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Suppression of natural cytotoxic cell activity by social aggressiveness in Tilapia
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 12, pp. 595-602, 1988 0145-305X/88 $3.00 + .OO Printed in the USA Copyright (c) 1988 Pergamon Press plc All rights reserved
SUPPRESSION OF NATURAL CYTOTOXIC CELL ACTIVITY SOCIAL AGGRESSIVENESS IN TILAPIA
M.
Ghoneum"*,
M. Faisa11'3, and E.L.
G. Peters4, Cooper
1.1.
BY
Ahmedls3,
of Comparative Immunology, Department 1. Laboratory Los Angeles, University of California, of Anatomy, Drew Otolaryngology, Department of CA 90024; 2. University of Medicine and Science, Los Angeles, CA Medicine, Veterinary Faculty of 90059; 3. fiir Institut University of Alexandria, Egypt; 4. Hydrobiologie und Fischereiwissenschaft, Universitat Hamburg, FRG.
ABSTRACT Our earlier observations revealed that social stress causes drastic effects on different physiological mechanisms and degenerative changes in leucocytes. In this preliminary work, we analyzed the effect of social aggressiveness on functional activites of leucocytes emphasizing (NCC) activity in Tilapia. At 10 hr post stress induction, fish could be differentiated into three categories: 1) dominants; 2) subordinates; and 3) determinants. Results of NCC as indicated by 4 hr. f? Cr-release assay, demonstrated a significant suppression in cytotoxic reactivity in the subordinates and indeterminants compared to dominants. This suppression appears to be due to a decrease in the binding capacity of effector cells to YAC-1 target cells as indicated by the decreased number of conjugate forming cells. This binding process is a key event in activating the fish equivalent of NK cells in mammals. Decreased NCC-activity in stressed fish suggests that aberrations in cell mediated immunity result from social aggressiveness.
INTRODUCTION Social stress causes drastic effects on different physiological mechanisms in fish (1,2). As a result, battle for rank increases the susceptibility of subordinate rainbow trout (Salmo gairdneri) to infection with the bacterium Aeromonas hydrophila (3) and induces degenerative changes in the leucocytes of Tilapia In fish a variety of leucocyte types are cytotoxic against (4). 595
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established mammalian and p i s c l n e cell lines (5-7). C a r l s o n et al. (8) w h o s t u d i e d the m e t a b o l i c requirements for l y s i s of target cells by n o n s p e c i f i c c y t o t o x l c cells f r o m c h a n n e l c a t f i s h i n d i c a t e d a s i m i l a r i t y to those of m a m m a l i a n n a t u r a l k i l l e r cells (NK). Many investigators consider mammalian NK cells to be the first llne of n a t u r a l immune defense against neoplasms and pathogens (9-12), in a d d i t i o n to t h e i r regulatory functions (13,14). Moreover, different stressors can suppress or e v e n inhibit NK-actlvity (15,16). We t h e r e f o r e wanted to d e t e r m i n e the e f f e c t of s o c i a l a g g r e s s i v e n e s s on N C C - a c t i v i t y in T i l a p i a . B e s i d e the i m p o r t a n c e of this s t u d y for a q u a c u l t u r e , it may aid in b e t t e r u n d e r s t a n d i n g h o w the p a t h o g e n e s i s of stress e v o l v e d . MATERIALS
AND
METHODS
Fish Hybrids
of
Tilapia
mossambica
x
Tilapia
honorum
of
both
sexes (237 g + 17; 23 cm + 5; one year old) were o b t a i n e d from the P a c i f i c Aquafarms, Niland, California, USA. A t o t a l of 25 f i s h w e r e m a i n t a i n e d for 4 w e e k s prior to the e x p e r i m e n t in 150 1 glass a q u a r i a at a t e m p e r a t u r e of 18+i°C. The diet c o n s i s t e d of p e l l e t e d f i s h feed ( P u r i n a C a t f i s h S t a r t e n a , R a l s t o n P u r i n a Co.). I n d u c t i o n of s o c i a l s t r e s s Social stresswas i n d u c e d a c c o r d i n g to the m e t h o d p r e v i o u s l y d e s c r i b e d (3). In brief, fish w e r e kept s e p a r a t e d for I week, by a partition ( p e r f o r a t e d d a r k p l a s t i c ) d i v i d i n g the tank in half. Thereafter, the p a r t i t i o n was r e m o v e d , thus b r i n g i n g the two fish t o g e t h e r for I0 hrs. D u r i n g their e n c o u n t e r , their b e h a v i o r was o b s e r v e d to d e t e r m i n e the r a n k o r d e r . Media All cell c u l t u r e s were performed in c o m p l e t e m e d i a (CM), that c o n s i s t e d of R P M I - 1 6 4 