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Immunology Letters, 40 (1994) 117 124

0165 2478 / 94 / $ 7.00 © 1994 Elsev ier Science B.V. All r ights reserv ed

I M L E T 2 1 3 2

A u t o c r i n e r e g u l a t i o n o f I C A M , 1 e x p r e s s io n o n b l a d d e r c a n c er c el l

li ne s: e v i d e n c e f o r t h e r o l e o f I L - l e

b *A n t o n B . A l e x a n d r o f f ~ ' , A n d r e w M . J a c k s o n a , K e s a v a n E s u v a r a n a t h a n a , S t e p h e n P r e s c o t t

a n d K e i t h J a m e s a

Department of Surgery (WGH), University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK, bResearch Institute of

Paediatric Haematology of Russia, Moscow, Russia

( Rece i ved 7 Mar ch 1994 ; accep t ed 18 Ma r ch 1994 )

I . Summary

W e h a v e s t u d i e d t h e a u t o c r i n e r e g u l a t i o n o f e s s e n -

t i al e x p r e s s i o n o f t h e i n t e r c e l lu l a r a d h e s i o n m o l e c u l e -

1 ( I C A M - 1 ) o n 8 t r a n s i ti o n a l c el l c a r c i n o m a ( T C C )

ce l l l i ne s (h i s topa tholog ica l g rades 1 -3) . The cons t i -

t u t i v e e x p r e s s i o n o f I C A M - 1 w a s r e g u l a t e d b y s o l u -

b l e f a c t o r s i n a n a u t o c r i n e f a s h i o n . T h e s e f a c t o r s

w e r e p r o d u c e d b y a l l c el l li n es , w i t h t h e e x c e p t i o n o f

t h e M G H - U 1 c e ll l in e . T h e e f f e c t s o b s e r v e d c o u l d b e

l a r g e l y a t t r i b u t e d t o I L - I ~ . H o w e v e r , t h e r e s i d u a l

I C A M - 1 i n d u c i n g a c t iv i t y ( u p t o 3 0 % o f IC A M - 1 i n-

d u c t i o n ) c o u l d n o t b e a s s o c i a t e d w i t h a n y k n o w n

I C A M - I i n d u c e r s ( I F N T , T N F ~ , T N F / ~ , I L - I ~ , I L -

1 /~ , IL -4 , r e t i no i c ac id , LPS) . In con t ras t t o r eco mb i -n a n t d e r i v e d c y t o k i n e s , t h e I L - I ~ p r e s e n t i n t i s s u e

c u l tu r e s u p e r n a t a n t w a s o n l y a b le t o i n d u c e I C A M - 1

o n h i g h - g r a d e t u r n o u t s a n d n o t l o w - g r a d e c e l l s . T h i s

d i s c r i m i n a t i v e e f f e c t i s s i m i l a r t o t h a t n o t e d f o l l o w i n g

i n v i t r o c u l t u r e o f t u m o u r c e l l s w i t h bac i l lu s C a lm -

e t t e -G ue r in o r g a n i sm s . W h e t h e r t h e p r o d u c t i o n o f s o -

lub l e fac tor s ( e .g . , IL - I~ ) by TCC ce l l l i ne s p l ays an

e s s e n t i a l a u t o c r i n e r o l e f o r b l a d d e r t u m o u r s a n d / o r

a f f e c t s t h e i n t e r a c t i o n w i t h c e l l s o f t h e i m m u n e s y s -

t e m n e e d s t o b e i n v e s t i g a t e d f u r t h e r .

2. Introduction

I n t e r c e l l u l a r a d h e s i o n m o l e c u l e - 1 ( I C A M - I ) i s a

Key words: I n t e r ce l l u l a r adhes i on mol ecu l e - l ; Adhes i on mol ecu l e ;

Cy t ok i ne ; B l adde r cance r ; l n t e r l euk i n - l c< Aut oc r i ne p r od uc t i on

*Corresponding author: Ant on B . A l exand r of f , Depar t men t o f S ur -

g e r y (W G H ) , U n i v e r s i t y o f E d i n b u r g h M e d i c a l S c h o o l, T e v i o tP l ace , Ed i nbur gh EH8 9AG, UK.

c e l l - s u r f a c e g l y c o p r o t e i n o f 9 5 k D a m o l e c u l a r w e i g h t

a n d a m e m b e r o f th e i m m u n o g l o b u l i n s u p e r f a m i ly[1 ,2 ] . I t f ac i l i t a t e s adhes ion of l eukocyte s t o each

o t h e r a n d t o o t h e r c e l l s . T h e d e s c r i b e d l i g a n d s o f

I C A M - 1 ( L F A - 1 , M A C - 1 a n d C D 4 3 ) a r e e x p r e s s e d

o n d i f f e r e n t t y p e s o f h e m a t o p o i e t i c c e l l s . R e c e n t l y i t

h a s b e e n s h o w n t h a t I C A M - 1 i s a s i g n a l m o l e c u l e

c a p a b l e o f a c t i v a t i o n o f l y m p h o c y t e s [ 3 , 4 ] . I t p l a y s

a n i m p o r t a n t r o l e i n m a n y i m m u n e r e a c t i o n s a n d

p a t h o l o g i c a l c o n d i t i o n s i n c l u d i n g l e u k o c y t e m i g r a -

t i o n , a n t i - t u m o u r i m m u n i t y , a u t o i m m u n e d i se a se , a l-

l ogra f t r e j ec t ion , e t c . [2 ,5 -8] . ICAM-1 i s cons t i t u-

t i v e l y e x p r e s s e d o n v a s c u l a r e n d o t h e l i u m , e p i t h e l i a l

c e ll s, f ib r o b l a s t s a n d a l i m i t e d n u m b e r o f h e m a t o -

