulation

509

FMLP

U,

a)

Cl)

C

a) 0 0 Cl)

-J

0

10

10

10’U/ml

0

0.1

1

10

ng/mI

0

1

10

lOOnM

Fig. 3. Effect of pertussis toxin on lactoferrin plaque formation induced by LT, PMA, and FMLP. Neutrophils were preincubated for 2 hr at 37#{176} C in the absence of (L]-Li) and in the presence of 300 ng/ml of pertussis toxin (A-A) before the assay of degranulation. Each point represents the mean ± SEM of at least three different determinations. SEM not marked in the figure faIls within the corresponding symbol.

LT

PMA

FMLP

100

U,

a) Cl)

50

0

i02

104U/mI

0.3

0

3

30 ng/ml

0

10

lOOnM

Fig. 4. Effect of pertussis toxin on MPO plaque formation induced by LT, PMA, and FMLP. Neutrophils were preincubated for 2 hr at 37#{176} C in the absence (Li-Li) and in the presence of 300 ng/ml of pertussis toxin (A-A) before assay of degranulation. Cytochalasin B (0.3 p.g/ml) was added in the assay of degranulation. Each point represents the mean ± SEM of at least three different determinations. SEM not marked in the figure falls within the corresponding symbol.

or fura-2 LT,

loaded

like

the

changes

cells. other

in cytosolic

suspension

(data

In the present two

cytokines,

free

Ca2

did

we found not

in fura-2

not shown).

used not only as a calcium changes in the cytoplasmic

work.

loaded quin-2

However, indicator but free calcium

cause

that

investigated

by using

any

(TFP)

In

in be

more

than a 50%

plaques

induced

cells can

also to buffer concentration

[19. 33]. When neutrophils were loaded with I jiM quin2 for 1 hr at a cell concentration of 10#{176}/mIand subsequently used in the plaque assay, the secretion of both MPO and lacoferrin following LT stimulation was almost totally inhibited (Figs. 5. 6). FMLPand PMA-

induced

exocytosis

was

not

Calmodulin-dependent mechanisms

the

the phenotiazine plaque

assay,

trifluoroperazine 15 jiM

reduction in the number by LT. following a S mm

TFP

caused

of lactoferrin preincubation

of the granulocytes with TFP at the same concentration. PMA-induced plaque formation was not affected under these used

conditions. TFP in concentrations

has been reported above 10 p.M

to be toxic when [21]. We did not.

however, notice any toxic effects on neutrophils 2.5 hr incubation with 15 jiM TFP. asjudged blue exclusion and release of LDH.

during a by trypan-

affected.

DISCUSSION

Effect of Trifluoroperazine duction

[31].

processes

in neutrophils

have

in signal-transin the past been

We have induce

previously

secretion

shown

of lactoferrin

that and

TNF MPO

and

GM-CSF

from

single

can ad-

510

Richter

herent also

granulocytes can

[27].

we

LT

neutrophils

is well data).

of MPO was weak and required the presence lasin B, which was also the case with TNF

of cytochaand GM-CSF

acterizing

superoxide

than

sponse

and

Superoxide anion production, tion, and granule enzyme secretion

the

hemolytic investigators

by the from

plaque assay. [9] observed

data

for

maximal

dose-response secretion

recurves

of lysozyme

(an enzyme found both in primary and secondary granules) following incubation with LT only if neutrophils were treated with cytochalasin B, whereas we found that lactoferrin,

which is localized solely in the secondary was released even in the absence of cytocha-

granules, lasin B. No significant release of beta-glucuronidase, which is localized in primary granules, was noted by the same

investigators

even work,

[9]. In the present secretion The

of

MPO

previous difference

Nathan CSF, from

primary

B treatment weak, of LT on

granules,

was

investigated

the effects

of TNF,

LT

[22] , GM-

and G-CSF [23] on superoxide anion adherent neutrophils. All four cytokines

to induce a massive herent cells following effects greater

found.

observations [9] , however, were performed in suspension, which probably explains from our results for adherent neutrophils.

on neutrophils the

from

after cytochalasin an effect, albeit

on than

superoxide

anion

production were found

production

a lag period of 30-45 adherent cells were orders of the effects on cells in suspension,

in admm. The magnitude and they

could be inhibited if cytochalasin B was added during the lag period. A model for the effects of cytokines on neutrophil granulocytes was suggested based on a priming

effect and a triggering effect of the cytokines [23] . When acting on a cell in suspension, a cytokine alone would induce a minor functional response (or none at all), but would instead prime the cell to react with an enhanced response to a second stimulus added later. The triggering effect of a cytokine receptors are ligated binds to its receptor,

should be expressed if cell adhesion at the same time as the cytokine resulting in a massive functional response. According to Nathan [23], the long lag period and the ablative effect of cytochalasins are characteristic for cytokine triggering of neutrophil functions. Although

this may be true for superoxide anion production induced by cytokines, the findings cannot be generalised to all functions of adherent neutrophils activated by cytokines. We

previously

granule

protein

observed

that

secretion

elicited

cytochalasin by TNF

B enhanced and

GM-CSF

[27] and, in the present work, it was shown that the same is true for secretion in response to LT. We have also observed that the secretory response to TNF in adherent

vation

adding

of these

cytochalasin

B,

functions

by

(unchar-

granule

opposite

mechanisms

two

I 5 mm kinetics

and

diametrically

potent

obtained

and

after

different

production

the

intracellular

as judged

treatment

anion

and

by

EC5O

toxin

secretion,

different

Other

pertussis

already the

obtained

TNF

both

tein

developed

Considering

loading with quin-2 inhibited MPO secretion makes it unlikely that this small response solely reflects a toxic effect of LT on the neutrophils. LT was slightly less

using

that

The

that

published

fact

secretion.

demonstrate

on secretion

The

granule

Here

effect

[27].

elicit

proeffects

it seems likely that are involved in acti-

cytokines

in adherent

neutrophils. chemotaxis, in response

are inhibited in a doseand lowing exposure to pertussis

time-dependent toxin [18].

granule

to CSa,

proteins

in response

aggregato FMLP manner Secretion

leukotriene

B4,

platelet-activating factor can also be inhibited tussis toxin [3]. We have shown that the same GM-CSF induced secretion of both lactoferrin [27].