0 (Flow L a b o r a t o r i e s , R o c k v i l l e , MD.), 10% - heat i n a c t i v a t e d f e t a l calf s e r u m (FCS) and 1% a n t i b i o t i c s . [(50 u n i t s penicillin and 50 ug s t r e p t o m y c i n per ml.) (Grand Island Biological, Santa Clara, CA)]. Hank's balanced salt solution (HBSS) was used to wash fish leucocytes before e s t a b l i s h i n g c u l t u r e s w i t h tumor t a r g e t s . Cell p r e p a r a t i o n Fish were captured, anestheslzed in 200 mg/l M S - 2 2 2 (Sandoz, Swltzerland) and the head kidney (pronephros) removed. Head kidney cell suspensions were prepared by g e n t l y pressing the tissues through 80-gauge mesh stainless steel sieves. C e l l s were c o l l e c t e d and w a s h e d w i t h H B S S and r e s u s p e n d e d in CM. T a r g e t cells YAC-I, a v i r u s i n d u c e d l y m p h o m a of A/Sn m o u s e o r i g i n was ~Intalned in a s u s p e n s i o n c u l t u r e at 37°C in CM. -Cr R e l e a s e A s s a y Y A C - I cells (5xi06) were l a b e l l e d w i t h 100 uCi of 5 1 C r - N a ^ Cr0~ (New England Nuclear, Boston, MA) f o r o n e 5 h o u r at 2 5 ° C ~ Ce~s w e r e w a s h e d 3 times in CM d i l u t e d to 1 X I0 c e l l s / m l , and 101 cells w e r e d e l i v e r e d to each w e l l of a 96 well r o u n d b o t t o m tissue culture plates (Linbro Plastics). Fish effector cells were then a d d e d in I00 ul v o l u m e s at I00, 50 and 25:1 e f f e c t o r to target cell r a t i o s . Following a 4hrs. i n c u b a t i o n at 22°C, the L i n b r o p l a t e s were c e n t r i f u g e d (1600 rpm) for 5 m i n u t e s , and 0.I ml of s u p e r n a t a n t f r o m e a c h w e l l was c o l l e c t e d and c o u n t e d in a
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597
60
50 I
I
40
cl
a n=9
@j
I
n=IO
m
p
n=6
1
s F
30-
t ii5 z
20-
s 10-
L_
liLlL 25:1
Effector: Target Cell Ratio Figure (Linear
1. Non-specific cytotoxicity bars represent mean + SD) .
in
the
three
fish
%
50
I
40.
n=S
30, 20
n=O
I
T
10
Percentage
of conjugate
forming
cells.
groups
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FIGHTING BLOCKS CYTOTOXICITY
gamma counter. follows:
Percent
%speciflc
=
(test release
specific
release
Vol.
was
release)-(spontaneous
12, No. 3
calculated
as
release) x
(total
release)-(spontaneous
I00
release)
Spontaneous release (sp) f r o m the t a r g e t cells was always 8 - 1 0 % of the t o t a l r e l e a s e . T o t a l r e l e a s e was m e a s u r e d by a d d i n g 0.I ml T r i t o n X I 0 0 to d e s i g n a t e d wells. Conjugate formation Binding between effector cells and target tumor cells represents an i n i t i a l s t a g e in t a r g e t c e l l l y s i s . The c a p a c i t y of e f f e c t o r cells from the three different fish groups was examined as d e s c r i b e d by G h o n e ~ m et a l . ( l l ) . Effector cells (Ixl0") were incubated with I x l 0 ~ Y A C - I t a r g e t c e l l s in i ml of CM in 12 x 75 mm g l a s s t u b e s , s e d l m e n t e d by c e n t r i f u g a t l o n for 5 m i n and i n c u b a t e d for I hr at 4°C. Pellets were resuspended and cytocentrlfuged smears were stained with Giemsa. P e r c e n t of c o n jugate forming cells (CFCs) was determined by counting 200 leucocytes ( b o u n d and f r e e ) in t r i p l i c a t e samples. Statistical analysis For the statistical evaluation, the mean (X), standard deviation (SD), and coefficient of variation (V=SD/x) were determined, and the d a t a a n a l y z e d u s i n g the M a n n W h i t n e y U - t e s t . RESULTS Rank
order As a r e s u l t of b a t t l e for r a n k , t h r e e b e h a v i o r a l l y different g r o u p s of f i s h w e r e o b s e r v e d : dominants ( a l p h a ) that w e r e a g g r e s sive against their partners, subordinates (beta) that showed passive behavior and t r i e d to e s c a p e f r o m t h e i r a l p h a c o h o r t s and indeterminates (I) w h o s e status c o u l d n o t be d i s c e r n e d clearly. We classified nine fish as alpha, six as beta and ten as indeterminate or i n d i f f e r e n t . Non specific cytotoxiclty (NCC) NCC a c t i v i t i e s have been examined at ~ f f e r e n t effector: t a r g e t (E:T) r a t i o s u s i n g the s t a n d a r d 4 hrs. Cr-release assay D a t a in Fig. 1 reveal a considerable difference in NCC a c t i v i t y between the 3 fish groups. Alphas showed a high cytotoxic reactivity at all E:T ratios and the mean percentages of cytotoxlcity w e r e 40%, 42% and 44% at 