p o i e t ic c e ll s s u c h a s t i s s u e m a c r o p h a g e s [ 1 ,2 ] . H o w -

e v e r , it s e x p r e s s i o n o n m a n y t y p e s o f ce ll s i n c l u d i n g

t u m o u r s c a n b e g r e a t l y i n d u c e d a n d a u g m e n t e d b y a

n u m b e r o f p r o - i n f l a m m a t o r y c y t o k i n e s a n d a g e n t s ,

n a m e l y i n t e r f e r o n - g a m m a ( I F N T ) , t u m o u r n e c r o s i s

f a c t o r - a l p h a a n d - b e t a ( T N F ~ a n d T N F / ~ ) , i n t e r l e u -

k i n - a l p h a a n d - b e t a ( I L - I ~ a n d I L - / ~ ) , I L - 4 , r e t i n o i c

a c i d , l i p o p o l y s a c c h a r i d e s ( L P S ) a n d bac i l lu s C a lm -

e t t e -G ue r in o r g a n i s m s ( B C G ) [ 1 ,2 , 9- 1 3 ].

R e c e n t ly w e h a v e s h o w n t h a t 4 o f 8 b l a d d e r c a n c e r

c e l l l i n e s c o n s t i t u t i v e l y e x p r e s s d i f f e r i n g a m o u n t s o f

I C A M - l [ 1 4 ] . T h e e x p r e s s i o n o f th i s m o l e c u l e is in -

d u c e d a n d a u g m e n t e d b o t h i n v i v o a n d i n v i t r o o nT C C c el ls b y c y t o k i n e s p r o d u c e d d u r i n g i m m u -

nothe rapy [14-19] . Th i s confe r s i nc reased suscept ib i l -

i t y o f t u r n o u t c e l l s t o n o n - M H C - r e s t r i c t e d - l y s i s i n

v i t r o [ 5 ] . I n t h e p r e s e n t s t u d y , w e i n v e s t i g a t e d t h e

c a u s e o f c o n s ti t u ti v e I C A M - 1 e x p r es s io n o n b l a d d e r

c a n c e r ce ll s - - a p h e n o m e n o n w h i c h c o u l d p la y a n

i m p o r t a n t r o le in i m m u n o t h e r a p y o f b l a d d e r a n d

o t h e r t y p e s o f m a l i g n a n c y .

SSDI 0 1 6 5 - 2 4 7 8 ( 9 4 ) 0 0 0 4 7 - U

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3 . M a t e r i a l s a n d M e t h o d s

3.1. Cell lines

In the study 8 human transitional-cell carcinoma

lines, namely RT4 (histopathological tumour grade

I), RT112 (grade II), MGH-U1 (grade III, supplied

by Dr. J. Masters, Institute of Urology, London),

UMUC3 (grade I), SD, 5637, EJ18, J82 (grade III,

supplied by Dr. P. Perlmann) were used. These celllines have proved to be stable and their cytological

appearance and growth characteristics have re-

mained representative of the parent tumours.

The cells were routinely grown in RPMI-1640

medium (Gibco, Uxbridge, UK) supplemented with

5% foetal calf-serum (Sera-Lab, Crawley Down,

UK), sodium pyruvate (5 mM) and L-glutamine (2

raM). For routine purposes and analysis, cells were

recovered by trypsinization (trypsin/EDTA, 0.5 g/1

trypsin and 0.2 g/1 disodium EDTA).

3.2. Cytokines polyclonal and monoclonal antibodies

Highly purified recombinant IFN7 (rINFT) was

purchased from Boehringer Mannheim (Lewes, UK)

and contained 2.5 × 107 U/mg protein and less than 2

endotoxin U/mg. rIFNc~2a was the generous gift of

Dr. S. Langden, ICRF, Edinburgh, and contained

2 × 107 U/mg protein, rIL-l~ was obtained from Brit-

ish Biotechnology and contained 5 × l0 s U/mg pro-

tein. IL-2 was generously provided by Euro-Cetus

and contained 1.8 × 10 7 U/mg protein. Highly puri-

fied rTNF~, IL-3, IL-4, IL-7, granulocyte-macro-

phages colony stimulating factor (GM-CSF) were

the kind gift of Behringwerke (Marburg/Lahn, Ger-

many). Recombinant transforming growth factor-

beta (rTGFb2) containing 5 × 107 U/mg protein and

IL-8 were purchased from Genzyme (West Malling,

UK). Highly purified murine epidermal growth fac-

tor (EGF) was generously donated by Dr. F.K. Ha-

bib (Surgery, WGH, Edinburgh University) and ori-

ginally was obtained from Collaborative Research

(Bedford, MA). Retinoic acid was obtained from Sig-

ma (Poole, UK).

Neutralising sheep polyclonal antibodies to human

IFNT, TNF~, TNF/3 were kindly provided by Dr. A.

Meager (NIBSC, Potters Bar, UK). The murinemonoclonal antibodies (mAb) t.o human ICAM-1

were generously provided by Dr. T.A. Springer

(Harvard Medical School, Boston, MA); this anti-

body is coded RRI /I and is of the IgG1 isotype. Poly-

clonal antibodies to IL-I~ and IL-I/~ were obtained

from the National Institute of Biological Standardi-

118

zation and Control (Potters Bar, UK). Sheep anti-

(mouse-IgG) conjugated to fluorescein isothiocya-

nate (FITC) was obtained from Sigma (Poole, UK).

