Rainbow trout macrophages in vitro: Morphology and phagocytic activity

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 6, pp. 281-291, 1982

0145-305X/82/020281-II$03.00/0 Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.

RAINBOW TROUT ~ C R O P H A G E S IN VITRO: MORPHOLOGY AND PHAGOCYTIC ACTIVITY

Rosemarie Braun-Nesje, Gilla Kaplan and Rolf Seljelid Institute of Medical Biology University of Troms~ and Department of Tumor Biology Norwegian Cancer Society Troms~, Norway

ABSTRACT The properties of macrophages from the pronephros of Ra-inbow trout (Salmo ~airdneri Richardson) were studied in vitro. We found that phagocytes obtained from the pronephros constitute a non-homogeneous cell population. Three populations with different adherence properties were examined with special emphasis on morphology and phagocytic capacity. The differentiation of the three populations in culture was similar morphologically, and their phagocytic activity showed only small variations. The methods for cell separation and culture reported here are a useful tool for gaining better understanding of how Rainbow trout macrophages function in the immune response.

INTRODUCTION To study fish leucocyte functions and their role in immunity, a method for separating and culturing these cells has been established (i). Fish leucocytes were obtained from the pronephros (2) of Rainbow trout, Salmo ~airdneri Richardson, and single cell suspensions prepare d . Different cell types were separated by density gradient centrifugation. The fraction of cells containing the phagocytes was identified and the cells cultured in vitro. To further characterize

and understand the functions of 281

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m a c r o p h a g e - l i k e cells of R a i n b o w trout, m o r p h o l o g y , p h a g o c y t i c activity, p r o t e i n content, and l y s o s o m a l e n z y m e a c t i v i t y of these cells w e r e e v a l u a t e d , and the p r e s e n c e of f u n c t i o n a l l y d i f f e r e n t groups of cells studied.

MATERIALS AND METHODS E x p e r i m e n t a l fish, c u l t u r e medium, and p u r i f i c a t i o n of leucocytes. R a i n b o w trout (20-100 g) w e r e k e p t in fresh w a t e r at 5-7~C and fed until used. The m e d i u m for cell s u s p e n s i o n s and m a i n t e n a n c e in c u l t u r e was L-15 (Leibovitz, G r a n d Island) s u p p l e m e n t e d w i t h 0,33 g glucose, I00 I.U. p e n i c i l l i n - s t r e p t o m y c i n (Gibco, B i o - c u l t , Scotland) and i0 % foetal calf s e r u m (Gibco) (FCS). S i n g l e cell s u s p e n s i o n s w e r e p r e p a r e d and s e p a r a t e d as d e s c r i b e d (i). In short, the p r o n e p h r o s of R a i n b o w t r o u t was d i s s e c t e d out and the cell s u s p e n s i o n s o b t a i n e d s e p a r a t e d on a d i s c o n t i n u o u s P e r c o l l g r a d i e n t r e p r e s e n t e d s c h e m a t i c a l l y in Fig. i. The g r a d i e n t s w e r e c e n t r i f u g e d for 40 min. at 400 g and the cells f o u n d in b a n d 3 w e r e collected. The e n t i r e p r o c e d u r e was c a r r i e d o u t in a 5°C l a b o r a t o r y or on ice (i). In v i t r o culture c o n d i t i o n s . Cell s u s p e n s i o n s f r o m b a n d N o 3 w e r e w a s h e d in L-15 m e d i u m s u p p l e m e n t e d w i t h FCS and c o u n t e d (~ig. i). O n e 6 h u n d r e d ~ i from s u s p e n s i o n s c o n t a i n i n g 0.75 x i0~ - 1.5 x 10 c e l l s / m l w e r e s e e d e d on 14 m m glass c o v e r s l i p s in c u l t u r e w e l l s (Linbro or C o s t a r Plates) contain i n g 1 ml L-15 w i t h i0 % FCS. The cells w e r e m a i n t a i n e d in c u l t u r e at 10°C in a i r from 1 day to 3 m o n t h s as d e s c r i b e d (i). Cell m o r p h o l o g y . F o r p h a s e - c o n t r a s t m i c r o s c o p y the cells on cover slips w e r e f i x e d in 2.5 % g l u t a r a l d e h y d e in 0.1 M c a c o d y l a t e b u f f e r w i t h 0.i M s u c r o s e (pH 7.4) for at least 24 hours. The c o v e r s l i p s w e r e m o u n t e d and e x a m i n e d w i t h a Zeiss P h o t o m i c r o s c o p e . For s c a n n i n g e l e c t r o n m i c r o s c o p y f i x e d cells w e r e d e h y d r a t e d in alcohol, t r a n s f e r r e d to amyl acetate, and c r i t i c a l p o i n t d r i e d (Hitachi CPI, Tokyo, Japan) in c a r b o n dioxyde. The s p e c i m e n s w e r e c o a t e d w i t h g o l d (Polaron S E M c o a t i n g U n i t E 5000) and e x a m i n e d w i t h a h i g h - r e s o l u t i o n H i t a c h i S E M (HHS/2R) at 20 k V and a tilt angle of 30 v. A t t a c h m e n t and i n @ e s t i o n of p a r t i c l e s by p h a g o c y t e s . In p h a g o c y t o s i s studies the c u l t u r e m e d i u m was c a r e f u l l y r e m o v e d and n e w m e d i u m c o n t a i n i n g v a r i o u s n u m b e r s of g l u t a r a l d e h y d e f i x e d s h e e p red b l o o d cells (GA-SRBC) was added (i). M a c r o p h a g e s w e r e i n c u b a t e d at 10°C for a t t a c h m e n t and i n g e s t i o n A t the e n d of the a t t a c h m e n t time the c o v e r s l i p s w e r e w a s h e d by d i p p i n g in c o l d m e d i u m (10-20x). In i n g e s t i o n s t u d i e s the cells w e r e c u l t u r e d in new m e d i u m after w a s h i n g away the n o n - a t t a c h e d GASRBC. A t the end of the i n g e s t i o n time the p h a g o c y t e s w e r e w a s h e d and p r e p a r e d for o b s e r v a t i o n as d e s c r i b e d above. P r o t e i n c o n t e n t and l y s o s o m a l e n z y m e activity. For bioc h e m i c a l d e t e r m i n a t i o n s cells w e r e seeded, w a s h e d after 20 h,