25:1, 50:1 and I00:I r a t i o s respectively. In contrast, betas had very low levels of activities; 3% at 25:1, 4% at 50:1 and 9% at I 0 0 : I . The c e l l s from indeterminate fish showed cytotoxlc activities in b e t w e e n a l p h a s a n d b e t a s and b o t h p a r t n e r s showed equivalent values; 10%, 17% and 24% at 25:1, 50:1 a n d I00:I r e s p e c t i v e l y . Statistical analysis indicated a significant decrease of N C C a c t i v i t y between a l p h a s and b o t h b e t a s (p<0.001) and indeterminate fish (p<0.01). Conjugate formation Cytospin preparations showed three different effector cell types in c l o s e contact with the target tumor cells. These include lymphoid cells, macrophages and granulocytes ( P M N and pro-PMN). Data in F i g . 2 show that the percentage of c e l l s forming conjugates averaged 32% in a l p h a s , 20% in b e t a s w h i l e the percentage in i n d e t e r m i n a t e s represented a value between alphas
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and betas (26%). Similarly, statistical analysis showed significant differences b e t w e e n a l p h a s and b o t h betas (P< 0.001) and indetermlnates (P~0.01). In a l p h a fish, the c o n j u g a t e f o r m i n g cells (CFCs) w e r e m o s t l y m a c r o p h a g e s f o l l o w e d by g r a n u l o cytes and to a l e s s e r e x t e n t l y m p h o c y t e s . In b o t h beta and indeterminate fish, the CFCs w e r e m o s t l y g r a n u l o c y t e s f o l l o w e d by macrophages but rarely lymphocytes. CFCs of s t r e s s e d fish a p p e a r e d h y p e r t r o p h i c and d i s t o r t e d . DISCUSSION Stressed fish are more vulnerable to diseases (3,17), however, the e x a c t m e c h a n i s m s of this r e l a t i o n s h i p are not yet fully understood. In p r e v i o u s reports (4,18), degenerative c h a n g e s in the l e u c o c y t e s of s o c i a l l y s t r e s s e d r a i n b o w trout and Tilapia following relatively short stress periods were found. These alterations were a c c o m p a n i e d by l y m p h o p e n i a and g r a n u l o cytosis. Hence in t h e p r e s e n t study we w i s h e d to e x a m i n e , whether or not these morphological alterations are also associated with impaired functions of certain immunocompetent cells. As n o n s p e c l f i c cytotoxic cells of fish are c o n s i d e r e d functionally analogous to m a m m a l i a n natural killer cells (5), their activity may r e p r e s e n t the first llne of d e f e n s e against p a t h o g e n s and n e o p l a s m s in fish (I0). Our r e s u l t s d e m o n s t r a t e d a significant decrease in b o t h c y t o t o x l c i t y a n d the n u m b e r of conjugate f o r m i n g cells in s u b o r d i n a t e and i n d e t e r m i n a t e fish as c o m p a r e d to d o m i n a n t fish. Recognition and lysis of target cells by e f f e c t o r c y t o t o x i c cells i n v o l v e a s e q u e n c e of e v e n t s that has been a n a l y z e d r a t h e r extensively in m a m m a l s (19). First, the e f f e c t o r cell must recognize and bind to a t a r g e t cell, w h i c h is then f o l l o w e d by activation of e f f e c t o r cells and the i n i t i a t i o n of their p r o g r a m for t a r g e t cell l y s l s . Later, r e l e a s e of c y t o t o x i c f a c t o r s and lysis of the t a r g e t cells occur. The e f f e c t o r cells can then recycle a n d b i n d to new target cells. The d e c r e a s e d NCC activity of b e t a and i n d e t e r m i n a t e fish a p p e a r s to be due to a decrease in the b i n d i n g capacity of e f f e c t o r cells to Y A C - I target cells as i n d i c a t e d by the d e c r e a s e d number of c o n j u g a t e forming cells. This binding process is a k e y e v e n t in the a c t i v a t i o n of these cells (20). Although there appears to be no p r e v i o u s studies on the e f f e c t of s t r e s s on the N C C - a c t i v i t y of fish, there are s i m i l a r studies using other vertebrates. In this r e s p e c t , A a r s t a d et al. (21); M a t h e w s et al (22) and O e h l e r and H e r b e r m a n (23) r e p o r t e d that increased corticosteroid levels, associated with stress, directly suppresses the N K - a c t i v l t y . Other mechanisms that may be i n v o l v e d in the s u p p r e s s i o n of N K - a c t i v l t y by s t r e s s are due to o p l o l d s and m o r p h i n e . Shavit et al. (16) r e p o r t e d that o p i o l d peptides