3.3. Quantification o f surface antigen expression

Single-colour immunofluorescence was performed

on bladder cancer cell lines stained indirectly with

mAbs. For each analysis 5 × 105 cells were washed

with washing buffer (phosphate-buffered saline, 1%foetal calf serum, 0.01% sodium azide) and incu-

bated at 4°C for 30 min with optimal concentrations

of primary mAb. Following two washings, the bind-

ing of unconjugated mAbs was detected by incubating

with a F(ab')2 fragment of sheep anti-(mouse IgG)-

FITC conjugate for a further 30 rain at 4°C. The cells

were washed twice more and resuspended in 1% for-

maldehyde prior to analysis using a flow micro-fluori-

meter (EPICS-C). Non-viable cells were gated out of

the window and at least 5000 events were accumulated

using linear amplification of fluorescence.

3.4. Experimental s trategy

The TCC cells growing in mid-log phase in 24-well

tissue-culture plates were stimulated with superna-

tants from different bladder cancer cell lines (diluted

in complete medium 1:1) and recombinant cytokines.

After 24 h incubation cells were recovered using tryp-

sin/EDTA, stained with mAbs and analysed by

fluorocytometry. The supernatants of TCC cell lines

were collected from a confluent flasks after at least 24

h of incubation and kept at -20°C.

In order to correct for the influence of cell density

on ICAM-1 expression the following formula was

used: the percentage o f ICAM-l-pos itive cells was di-

vided by a coefficient K=A/Bo .18 where A was the

number of cells/well for an experimental cell density

and B0.18 was the number of cells/well for the cell

density of 0.18 × 105 cells/cm 2 (the lowest cell density).

For neutralising experiments undiluted samples of

supernatants were incubated at 20°C for 1 h with the

following dilution of neutralising antibodies: IFNy

(1:200), TNF~ (1:200), TNF/~ (1:500), IL-l~

(1:1000), IL-1/3 (1:1000) prior to applying to cells.

These concentrations of the antibodies were enough

to neutralise cytokines at the concentrations at least500 U/ml.

3.5. Statistical analysis

Statistical analysis was performed using the non-

parametric Mann-Whitney U test provided in Stat-

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v i e w 51 2 s o f t w a r e a n d u s i n g a M a c i n t o s h c o m p u t e r .

4. R e su l t s

4 .1 . Cons t i tu t ive express ion o f ICA M -1 depends on

product ion o f a so lub le fac to r

P r e v i o u s l y w e h a v e s h o w n t h a t b l a d d e r c a n c e r c e ll

l in e s c o n s t i t u t i v e l y e x p r e s s e d v a r y i n g a m o u n t s o f th ea d h e s i o n m o l e c u l e I C A M - 1 ( r an g e : 4 - 8 0 % c e ll s p o s i -

t i v e ) [1 4] . O n e c e l l li n e ( S D ) , w h i c h e s s e n t i a l l y e x -

p r e s s e d m o d e r a t e a m o u n t s o f s u r fa c e I C A M - 1 , w a s

c h o s e n f o r f u r t h e r in v e s t i g a t i o n s . T h e I C A M - 1 e x -

1

t / ) 0

t , . 5 0 .

._>

U )o

<(..)

o

5 0o l

a.)oa )

D..

j . A

B

C

C e l l d e n s i ~ x l O 5 c e l l ~ m 2

Fig. I. Density-dependent expression of ICAM-1 on two bladdercancer cell lines. Cells were seeded at different densities, grown,recovered and stained with anti-lCAM-1 antibodies. The numberof cel ls was counted and ICA M-I expression was analysed using

flow cytometry. A: dependence of ICAM-1 expression on the celldensity for SD cell l ine. On the upper graph the correlat ion be-tween cell density and ICAM-I expression is shown. B: depen-dence of ICAM-I express ion on the ce l l dens i ty for SD af te rcorrection according to number of cells per well (see Methods). C:dependence o f ICAM-1 expression on the cell density for MG H-U I cel l l ine. Bars indicate the m ean o f tr ipl icate determin ationsand error bars indicate 1 SD from the mean. *SD <0.5% .

p r e s s i o n o n t h i s ce l l l in e w a s f o u n d t o b e c e l l - d e n s i t y

d e p e n d e n t a n d t o i n c r e a s e in p a r a l l e l w i t h i n c r e a s i n g

c e l l d e n s i t y a f t e r r e a c h i n g a c e l l d e n s i t y o f 0 .6 2 × 10 6

c e l l/ c m 2 ( s im p l e c o r r e l at i o n : r = 0 . 9 8 4 , P < 0 . 0 0 1 )

( F i g s . la a n d 2 ). H o w e v e r , w h e n t h e I C A M - 1 e x p r e s -

s i o n w a s c o r r e c t e d a c c o r d i n g t o th e n u m b e r o f c e ll s

p e r w e l l , i t a p p e a r e d t h a t t h e a b i l i t y o f c el ls t o r e g u -

l a t e I C A M - 1 e x p r e s s i o n i n a u t o c r i n e f a s h i o n w a s i n -

v e r s e l y p r o p o r t i o n a l t o c e ll d e n s i t y ( u n t il s t a b i l i s i n g

a t a c e l l d e n s i t y o f 0 . 6 2 x 1 06 c e ll / c m 2 ; l o g a r i t h m i cc o r r e l a ti o n : r : 0 . 8 8 5 , P < 0 . 0 1 ) ( F i g. lb ) . I n c o n -

t r a s t , f o r a c e l l l i n e t h a t e x p r e s s e d l o w l e v e ls o f s u r -

f a ce I C A M - 1 ( n a m e l y M G H - U 1 ) c h a n g e s in e x p r e s -

s i o n d u e t o c e l l d e n s i t y w e r e m i n o r o r a b s e n t ( F i g .

l c ) . S i m i l a r e f fe c t s w e r e a l s o n o t e d w i t h R T 4 a n d

R T 1 1 2 c e ll l in e s ( d a t a n o t p r e s e n t e d ) .