The

tion, was was

effect

of pertussis

be affected which would mon

receptor

evidence teins

[ 1 3]

. So

indicating

are

signal

in the same be expected,

not

involved

in neutrophils

than

secretion

Our

finding

different

far,

of that

granule

most

reports

pertussis in the

[5,

per-

as lactoferrin secretion whereas MPO secretion TNF has been reported to

way by pertussis as LT and TNF

that

and

with

is true for and MPO on LT-induced secre-

toxin

however, was ambiguous, not inhibited significantly, almost totally abolished.

folof

20].

have

turned

toxin-sensitive transduction

granule

enzymes of

TNF

not

other

investigated.

proteins was

the

functions were

up

G-proof

However,

exocytosis populations

toxin [27], share a com-

from

the

affected

two

in

the

same

way by pertussis toxin has a parallel case in observations of neutrophil activation via the 42 kDa Ig-Fc receptor [8]. The secretion of different granule enzymes were not inhibited to the same extent by pertussis response to stimulation of the Fc receptor with ments suggest and

of antireceptor mAb that TNF/LT-induced

secondary

granules

diated via two different may involve a pertussis Even though LT did cytosplasmic

free

calcium,

and

toxin in Fab frag-

(Fab’)2 anti-Ig. Our data degranulation of primary

in adherent

neutrophils

are

me-

pathways, only one of which toxin sensitive G-protein. not induce detectable changes of we found

trophils with quin-2, both lactoferrin could be blocked. There are three

that

by loading

neu-

and MPO secretion possible explanations

to this finding. One is that the effect of quin-2 is not due to its calcium buffering capacity, but rather to a nonspecific effect on the neutrophils. This is unlikely, as the responses to FMLP and PMA were not altered, suggesting mised would

that

the

neutrophils

by quin-2 loading. be that adherence

sponse so that the addition cytoplasmic free calcium cells third

in suspension, explanation

were

not

A second modulated

functionally

possible explanation the neutrophil re-

of LT resulted in adherent

compro-

in a change cells,

but

not

of in

which we have investigated so far. A would be that adherence itself modu-

Lymphotoxin

Induces

Neutrophil

PMA

LT

Degranulation

511

FMLP

U,

a)

Cl)

C a) 0 0 Cl)

-J

0

10

10’

10U/mI

0

0.1

1

10

ng/mI

0

1

10

lOOnM

Fig. 5.

Effect of intracellular quin-2 on Iactoferrin plaque formation induced by LT, PMA, and FMLP. Neutrophils were incubated for 1 hr at 37#{176} C in the absence (LI-Eli) and in the presence of 1 .0 pM quin-2/AM (A-A) before assay of degranulation. Each point represents the mean ± SEM of at least three different determinations. SEM not marked in the figure falls within the corresponding symbol.

LT

PMA

100

U,

a)

Cl)

50

0

0

Fig. 6.

Effect

Neutrophils

-

;;::r-r----

(1

0

i02

iO4U/mI

of intracellular

quin-2

0

0.3

on MPO

3

plaque

30

ng/mI

formation

0

induced

1

10

lOOnM

by LT, PMA,

and FMLP.

were incubated

for 1 hr at 37#{176} C in the absence of (U-U) and in the presence of 1.0 M quin-2/AM (A-A) before assay of degranulation. Cytochalasin B (0.3 gig/mI) was added in assay of degranulation. Each point represents the mean ± SEM of at least three different determinations.

lated uisite

the intracellular for the activating

SEM

calcium effect

not marked

homeostasis of cytokines.

in the figure

as a prereqIt has for

falls within

the corresponding

mediated

secretion

of a calcium-calmodulin and GM-CSF-mediated

example been shown that in the intracellular [Ca2 Also, the fact that quin-2

adherence induced oscillations ] of human neutrophils [14J. inhibited secretion induced by

hibited Taken

TNF,

in a similar

as minute

LT,

and

GM-CSF

gests that the effect a point in a common The

results

interpreted

of the calcium signal-transduction

manner chelator

obtained using trifluoroperazine with caution, as TFP has been

[27]

sug-

is exerted pathway.

at

have to be shown to in-

hibit protein kinase C as well as calmodulin, thereby not acting as a specific inhibitor [36]. It seems likely. though, that under the experimental conditions in question, TFP does not act as a protein kinase C inhibitor as PMA-induced it is conceivable

plaque that

formation the effect

was not affected. Thus, of TFP on lymphotoxin-

to a similar together, our changes

symbol.

of lactoferrin

is caused

dependent secretion

by

inhibition

process. Also, TNFof lactoferrin was in-

extent by TFP (data not published). data suggest that calmodulin, as well in cytoplasmic

free

part of a common signal-transduction in activation of adherent neutrophils

calcium,

may

be

pathway engaged by cytokines.

ACKNOWLEDGMENTS The

technical

author

wishes

assistance

to thank

Karin

Vegas

for

and Dr. Inge Olsson, who a scientific adviser. This work was supported Swedish Cancer Society, the Alfred Osterlund tion and the Medical Faculty of Lund.

excellent served as by the Founda-

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