and morphine released under stress might suppress NK-activity d i r e c t l y or i n d i r e c t l y by m o d u l a t i n g the r e l e a s e of cortlcosterolds. On the o t h e r hand, G h o n e u m et al. (24) s u g g e s t e d a p o s s i b l e redistribution of N K - a c t l v l t y b e t w e e n the d i f f e r e n t body c o m p a r t ments under stress. They demonstrated a remarkable suppression in s p l e n i c and p e r i p h e r a l blood N K - c y t o t o x l c l t y which coincides w i t h an e l e v a t e d bone m a r r o w N K - a c t l v l t y in s t r e s s e d rats. In this r e s p e c t , w h e t h e r the d e c r e a s e of s t r e s s e d T i l a p i a p r o n e p h r o s
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NCC is a part of an o v e r a l l s u p p r e s s i o n of the i m m u n e system, or there is a p o s s i b l e r e d i s t r i b u t i o n of NCC b e t w e e n the d i f f e r e n t hemopoietic t i s s u e s is q u e s t i o n a b l e . However, our p r e l i m i n a r y studies (data not shown) show a consistent decrease in NCC a c t i v i t y of p e r i p h e r a l blood, spleen, trunk k i d n e y in a d d i t i o n to pronephros of s u b o r d i n a t e s . Further studies are needed to c l a r i f y this point. An i n t e r e s t i n g r e s u l t was the c h a n g e of c o n j u g a t e f o r m i n g cell types in s t r e s s e d fish. We o b s e r v e d an i n c r e a s e in g r a n u l o c y t e s and a d e c r e a s e in l y m p h o c y t e s and m a c r o p h a g e s . This is in a c c o r d a n c e with the m a r k e d g r a n u l o c y t o s i s and l y m p h o p e n i a observed in socially stressed Tilapia (4) and trout (18). Furthermore, the r e m a i n i n g l e u c o c y t e s were r e p o r t e d to d i s p l a y h y p e r t r o p h y and a l t e r a t i o n s in cell m o r p h o l o g y . These m i g h t be r e s p o n s i b l e for the d i s t u r b e d b i n d i n g c a p a c i t y a f t e r stress. In c o n c l u s i o n , decreased NCC-activity in s t r e s s e d fish s u g g e s t s that a b e r r a t i o n s in cell m e d i a t e d i m m u n i t y are e v i d e n t under s o c i a l s t r e s s . This is a u g m e n t e d by the d e m o n s t r a t i o n of the p o s s i b l e importance of m a m m a l i a n N K c e l l s in h e m o p o i e t i c regulation (25). This d e f e c t may c e r t a i n l y play a s i g n i f i c a n t role in the o b s e r v e d i n c r e a s e d s u s c e p t i b i l i t y of s t r e s s e d fish to d i s e a s e s and n e o p l a s m s . ACKNOWLEDGMENT The a u t h o r s e x p r e s s a p p r e c i a t i o n to Mr. W i l l i a m Engler, who k i n d l y p r o v i d e d the fish used for this s t u d y in the a b s e n c e of grant support. The B i n a t i o n a l Fulbright Commission, Cairo, Egypt, and the B u n d e s m i n l s t e r i u m f~r F o r s c h u n g und T e c h n o l o g i e , FRG (MFE) p r o v i d e d l a b o r a t o r y , travel and f i n a n c i a l a s s i s t a n c e . REFERENCES I.
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MOODY, C.E., SERREZE, D . V . A N D R E N O , P . W . Non s p e c i f i c c y t o t o x i c a c t i v i t y of t e l e o s t l e u c o c y t e s . Dev. Comp. I m m u n o l . 9, 51, 1985. C A R L S O N , R.L., EVANS, D.L. & GRAVES, S.S. Nonspecific c y t o t o x l c cells in fish ( I c t a l u r u s p u n c t a t u s ) V. M e t a b o l i c r e q u i r e m e n t s of lysis. Dev. Comp. I m m u n o l . 9 , 271, 1985. E H R L I C H , R., E ~ R A T I , M., M A L A T S K Y , E., S C H O C H A T L., BAR-EYAL, AL. & WITZ, I.P. N a t u r a l host d e f e n s e d u r i n g o n c o g e n e s i s : NK a c t i v i t y and d i m e t h y l b e n z a n t h r a c e n e carcinogenesis. Int. J. Cancer, 31, 67, 1983. HERBERMAN, R.B. P o s s i b l e role of n a t u r a l k i l l e r cells in h o s t r e s i s t a n c e a g a i n s t tumors and other d i s e a s e . Clin. I m m u n o l - A l l e r g y 3,479, 1983. G H O N E U M , M., GILL, G., W O J D A N I , A., PAYNE, C. & A L F R E D , L. S u p p r e s s i o n of basal and C o r y n e b a c t e r l u m p a r v u m augmented NK a c t i v i t y during chemically induced tumour development. Int. J. I m m u n o p h a r m . , 9, 71, 1987. HELFLAND, S.L., W E R K M E I S T E R , J., & RODER, J.C. C h e m i l u m i n e s c e n c e r e p o n s e of h u m a n n a t u r a l k i l l e r cell. I. The r e l a t i o n s h i p b e t w e e n target cell b i n d i n g , chemilumin e s c e n c e and c y t o l y s i s . J. Exp. Med., 156, 92, 1982. HANSSON, M., BERAN, M., A N D E R S S O N , B., and K I E S S L I N G , B. Inhibition of in vitro granulopoiesis by autologous a l l o g e n e i c h u m a n NK cells. J. I m m u n o l . 