F u r t h e r s t u d ie s s h o w e d t h a t t h e in d u c t i o n o f

I C A M - I w a s d e p e n d e n t o n t h e re l ea s e o f a s o lu b l e

f a c t o r , s e c r e t e d b y a l l ce ll li n e s e x c e p t o n e ( M G H -

U 1 , F i g . 3 a ). S u c h s e c r e t i o n w a s n o t d e p e n d e n t o n

t h e c o n s t i t u t i v e e x p r e s s i o n o f I C A M - 1 ( d a t a n o t p r e -

s e n t e d ) . I n t e r e s t i n g l y , t h i s f a c t o r w a s a b l e t o i n d u c e

Z

A B

E

Fluorescence i n t e n s i t y l o g. cale

Fig. 2. Density dependent expression of ICAM-1 on SD cell line.Shown is the change in the profile of fluorescence intensity asso-ciated with data in Fig. IA. Profiles correspond to the followingdata : 0.96 (A), 1.6 (B), 2.1 (C), 2.9 (D) x l0 s cells/cm2, non-sp ecific

binding of FITC-conjugated antib odie s to the cells (E). Three dec-ades of logarithmic am plifications ar c presented.

119

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I C A M - l o n t h e c e ll l in e s t h a t p e r m a n e n t l y e x p r e s s e d

s u r f a c e I C A M - 1 , b u t i t f a il e d to h a v e a s i m i l a r e f f ec t

o n t h o s e w h i c h d i d n o t ( R T 4 a n d R T l l 2 ) ( F i gs . 3

a n d 4 ) . I C A M - 1 i n d u c t i o n b y th i s s o l u b l e f a c t o r a n d

i t s k i n e t i c s w a s s i m i l a r t o t h a t o b s e r v e d f o l l o w i n g

s t i m u l a t i o n w i t h 1 0 0 U / m l o f IF N ~ / ( F i g . 5 ), It

s h o u l d b e n o t e d t h a t t h e r e w a s a n i n c r e a s e i n b o t h

t h e n u m b e r o f p o s i t i v e c e ll s a n d t h e f l u o r e s c e n c e i n -

t e n s i t y .

4 . 2 . I L - lc~ exer t s the major ro le in the I CAM- 1

induc t ion by ce l l l ine supernatan ts

T o d e t e r m i n e t h e n a t u r e o f t h e s o l u b l e f a c t o r w e

p e r f o r m e d n e u t r a l i s i n g e x p e r i m e n t s o n S D c e l l l in e

s u p e r n a t a n t s u s i ng n e u t r a l i si n g p o l y c l o n a l a n t i b o d i e s

t o t h e d i f f e r e n t c y t o k i n e s w h i c h a r e k n o w n t o b e p o -

t e n t in d u c e rs o f I C A M - 1 . F o r t h e se e x p e r i m e n t s w e

Q )(0Q )

Q .

. r -

Q)

C ~

t -o)

Q~

13_

1 0 0 -

5 0 -

0

5 0 -

A

r - - - 1 [ - ~ I ' ~ - I , ND ( .- . -. - m i . . ~ , [ - ' - - ~ i ' - ' - 1 ND

, , , , i

> 2 0 0 -

( , 1

~ 100

,'r0 N N i i n i i i

2 3 4 5 6 7 8 9 10

T r e a t m e n t

Fig. 3. Supernatants of bladd er cancer cell lines induce IC AM -I onM GH -UI but not on RTI12 ce l l l ines. MGH -U1 (A, C) and

RT112 (B) were st imulated with medium alone (1), 100 U/mlIFN 7 (2), supernatants (diluted in medium 1:1) of following celll ines: MGH-UI (3), RT4 (4), UMUC3 (5), RTI12 (6), EJ18 (7),5637 (8), SD (9) and J82 (10). After 24 h incubation cells wererecovered, stained with anti-lCAM-1 antibodies and analysed byflow cytometry. Bars indicate the m ean o f triplicate determinationsand erro r bars indicate the level of 1 SD. *SD< 1%. ND, data n otdetermined.

120

u s e d M G H - U 1 c e ll s a s th e t e s t l in e b e c a u s e o f i t s

m a r k e d r e s p o n s e t o I C A M - 1 i n d u c t i o n by tu m o u r

c e ll s u p e r n a t a n t s .

T h e m o s t p o w e r f u l i n h i b i t o r o f t h e s o l u bl e f a c to r

a c t i v i t y w e r e f o u n d t o b e a n t i - IL - l c ~ a n t i b o d i e s ; h o w -

e v e r , th e i r e f fe c t w a s h e t e r o g e n e o u s . I n t w o e x p e r i -

m e n t s t h es e a n t i b o d i e s c o m p l e t e l y a b o l i s h e d t h e

I C A M - 1 i n d u c t i o n b y th e s o l u b l e f a c t o r s , w h i l e i n

t w o o t h e r s t h e y h a d a d r a m a t i c , y e t i n c o m p l e t e , s u p -

p r e s s i v e e f f e ct ( t he i n h i b i t i o n o f I C A M - I i n d u c t i o nr e a c h e d 4 4 .8 a n d 7 3 . 5 % ) . T h e a v e r a g e r e d u c t i o n o f

I C A M - 1 i n d u c t i o n i n 4 ex p e r i m e n t s w a s 8 0 .3 % ( F ig .