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m a i n t a i n e d in c u l t u r e for 3 days, and then w a s h e d 3 times in c o l d PBS. For p r o t e i n and l y s o s o m a l e n z y m e e s t i m a t i o n 1 ml of 0.i N N a O H or 0.i % T r i t o n w e r e a d d e d to the w e l l s r e s p e c t i v e l y and the cells w e r e frozen and t h a w e d twice. The p r o t e i n c o n t e n t of cell lysates was m e a s u r e d by the m e t h o d of Lowry et al. 1951 (3). A c i d p h o s p h a t a s e (E.C.3.1.3.2) (24 h incubation) was a s s a y e d by the m e t h o d s of G i a n e t t o and de Duve 1955 (4).

RESULTS Cell cultures. D e n s i t y c e n t r i f u g a t i o n of p r o n e p h r o s cell s u s p e n s i o n s gave rise to four d i f f e r e n t f r a c t i o n s (bands) of f a i r l y h o m o g e n e o u s s u b p o p u l a t i o n s : the m a c r o p h a g e s , lymphocytes, e r y t h r o c y t e s , and (in some cases) m e l a n o c y t e s (Fig. i). The cells o b t a i n e d f r o m b a n d 3 w e r e i n t r o d u c e d into c u l t u r e in L-15 m e d i u m s u p p l e m e n t e d w i t h g l u c o s e and FCS. G i e m s a s t a i n e d s m e a r s of the cells in b a n d 3 s h o w e d a m a j o r i t y of m a c r o p h a g e like cells w i t h a d i a m e t e r of 10-12 ~m. The n u c l e u s was b e a n s h a p e d and o c c u p i e d a b o u t 1/3 of the cell v o l u m e (5). The macrophages were contaminated with about 5 % polymorphonuclear l e u c o c y t e s (6 - 8 ~tm in diameter) (2), 5 % l y m p h o c y t e s w i t h a dense r o u n d n u c l e u s (6 ~m) and p o s s i b l y 5 % t h r o m b o c y t e s (4 6 ~m) w i t h an i n d e n t e d n u c l e u s and a very s m a l l a m o u n t of cytoplasm.

FIG.