129, 126, 1982. MATERA, L., SANTOLI, D., GARBARINO, G., FEGORARO, L., DELLONE, G., and P A G L I A R D I , G. Modulation of in vitro m y e l o p o i e s l s by LGL: d i f f e r e n t e f f e c t s on e a r l y and late p r o g e n i t o r cells. J. I m m u n o l . 136, 1260, 1986. LOCKE, S.E. S t r e s s , a d a p t a t i o n and i m m u n i t y : studies in humans. Gen. Hosp. P s y c h i a t r y , 4, 49, 1982. SHAVIT, Y., LEWIS, J.W., TERMAN, G.W., GALE, R.P. & LIEBESKIND, J.C. Opioid peptldes mediate the s u p p r e s sive e f f e c t of stress on n a t u r a l k i l l e r cell e y t o t o x i city. Science 223, 188, 1984. W E D E M E Y E R , G.A., MEYER, F.P. & SMITH, L. Environmental s t r e s s and fish d i s e a s e s . In: D i s e a s e s of F i s h e s . (S.F. S N I E S Z K O and H.R. A X E L R O D , Eds.) T.F.H. P u b l i c I n c . N e p t u n e , 1-181, 1976. P E T E R S , G. & S C H W A R Z E R , R. C h a n g e s in h e m o p o i e t l c tissue of r a i n b o w trout u n d e r i n f l u e n c e of s t r e s s . Dis. A q u a t . Org. I, I, 1985. RODER, J . C . , K I E S S L I N G , R., B I B E R F E I D , P. & A N D E R S O N , B. T a r g e t - e f f e c t o r i n t e r a c t i o n in the n a t u r a l (NK) cell system. II. The i s o l a t i o n of NK cells and s t u d i e s on the m e c h a n i s m of k i l l i n g . J. I m m u n o l . 121, 2509, 1978. G H O N E U M , M., G R I M E S , P.E., GILL, G., & P A U L - K E L L Y , A. N a t u r a l c e l l - m e d l a t e d c y t o t o x l c i t y in v l t i l a g o . D e r m a t o l o g y , in p r e s s . A A R S T A D , H.J, G A U D E R N A K , G. & S E L J E L I D , R. Stress causes r e d u c e d n a t u r a l k i l l e r a c t i v i t y in m i c e . Scand. J. I m m u n o l . , 18, 461, 1984.
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MATHEWS, P.M., F R O E L I C H , J.C., SIBBIT, W.L. & B A N K H U R S T , A.D. Enhancement of n a t u r a l cytotoxlcity by B-endorphin. J. I m m u n o l . 9, 51, 1985. 23. O E H L E R , J.R. & H E R B E R M A N , R.B. N a t u r a l c e l l - m e d l a t e d cytotoxiclty in rats. III. Effect of immunopharmacologlc treatment on n a t u r a l reactivity augumented by polyinosinic-polycytidyllc acid. Int. J. C a n c e r 21, 221, 1978. 24. G H O N E U M , M., GILL, G., A S S A N A H , P., & S T E V E N S , E. S u s c e p t i b i l i t y of n a t u r a l k i l l e r c e l l a c t i v i t y of old rats to s t r e s s . I m m u n o l . , 60, 461, 1987. 25. H E R B E R M A N , R.B. NK cells and other n a t u r a l e f f e c t o r cells. New York, A c a d e m i c Press. 1167, 1982. Received: August, 1987 Accepted: March, 1988
SUPPRESSION OF NATURAL CYTOTOXIC CELL ACTIVITY SOCIAL AGGRESSIVENESS IN TILAPIA
M.
Ghoneum"*,
M. Faisa11'3, and E.L.
G. Peters4, Cooper
1.1.
BY
Ahmedls3,
of Comparative Immunology, Department 1. Laboratory Los Angeles, University of California, of Anatomy, Drew Otolaryngology, Department of CA 90024; 2. University of Medicine and Science, Los Angeles, CA Medicine, Veterinary Faculty of 90059; 3. fiir Institut University of Alexandria, Egypt; 4. Hydrobiologie und Fischereiwissenschaft, Universitat Hamburg, FRG.
ABSTRACT Our earlier observations revealed that social stress causes drastic effects on different physiological mechanisms and degenerative changes in leucocytes. In this preliminary work, we analyzed the effect of social aggressiveness on functional activites of leucocytes emphasizing (NCC) activity in Tilapia. At 10 hr post stress induction, fish could be differentiated into three categories: 1) dominants; 2) subordinates; and 3) determinants. Results of NCC as indicated by 4 hr. f? Cr-release assay, demonstrated a significant suppression in cytotoxic reactivity in the subordinates and indeterminants compared to dominants. This suppression appears to be due to a decrease in the binding capacity of effector cells to YAC-1 target cells as indicated by the decreased number of conjugate forming cells. This binding process is a key event in activating the fish equivalent of NK cells in mammals. Decreased NCC-activity in stressed fish suggests that aberrations in cell mediated immunity result from social aggressiveness.