6 a ). I t i s n o t a b l e t h a t a n t i -I L - l c ~ a n t i b o d i e s a l s o

m a r k e d l y m o d u l a t e d t h e ef fe c t o f t h e s u p e r n a t a n t s

o f t w o o t h e r t e s t e d c el l l in e s : R T 4 a n d R T 1 1 2 ( r e-

s p e c t iv e l y , g r a d e s I a n d I I ). T h e s e a n t i b o d i e s d e -

c r e a s e d t h e a c ti v i t y o f th e s u p e r n a t a n t s d r a m a t i c a l l y

b u t n o t c o m p l e t e l y ; th e r e d u c t i o n o f th e I C A M - I i n-

d u c t i o n r e a c h e d 5 8 . 7 % a n d 6 3 . 5 % , r e s p e c t i v e ly , f o r

R T 4 a n d R T 1 1 2 ( d a t a n o t p re s e n te d ) .

1 0 0 - • C o n t r o l A

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m l

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p

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C e l l l i n e s

Fig. 4. Supernatant of SD cell line induces ICAM-1 on differentbladder cancer cell lines. Shown are data for following cell lines:RT4 (1, Grade 1), RTII2 (2, G2), MGH-U1 (3, G3), SD (4, G3),5637 (5, G3), EJI8 (6, G3). Cells were stimulated with the super-natant for 24 h and then analysed as described in Materials andMethods. Bars indicate the mean of triplicate determinations anderror ba rs indicate the level of 1 SD.

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F i g . 5 . D o s e - d e p e n d e n t e f f e c t o f S D c e ll l in e s u p e r n a t a n t o n

I C A M-I i n d u ct i o n o n th e M G H -U I ce l l l i n e. C e l l s w ere s ti m u -

l a t ed w i th m ed i u m a l o n e (1 ) , I F N ? ( 1 , 1 0 , 1 0 0 U / m l , respect ively,

o f I F N 7 (2 -4 ) ) o r S D s u p ern a ta n t ( re s p ec ti v e l y d i lu ted i n m ed i u m

1 : 8 , 1 : 4 , 1 : 2 , 1 :1 ( 5 8 ) ) f o r 24 h , reco v ered a n d I C A M-1 ex p res s i o n

w a s a n a l ys ed u s i n g f l o w cy to m etry . Ba rs i n d i ca te th e m ea n o f t r i -

p l icate determinations and error bars ind icate the l evel of 1 S D .* S D < 0 . 5 % .

Interest ingly, s imu ltaneou s neutra l i sat ion w ithant i bodi es aga i ns t a l l po tent cytok i ne- i nducers o f

ICAM -1 t es t ed ( namel y IFN 7 , TNF c( , TN Ff i , IL-l c( ,

IL-1/ / ) were st i l l not able to completely prevent the

i nduct i on o f ICAM -1 by the SD ce l l l ine supernatant

( F i g . 6b) . Thus ant i - IFNT, TNF~ , TNFf l ant i bodi es

fai led to inf luence the ICAM-1 inducing act iv i ty of

supernatant w hi le ant ibodie s to IL-1]? had a smal l

b u t s i g n i f i c a n t e f f e c t ( 7 . 7 % , P < 0 . 0 5 ) . H o w e v e r , t h e

com bin at ion of a l l ant ibod ies had a synergist ic sup-

press ive e f f ect ( to ta l reduct i on o f ICAM -1 i nduct i on

by the ant ibodies to a l l cytokines reached 68.6 vs.

4 4 . 8 % b y a n t i - I L - l ~ a n t i b o d i e s a l o n e , P < 0 . 0 5 ) .N everthe l ess , the resi dual i nduc t i on o f ICAM -1 a f t er

neutral i sat ion by al l tested ant ibodies reached 31.4%

( P <0 .05 ) . S imi lar resu l ts were obta i ned for the

supernatants of tw o other cel l l ines (J82, 5637, data

n o t s h o w n ) .

To prov i de further i n format i on on the na ture o f

the residual ind ucing act iv ity w e invest igated the abi l-

i ty o f d i f f erent recombi nant cytok i nes to i nduce

ICAM-1 on TCC cel l l ines. Several cytokines includ-

i n g I L - 2 , I L - 3 , I L - 7 , I L - 8 , I F N ~ , T G F / ~ , G M - C S F ,

E G F ( i n d o s e s o f 1 - 1 0 0 0 U / m l f o r 2 4 h ) f a i l e d t o

i nduce ICAM -1 on the M G H -U 1 ce l l l ine . Interes t-

i ngl y, o ther know n regul ators o f ICAM-1 express i on

- - I L - 4 ( d o s e 0 - 1 0 0 0 U / m l , 2 4 h s t i m u l a t i o n ) a n d

ret i no ic ac i d ( 0 .1 -100 g /ml , 24-96 h s t i mul a t ion) - -

w e r e a l s o u n a b l e t o i n d u c e I C A M - 1 o n b o t h R T 4a n d M G H - U 1 c e l l l in e s (d a ta n o t s h o w n ) .

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F i g . 6 . N e u tra l i s a t i o n o f cy to k i n es i n t is s u e cu ltu re s u p ern a ta n t s .

M G H -U I TC C ce l l l i n e w a s s ti m u l a ted w i th S D ce l l l i n e s u p ern a -

ta n t w h i ch h a d b een p re - i n cu b a ted w i th n eu tra l i s i n g a n t i b o d i e s tod i f f eren t cy to k i n es ( s ee Ma ter i a l s a n d M eth o d s ) . A : e f fec t o f a n t i -

I L -l c~ a n t i b o d ie s o n th e i n h i b it i o n o f I C A M-1 i n d u ct i o n b y fo u r S D

s u p ern a ta n t s ( 1 -4 ) a n d th e m ea n i n h i b i t i o n o f f o u r ex p er i m en t s ( 5 ) .