INTRODUCTION Social stress causes drastic effects on different physiological mechanisms in fish (1,2). As a result, battle for rank increases the susceptibility of subordinate rainbow trout (Salmo gairdneri) to infection with the bacterium Aeromonas hydrophila (3) and induces degenerative changes in the leucocytes of Tilapia In fish a variety of leucocyte types are cytotoxic against (4). 595
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established mammalian and p i s c l n e cell lines (5-7). C a r l s o n et al. (8) w h o s t u d i e d the m e t a b o l i c requirements for l y s i s of target cells by n o n s p e c i f i c c y t o t o x l c cells f r o m c h a n n e l c a t f i s h i n d i c a t e d a s i m i l a r i t y to those of m a m m a l i a n n a t u r a l k i l l e r cells (NK). Many investigators consider mammalian NK cells to be the first llne of n a t u r a l immune defense against neoplasms and pathogens (9-12), in a d d i t i o n to t h e i r regulatory functions (13,14). Moreover, different stressors can suppress or e v e n inhibit NK-actlvity (15,16). We t h e r e f o r e wanted to d e t e r m i n e the e f f e c t of s o c i a l a g g r e s s i v e n e s s on N C C - a c t i v i t y in T i l a p i a . B e s i d e the i m p o r t a n c e of this s t u d y for a q u a c u l t u r e , it may aid in b e t t e r u n d e r s t a n d i n g h o w the p a t h o g e n e s i s of stress e v o l v e d . MATERIALS
AND
METHODS
Fish Hybrids
of
Tilapia
mossambica
x
Tilapia
honorum
of
both
sexes (237 g + 17; 23 cm + 5; one year old) were o b t a i n e d from the P a c i f i c Aquafarms, Niland, California, USA. A t o t a l of 25 f i s h w e r e m a i n t a i n e d for 4 w e e k s prior to the e x p e r i m e n t in 150 1 glass a q u a r i a at a t e m p e r a t u r e of 18+i°C. The diet c o n s i s t e d of p e l l e t e d f i s h feed ( P u r i n a C a t f i s h S t a r t e n a , R a l s t o n P u r i n a Co.). I n d u c t i o n of s o c i a l s t r e s s Social stresswas i n d u c e d a c c o r d i n g to the m e t h o d p r e v i o u s l y d e s c r i b e d (3). In brief, fish w e r e kept s e p a r a t e d for I week, by a partition ( p e r f o r a t e d d a r k p l a s t i c ) d i v i d i n g the tank in half. Thereafter, the p a r t i t i o n was r e m o v e d , thus b r i n g i n g the two fish t o g e t h e r for I0 hrs. D u r i n g their e n c o u n t e r , their b e h a v i o r was o b s e r v e d to d e t e r m i n e the r a n k o r d e r . Media All cell c u l t u r e s were performed in c o m p l e t e m e d i a (CM), that c o n s i s t e d of R P M I - 1 6 4 0 (Flow L a b o r a t o r i e s , R o c k v i l l e , MD.), 10% - heat i n a c t i v a t e d f e t a l calf s e r u m (FCS) and 1% a n t i b i o t i c s . [(50 u n i t s penicillin and 50 ug s t r e p t o m y c i n per ml.) (Grand Island Biological, Santa Clara, CA)]. Hank's balanced salt solution (HBSS) was used to wash fish leucocytes before e s t a b l i s h i n g c u l t u r e s w i t h tumor t a r g e t s . Cell p r e p a r a t i o n Fish were captured, anestheslzed in 200 mg/l M S - 2 2 2 (Sandoz, Swltzerland) and the head kidney (pronephros) removed. Head kidney cell suspensions were prepared by g e n t l y pressing the tissues through 80-gauge mesh stainless steel sieves. C e l l s were c o l l e c t e d and w a s h e d w i t h H B S S and r e s u s p e n d e d in CM. T a r g e t cells YAC-I, a v i r u s i n d u c e d l y m p h o m a of A/Sn m o u s e o r i g i n was ~Intalned in a s u s p e n s i o n c u l t u r e at 37°C in CM. -Cr R e l e a s e A s s a y Y A C - I cells (5xi06) were l a b e l l e d w i t h 100 uCi of 5 1 C r - N a ^ Cr0~ (New England Nuclear, Boston, MA) f o r o n e 5 h o u r at 2 5 ° C ~ Ce~s w e r e w a s h e d 3 times in CM d i l u t e d to 1 X I0 c e l l s / m l , and 101 cells w e r e d e l i v e r e d to each w e l l of a 96 well r o u n d b o t t o m tissue culture plates (Linbro Plastics). Fish effector cells were then a d d e d in I00 ul v o l u m e s at I00, 50 and 25:1 e f f e c t o r to target cell r a t i o s . Following a 4hrs. i n c u b a t i o n at 22°C, the L i n b r o p l a t e s were c e n t r i f u g e d (1600 rpm) for 5 m i n u t e s , and 0.I ml of s u p e r n a t a n t f r o m e a c h w e l l was c o l l e c t e d and c o u n t e d in a
FIGHTING BLOCKS CYTOTOXICITY
Vol. 12, No. 3
597
60
50 I
I
40
cl
a n=9
@j
I
n=IO
m
p
n=6
1
s F
30-
t ii5 z
20-
s 10-
L_
liLlL 25:1
Effector: Target Cell Ratio Figure (Linear
1. Non-specific cytotoxicity bars represent mean + SD) .
in
the
three
fish
%
50
I
40.
n=S
30, 20
n=O
I
T
10
Percentage
of conjugate
forming
cells.
groups
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FIGHTING BLOCKS CYTOTOXICITY
gamma counter. follows:
Percent
%speciflc
=
(test release
specific
release
Vol.