B : e f f ec t o f n eu tra l i s i n g a n t i b o d i e s to d i f f eren t cy to k i n es o n th e

i n h i b it i o n o f 1 C A M -I i n d u ct i o n b y S D s u p ern a ta n t . A n t i b o d i e s to

f o l l o w i n g c y t o k i n e s w e r e u se d : I L - I ~ ( ( 1 ) , I L -1 /~ (2 ) , T N F c ( ( 3 ) ,

TN F / ~ (4 ) , 1 F N 7 ( 5 ) , th e m i x tu re o f a ll n a m ed cy to k i n es (6 ) .

121

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F i g. 7 . C o m p a r i s o n o f th e I C A M - 1 i n d u c i n g p r o p e r t ie s o f T C C

c u l t u r e s u p e r n a t a n t s o n d i f f e r e n t t u m o u r c e l l li n e s. F o u r c e ll li n e s

w e r e i n c u b a t e d w i t h m e d i u m a l o n e ( F q ) , I F N 2 ( D ) , T N F ~ ( g ] ) , I L -

1 ~ ( 1 1 ) o r a s u p e r n a t a n t o f S D c e l l l in e ( [ 3 ) f o r 2 4 h , r e c o v e r e d a n d

s t a in e d w i th a n t i - l C A M - 1 a n t i b o d ie s . T h e i n d u c t i o n o f I C A M - 1

e x p r e s s i o n w a s s t u d i e d u s i n g f l o w c y t o m e t r y , C y t o k i n e s w e r e a p -

p l i ed a t 1 00 U / m l a n d t h e s u p e r n a t a n t a p p l i e d t o c e l l s i n a d i lu t i o n

o f I : l i n c o m p l e t e m e d i u m . B a r s i n d i c a t e t h e m e a n o f t r i p li c a t e

d e t e r m i n a t i o n s a n d e r r o r b a r s i n d i c a t e t h e l e v e l o f 1 S D .

* S D < 0 . 5 % . N o t e s u p e r n a t a n t s o n l y i n d u c e I C A M - 1 i n h i g h -

g r a d e t u m o u r c e ll l in e s .

4.3. Comparison of ICA M -1 induct ion by cel l l ine

supernatants and recombinant cy tokines

P r e v i o u s l y w e d e m o n s t r a t e d a d i f f er e n t r e s p o n s e

o f h i g h - a n d l o w - g r a d e c e l l l i n e s t o I C A M - 1 i n d u c -

t i o n b y B C G ( J a c k s o n , A . M . , s u b m i t t e d ) . T o f u r t h e r

i n v e s t i g a t e t h e i n t e r a c t i o n b e t w e e n t u m o u r c e l l s a n d

s o l u b l e f a c t o r s p r e s e n t i n t h e s u p e r n a t a n t s w e c o m -

p a r e d t h e e ff e c t o f r e c o m b i n a n t c y t o k i n e s a n d S D

c e ll l in e s u p e r n a t a n t o n I C A M - 1 i n d u c t i o n .

F o u r T C C c e l l l i n e s w e r e s t i m u l a t e d w i t h I L - l c ~ ,

T N F c ~ , I F N } , o r t h e s u p e r n a t a n t . A l l r e c o m b i n a n t

c y t o k i n e s s t r o n g l y i n d u c e d I C A M - I o n b o t h h i g h -

1 0 0

8 0

8

6o

5: o 4o

2o#.

R T 4 R T 1 1 2 M G H - U 1

F i g . 8 . E f fe c t o f I L - I ~ s t i m u l a t i o n o n t h e e x p r e s s i o n o f I C A M - I b y

t h r e e T C C c e l l l i n e s ( R T 4 , R T 1 1 2 , M G H - U I ) . C e l l s w e r e e x p o s e d

t o 0 ( . ) , 1 ( Tq ), 1 0 ( g ] ) , a n d 1 00 ( D ) U / m l o f I L - I ~ f o r 2 4 h . B a r s

i n d i c a t e t h e m e a n o f t r i p li c a t e d e t e r m i n a t i o n s a n d e r r o r b a r s i n d i -

c a t e t h e l ev e l o f 1 S D . E r r o r b a r s r e p r e s e n t 1 S D .

1 7 5

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C e l l l i n e s

F i g . 9 . P M A i n d u c e s I C A M - 1 e x p r e s s i o n o n t h e g r a d e 3 b l a d d e r

c a n c e r c e l l li n e s. C e l l w e r e s t i m u l a t e d w i t h P M A ( 1 0 n g / m l ) f o r 2 4

h a n d t h e n a n a l y s e d a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . S h o w n

a r e d a t a f o r f o l l o w i n g ce ll l in e s : M G H - U I ( 1 ), S D ( 2 ), 5 6 3 7 ( 3) a n d

E J I 8 ( 4 ). B a r s i n d i c a te t h e m e a n o f t r ip l i c a te d e t e r m i n a t i o n s a n d

e r r o r b a r s i n d i c a t e t h e l e ve l o f 1 S D ,

g r a d e ( M G H - U 1 , E J 1 8 ) a n d l o w - g r a d e ( R T 4 a n d

R T l l 2 ) c e l l l i n e s ( F i g . 7 ) . A b o v e w e h a v e d e m o n -s t r a t e d t h a t S D c e l l l i n e s u p e r n a t a n t c o n t a i n s I L - I ~ .

N e v e r t h e l e s s , t h i s s u p e r n a t a n t f a i l e d t o i n d u c e

I C A M - 1 o n t h e l o w g r a d e T C C c e l l l i n e s a t a l l . I t

s h o u l d b e n o t e d t h a t t h e h i g h - g r a d e c e l l s w e r e m o r e

r e s p o n s i v e t o r I L - l c ~ t h a n t h e l o w - g r a d e c e l l l i n e s

(Fig. 8) .