was
release)-(spontaneous
12, No. 3
calculated
as
release) x
(total
release)-(spontaneous
I00
release)
Spontaneous release (sp) f r o m the t a r g e t cells was always 8 - 1 0 % of the t o t a l r e l e a s e . T o t a l r e l e a s e was m e a s u r e d by a d d i n g 0.I ml T r i t o n X I 0 0 to d e s i g n a t e d wells. Conjugate formation Binding between effector cells and target tumor cells represents an i n i t i a l s t a g e in t a r g e t c e l l l y s i s . The c a p a c i t y of e f f e c t o r cells from the three different fish groups was examined as d e s c r i b e d by G h o n e ~ m et a l . ( l l ) . Effector cells (Ixl0") were incubated with I x l 0 ~ Y A C - I t a r g e t c e l l s in i ml of CM in 12 x 75 mm g l a s s t u b e s , s e d l m e n t e d by c e n t r i f u g a t l o n for 5 m i n and i n c u b a t e d for I hr at 4°C. Pellets were resuspended and cytocentrlfuged smears were stained with Giemsa. P e r c e n t of c o n jugate forming cells (CFCs) was determined by counting 200 leucocytes ( b o u n d and f r e e ) in t r i p l i c a t e samples. Statistical analysis For the statistical evaluation, the mean (X), standard deviation (SD), and coefficient of variation (V=SD/x) were determined, and the d a t a a n a l y z e d u s i n g the M a n n W h i t n e y U - t e s t . RESULTS Rank
order As a r e s u l t of b a t t l e for r a n k , t h r e e b e h a v i o r a l l y different g r o u p s of f i s h w e r e o b s e r v e d : dominants ( a l p h a ) that w e r e a g g r e s sive against their partners, subordinates (beta) that showed passive behavior and t r i e d to e s c a p e f r o m t h e i r a l p h a c o h o r t s and indeterminates (I) w h o s e status c o u l d n o t be d i s c e r n e d clearly. We classified nine fish as alpha, six as beta and ten as indeterminate or i n d i f f e r e n t . Non specific cytotoxiclty (NCC) NCC a c t i v i t i e s have been examined at ~ f f e r e n t effector: t a r g e t (E:T) r a t i o s u s i n g the s t a n d a r d 4 hrs. Cr-release assay D a t a in Fig. 1 reveal a considerable difference in NCC a c t i v i t y between the 3 fish groups. Alphas showed a high cytotoxic reactivity at all E:T ratios and the mean percentages of cytotoxlcity w e r e 40%, 42% and 44% at 25:1, 50:1 and I00:I r a t i o s respectively. In contrast, betas had very low levels of activities; 3% at 25:1, 4% at 50:1 and 9% at I 0 0 : I . The c e l l s from indeterminate fish showed cytotoxlc activities in b e t w e e n a l p h a s a n d b e t a s and b o t h p a r t n e r s showed equivalent values; 10%, 17% and 24% at 25:1, 50:1 a n d I00:I r e s p e c t i v e l y . Statistical analysis indicated a significant decrease of N C C a c t i v i t y between a l p h a s and b o t h b e t a s (p<0.001) and indeterminate fish (p<0.01). Conjugate formation Cytospin preparations showed three different effector cell types in c l o s e contact with the target tumor cells. These include lymphoid cells, macrophages and granulocytes ( P M N and pro-PMN). Data in F i g . 2 show that the percentage of c e l l s forming conjugates averaged 32% in a l p h a s , 20% in b e t a s w h i l e the percentage in i n d e t e r m i n a t e s represented a value between alphas
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and betas (26%). Similarly, statistical analysis showed significant differences b e t w e e n a l p h a s and b o t h betas (P< 0.001) and indetermlnates (P~0.01). In a l p h a fish, the c o n j u g a t e f o r m i n g cells (CFCs) w e r e m o s t l y m a c r o p h a g e s f o l l o w e d by g r a n u l o cytes and to a l e s s e r e x t e n t l y m p h o c y t e s . In b o t h beta and indeterminate fish, the CFCs w e r e m o s t l y g r a n u l o c y t e s f o l l o w e d by macrophages but rarely lymphocytes. CFCs of s t r e s s e d fish a p p e a r e d h y p e r t r o p h i c and d i s t o r t e d . DISCUSSION Stressed fish are more vulnerable to diseases (3,17), however, the e x a c t m e c h a n i s m s of this r e l a t i o n s h i p are not yet fully understood. In p r e v i o u s reports (4,18), degenerative c h a n g e s in the l e u c o c y t e s of s o c i a l l y s t r e s s e d r a i n b o w trout and Tilapia following relatively short stress periods were found. These alterations were a c c o m p a n i e d by l y m p h o p e n i a and g r a n u l o cytosis. Hence in t h e p r e s e n t study we w i s h e d to e x a m i n e , whether or not these morphological alterations are also associated with impaired functions of certain immunocompetent cells. As n o n s p e c l f i c cytotoxic cells of fish are c o n s i d e r e d functionally analogous to m a m m a l i a n natural killer cells (5), their activity may r e p r e s e n t the first llne of d e f e n s e against p a t h o g e n s and n e o p l a s m s in fish (I0). Our r e s u l t s d e m o n s t r a t e d a significant decrease in b o t h c