I n a d d i t i o n , h i g h - g r a d e t u m o u r c e l l s r e s p o n d e d

w e l l t o p h o r b o l m y r i s t a t e a c e t a t e ( P M A ) i n d u c t i o n

o f 1 C A M - 1 e x p r e s s i o n ( F i g . 9 ) . H o w e v e r , l o w - g r a d e

t u m o u r c e l l s f a i l e d t o d o s o ( d a t a n o t s h o w n ) .

4 . D i s c u s s i o n

I n t h i s s t u d y , w e i n v e s t i g a t e d t h e c a u s e o f c o n s t i -

t u t i v e I C A M - I e x p r e s s i o n o n h i g h - g r a d e b l a d d e r

c a n c e r c e l l s , a p h e n o m e n o n w h i c h c o u l d p l a y a n i m -

p o r t a n t r o le in i m m u n o t h e r a p y o f b l a d d e r a n d o t h e r

122

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t y p e s o f m a l i g n a n c y . W e d e m o n s t r a t e d t h e f ol l o w i n g

p o i n t s. ( 1 ) T h e c o n s t i t u t iv e e x p r e s s i o n o f I C A M - 1 o n

b l a d d e r c a n c e r c e l l l i n e s i s r e g u l a t e d i n a n a u t o c r i n e

f a s h i o n . (2 ) T h e m a j o r i t y o f I C A M - l - i n d u c i n g a b i l it y

o f h u m a n c e ll c u l t u r e s u p e r n a t a n t s b e l o n g e d t o I L -

1 ~. ( 3) T h e r e s i d u a l I C A M - l - i n d u c i n g a c t i v i ty ( u p t o

3 0 % o f I C A M - 1 i n d u c t i o n ) c o u l d n o t b e a s s o c i a te d

w i t h a n y k n o w n I C A M - 1 i n d u c e r s ( i n c l u d i n g I F N T ,

T N F c ~ , T N F / ~ , I L - I ~ , I L - 1 / 3 , I L - 4 a n d r e t i n o i c a c i d ) .

( 4 ) A l t h o u g h l o w - g r a d e T C C c e l l l i n e s ( R T 4 , R T I 1 2 ,g r a d e s I a n d I I ) r e s p o n d e d t o r l L - l ~ a n d t h e r e f o r e

e x p r e s s e d i t s r e c e p t o r s , t h e y f a i l e d t o r e s p o n d t o I L -

1 ~ c o n t a i n e d i n t h e t i s s u e c u l t u r e s u p e r n a t a n t s .

S e v e r a l p o i n t s n e e d t o b e d i s c u s s e d i n m o r e d e t a i l .

P r e v i o u s l y , w e h a v e s h o w n t h a t i n v i v o b l a d d e r c a n -

c e r c e l l s e x p r e s s I C A M - 1 o n l y a f t e r i n t r a v e s i c a l B C G

i n s t il l a ti o n s b u t n o t b e f o r e ( in 2 2 e x a m i n e d t u m o u r s )

a n d t h a t i n v i t r o o n l y h i g h - g r a d e T C C c e l l l i n e s e x -

p r e s s e d I C A M - I c o n s t i t u t i v e l y [1 4 ,1 7] . H o w e v e r , in

o u r p r e s e n t s t u d y a l m o s t a l l T C C c e ll l i n es ( 7 o f 8 )

p r o d u c e d a s o l u b le f a c t o r c a p a b l e o f in d u c i n g

1 C A M - 1 i n d e p e n d e n t l y o f t h e i r h i s t o p a t h o l o g i c a lg r a d e a n d l e v e l o f c o n s t a n t I C A M - 1 e x p r e s s i o n . I t i s

p o s s i b l e t h a t t h i s f i n d i n g i s s i m p l y a n i n v i t r o a r t e f a c t

a n d d o e s n o t i n f l u e n c e th e b i o l o g y o f t u m o u r . N e v e r -

t h e le s s, s u c h a n o b l i g a t o r y p r o d u c t i o n o f t h e s o lu b l e

f a c t o r s b y m o s t T C C c e l l l i n e s l e a d s u s t o s p e c u l a t e

w h e t h e r t h e i r p r o d u c t i o n p l a y s a n e s s e n t i a l a u t o c r i n e

r o l e f o r T C C c e l l s i n v i t r o o r i n v i v o . T h i s p o s s i b i l i t y

s h o u l d b e i n v e s t i g a t e d f u r t h e r .

I t i s c l ea r tha t a t l eas t ha th ree T CC ce l l l i ne s the

m a j o r i t y o f t h e e f f e c t o f t h e s e s o l u b l e f a c t o r s b e l o n g s

t o t h e I L - I ~ . T h i s c y t o k i n e h a s b e e n s h o w n t o p l a y a

c r i t i c a l r o l e i n m a n y i m m u n e r e a c t i o n s i n c l u d i n g r e -

c r u i t m e n t o f i m m u n o c o m p e t e n t c el ls a n d e n h a n c i n g

t h e i r i n t e r a c t i o n s w i t h t u m o u r c e l l s [ 2 0 , 2 1 ] . T h e r e -

f o r e , p r o d u c t i o n o f I L - l~ t b y t u m o u r c e ll s m a y a f f ec t

a n t i - t u m o u r i m m u n e r e s p o n s e s .

T h e d a t a p r e s e n t e d s h o w t h a t a l t h o u g h l o w - g r a d e

c e ll li n es p r o d u c e d I L - I ~ a n d r e s p o n d e d t o r e c o m b i -

n a n t I L - I ~ ( i n d i c a t i n g t h e p r e s e n c e o f r e c e p t o r ) th e y

s u r p r i s i n g l y f a i l e d t o e x p r e s s I C A M - 1 c o n s t i t u t i v e l y

a n d , m o r e o v e r , t o r e s p o n d t o t h e I L - I ~ c o n t a i n e d i n

t h e c e l l c u l t u r e s u p e r n a t a n t . T h e r e a s o n f o r t h i s r e -

m a i n s u n c l e a r . I t i s p o s s i b l e t h a t g r a d e I c e l l l i n e s

a r e l e s s s e n s i t iv e t o I L - I ~ , w h i c h c o u l d b e a c c o u n t e d

f o r b y l o w e r a m o u n t s o f t h e c y t o k i n e r e c e p t o r s o r t h es e c r e t i o n o f I L - 1 f u n c t i o n a l i n h i b i t o r s [ 22 ]. H o w e v e r ,

i t i s i n t r i g u i n g t h a t a s w e p r e v i o u s l y h a v e s h o w n l o w -

g r a d e T C C c e l l l i n e s ( i n c o n t r a s t t o h i g h - g r a d e l i n e s )

a l s o fa i le d t o r e s p o n d t o P M A a n d t o a n o v e l i n d u c e r

o f I C A M - I , n a m e l y B C G o r g a n is m s ( se e R e s u lt s a n d

J a c k s o n , A . M . , u n p u b l i s h e d ) . W h e t h e r a s i m i la r m e -

c h a n i s m , i n d e p e n d e n t o f IL - 1 r e c e p t o r e x p r e ss i o n , is

i n v o l v e d i n b o t h p r o c e s s e s n e e d s t o b e i n v e s t i g a t e d

f u r t h e r .

F i n a l l y, th e o b s e r v a t i o n t h a t t h e I C A M - 1 i n d u c i n g

a c t i v i ty o f t is s ue c u l t u r e s u p e r n a t a n t s c o u l d n o t b e

c o m p l e t e l y i n h i b i t ed w i t h c o c k t a i l o f a n t i b o d i e s t o

v a r i o u s c y t o k i n e s i s o f in t e r e s t . I t s h o u l d , h o w e v e r ,

b e n o t e d t h a t t w o o t h e r m o d u l a t o r s o f I C A M - 1 e x -

p r e s s i o n ( I L - 4 a n d r e t i n o i c a c i d ) f a i l e d t o i n d u c e

I C A M - 1 o n T C C c e l l l i n e s [ 9 , 1 2 ] . F i n a l l y , L P Sk n o w n t o i n d u ce I C A M - 1 c a n n o t b e p r o d u c e d b y

m a m m a l i a n c e l l s a n d w a s n o t p r e s e n t i n t h e t i s s u e

c u l t u r e m e d i a a n d t h e r e f o r e c a n b e a l so e x c l u d e d

[ 1 3 , 2 3 ] . W e a l s o r e c e n t l y p e r f o r m e d p r e l i m i n a r y s t u -

d i e s o n t h e e f f e c t o f o t h e r c y t o k i n e s ( I L - 2 , I L - 3 , I L - 7 ,

I L -8 , IF N ~ , T G F / ~, G M - C S F , E G F ) o n I C A M - 1 e x -

p r e s s io n b y t h e se c el ls . N o n e o f t h es e i n d u c e d I C A M -

1 e x p r e s s i o n o n T C C c e ll s.

T h e n a t u r e o f I L - I ~ i n d u c i n g f a c t o r ( s ) r e s p o n s ib l e

f o r I C A M - 1 e x p r e s s i o n o n b l a d d e r t u m o u r c e l l s r e -

m a i n s t o b e d e t e r m i n e d ; h o w e v e r , a n o v e l I C A M - I

i n d u c i n g f a c t o r ( n e u d i f f e r e n t i a t i o n f a c t o r ( N D F ,h e r e g u l i n ) ) h a s r e c e n t l y b e e n d e s c r i b e d b y B a c u s e t

a l . [ 2 4 ] . T h i s f a c t o r i n d u c e s p h e n o t y p i c c h a n g e s i n

m a m m a r y c a n c e r c e l l s a n d a l s o a c t s a s a g r o w t h f a c -

t o r f o r t u m o u r c e l l s . W h e t h e r N D F i s s e c r e t e d b y

b l a d d e r t u m o u r c e l l s r e m a i n s t o b e d e t e r m i n e d .

Acknowledgements

T h e a u t h o r s a r e p a r t i c u l a r l y g r a t e f u l t o D r . A .

M e a g e r ( P o t t e r s B a r , U K ) f o r t h e g e n e r o u s g i f t o f

n e u t r a l i s i n g p o l y c l o n a l a n t i b o d i e s t o I F N T , T N F ~

a n d T N F / ~ , t o D r . T . A . S p r i n g e r ( H a r v a r d M e d i c a l

S c h o o l , B o s t o n , M A ) f o r t h e d o n a t i o n o f t h e m u r i n e

m o n o c l o n a l a n t ib o d i e s to h u m a n I C A M - I , t o B e h -

r i n g w e r k e ( M a r b u r g / L a h n , G e r m a n y ) f o r t h e g e n e r -

o u s d o n a t i o n o f t h e r e c o m b i n a n t T N F ~ , I L - 3 , I L - 4 ,

I L - 7 , G M - C S F a n d t o D r . P . P e r l m a n n a n d D r . J .

M a s t e r s f o r t h e k i n d g i f t o f t h e c e ll li n e s. T h e

a u t h o r s a l s o t h a n k M i s s S . E l l i o t t a n d M r . A . C u n -

n i n g h a m f o r t h e i r c ri t ic a l r e a d i n g o f m a n u s c r i p t .

T h i s w o r k w a s f u n d e d b y t h e C a n c e r R e s e a r c h

C a m p a i g n o f G r e a t B r i ta i n , b y th e S i n g a p o r e C a n -

c e r S o c i e ty , a n d t h e R o y a l C o l l e g e o f S u r g e o n s o f

E d i n g b u r g h .

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