y t o t o x l c i t y a n d the n u m b e r of conjugate f o r m i n g cells in s u b o r d i n a t e and i n d e t e r m i n a t e fish as c o m p a r e d to d o m i n a n t fish. Recognition and lysis of target cells by e f f e c t o r c y t o t o x i c cells i n v o l v e a s e q u e n c e of e v e n t s that has been a n a l y z e d r a t h e r extensively in m a m m a l s (19). First, the e f f e c t o r cell must recognize and bind to a t a r g e t cell, w h i c h is then f o l l o w e d by activation of e f f e c t o r cells and the i n i t i a t i o n of their p r o g r a m for t a r g e t cell l y s l s . Later, r e l e a s e of c y t o t o x i c f a c t o r s and lysis of the t a r g e t cells occur. The e f f e c t o r cells can then recycle a n d b i n d to new target cells. The d e c r e a s e d NCC activity of b e t a and i n d e t e r m i n a t e fish a p p e a r s to be due to a decrease in the b i n d i n g capacity of e f f e c t o r cells to Y A C - I target cells as i n d i c a t e d by the d e c r e a s e d number of c o n j u g a t e forming cells. This binding process is a k e y e v e n t in the a c t i v a t i o n of these cells (20). Although there appears to be no p r e v i o u s studies on the e f f e c t of s t r e s s on the N C C - a c t i v i t y of fish, there are s i m i l a r studies using other vertebrates. In this r e s p e c t , A a r s t a d et al. (21); M a t h e w s et al (22) and O e h l e r and H e r b e r m a n (23) r e p o r t e d that increased corticosteroid levels, associated with stress, directly suppresses the N K - a c t i v l t y . Other mechanisms that may be i n v o l v e d in the s u p p r e s s i o n of N K - a c t i v l t y by s t r e s s are due to o p l o l d s and m o r p h i n e . Shavit et al. (16) r e p o r t e d that o p i o l d peptides and morphine released under stress might suppress NK-activity d i r e c t l y or i n d i r e c t l y by m o d u l a t i n g the r e l e a s e of cortlcosterolds. On the o t h e r hand, G h o n e u m et al. (24) s u g g e s t e d a p o s s i b l e redistribution of N K - a c t l v l t y b e t w e e n the d i f f e r e n t body c o m p a r t ments under stress. They demonstrated a remarkable suppression in s p l e n i c and p e r i p h e r a l blood N K - c y t o t o x l c l t y which coincides w i t h an e l e v a t e d bone m a r r o w N K - a c t l v l t y in s t r e s s e d rats. In this r e s p e c t , w h e t h e r the d e c r e a s e of s t r e s s e d T i l a p i a p r o n e p h r o s
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NCC is a part of an o v e r a l l s u p p r e s s i o n of the i m m u n e system, or there is a p o s s i b l e r e d i s t r i b u t i o n of NCC b e t w e e n the d i f f e r e n t hemopoietic t i s s u e s is q u e s t i o n a b l e . However, our p r e l i m i n a r y studies (data not shown) show a consistent decrease in NCC a c t i v i t y of p e r i p h e r a l blood, spleen, trunk k i d n e y in a d d i t i o n to pronephros of s u b o r d i n a t e s . Further studies are needed to c l a r i f y this point. An i n t e r e s t i n g r e s u l t was the c h a n g e of c o n j u g a t e f o r m i n g cell types in s t r e s s e d fish. We o b s e r v e d an i n c r e a s e in g r a n u l o c y t e s and a d e c r e a s e in l y m p h o c y t e s and m a c r o p h a g e s . This is in a c c o r d a n c e with the m a r k e d g r a n u l o c y t o s i s and l y m p h o p e n i a observed in socially stressed Tilapia (4) and trout (18). Furthermore, the r e m a i n i n g l e u c o c y t e s were r e p o r t e d to d i s p l a y h y p e r t r o p h y and a l t e r a t i o n s in cell m o r p h o l o g y . These m i g h t be r e s p o n s i b l e for the d i s t u r b e d b i n d i n g c a p a c i t y a f t e r stress. In c o n c l u s i o n , decreased NCC-activity in s t r e s s e d fish s u g g e s t s that a b e r r a t i o n s in cell m e d i a t e d i m m u n i t y are e v i d e n t under s o c i a l s t r e s s . This is a u g m e n t e d by the d e m o n s t r a t i o n of the p o s s i b l e importance of m a m m a l i a n N K c e l l s in h e m o p o i e t i c regulation (25). This d e f e c t may c e r t a i n l y play a s i g n i f i c a n t role in the o b s e r v e d i n c r e a s e d s u s c e p t i b i l i t y of s t r e s s e d fish to d i s e a s e s and n e o p l a s m s . ACKNOWLEDGMENT The a u t h o r s e x p r e s s a p p r e c i a t i o n to Mr. W i l l i a m Engler, who k i n d l y p r o v i d e d the fish used for this s t u d y in the a b s e n c e of grant support. The B i n a t i o n a l Fulbright Commission, Cairo, Egypt, and the B u n d e s m i n l s t e r i u m f~r F o r s c h u n g und T e c h n o l o g i e , FRG (MFE) p r o v i d e d l a b o r a t o r y , travel and f i n a n c i a l a s s i s t a n c e . REFERENCES I.
2.
3.
4.
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