Fraction number

Density Volume loaded <108in mediumCellsI--7-mi---

~//22/-/12//////~I/////~ 2

1,075

!!r[

1,080

T 7 ml 4

1,100

1

S c h e m a t i c r e p r e s e n t a t i o n of the discontinuous density Percoll g r a d i e n t u s e d for cell s e p a r a t i o n . The cell s u s p e n s i o n is l a y e r e d on the top of the g r a d i e n t , w h i c h is then c e n t r i f u g e d at 400 g for 40 min. B a n d 1 c o n t a i n s debri, d e a d cells and cell clumps. B a n d 2 c o n t a i n s m o s t l y cell clumps, some m a c r o p h a g e s and s o m e t i m e s melanocytes. B a n d 3 c o n t a i n s the m a j o r i t y of m a c r o p h a g e s f r o m the p r o n e p h r o s , c o n t a m i n a t e d w i t h 5 - i0 % p o l y m o r p h o n u c l e a r cells, 5 % lymphocytes and p o s s i b l y 5 % t h r o m b o cytes. B a n d 4 contains 95 % cells m o r p h o logically resembling lymphocytes, and a few m a c r o p h a g e s . The b o t t o m s e d i m e n t c o n t a i n s >95 % e r y t h r o c y t e s , in m o s t cases in addition melanocytes.

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FIG.

2

Scanning electron microscopy m i c r o g r a p h s of R a i n b o w trout m a c r o p h a g e s in vitro. D i f f e r e n t stages of cell spreading. a) The cell on the left adh e r e d to the b l a s s but was still c o m p l e t e l y rounded. The cell on the r i g h t b e g a n to s p r e a d and d e v e l o p e d pseudopods (arrows). x 7200.

b) S p r e a d i n g d e v e l o p e d f u r t h e r in this cell. The p e r i n u c l e a r area was s t i l l rounded. Note the r u f f l e d m e m b r a n e (arrow). x 8800.

c) The cell seen in this p i c t u r e is s p r e a d and has an e l o n g a t e d shape, x 4700.

d) The cell is w e l l s p r e a d and round. The p e r i n u c l e a r area of this cell is flattened, too. N o t e r u f f l i n g of the m e n ~ r a n e (arrow). x 3800.

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A t t a c h m e n t and s p r e a d i n g of r a i n b o w t r o u t m a c r o p h a ~ e s on ~lass: P h a g o c y t e s p r e a d i n g in c u l t u r e was o b s e r v e d by s c a n n i n g e l e c t r o n m i c r o s c o p y . Fig. 2 d e m o n s t r a t e s the d i f f e r e n t stages of cell spreading. The cell on the left in Fig. 2a is f i x e d to the glass s u r f a c e and still c o m p l e t e l y rounded. The o t h e r cell has b e g u n s p r e a d i n g and small p s e u d o p o d s are observed. W i t h time the cells s p r e a d m o r e (2b). In 2a, b and c the p e r i n u c l e a r area of the cell has n o t y e t f l a t t e n e d w h i l e in 2d the cell is c o m p l e t e l y s p r e a d o u t and flat. W h e n a p o p u l a t i o n of cells is e x a m i n e d , the cells s h o w d i f f e r e n t e x t e n t s of s p r e a d i n g w i t h a m a j o r i t y of cells s p r e a d to one d e g r e e or the other. W h e n fish p h a g o c y t e s w e r e i n t r o d u c e d i n t o culture, some c o u l d be s e e n to adhere to the glass and s p r e a d v e r y q u i c k l y (about i0 - 30 %). If the n o n - a d h e r e n t cells w e r e w a s h e d away after 30 - 60 m i n u t e s , a p o p u l a t i o n of cells r e m a i n e d b o u n d to the c o v e r s l i p , w h i c h w e call the e a r l y a d h e r e n t cells. W h e n the cells t h a t did n o t adhere w e r e r e i n t r o d u c e d into c u l t u r e again, a s e c o n d p o p u l a t i o n of a d h e r e n t cells was obtained. T h e s e w e r e a d h e r e d w i t h i n 1 - 6 hours in culture. R e - s e e d i n g the cells n o t a d h e r e n t by 6 h o u r s on an a d d i t i o n a l c o v e r s l i p , g a v e rise to a t h i r d p o p u l a t i o n of cells, a d h e r e n t by 20 hours. T h e s e we r e f e r to as s l o w (late) a d h e r e n t cells. A d h e r e n t cells f r o m c u l t u r e s w a s h e d at 60 min, 6 h, and 20 h w e r e m a i n t a i n e d in v i t r o for s e v e r a l months. The total p h a g o c y t e r e c o v e r y o b t a i n e d in d e s c r i b e d above is p r e s e n t e d in T a b l e I. W h e n one m i l l i o n cells, the total r e c o v e r y o b t a i n e d F o r n o r m a l f u n c t i o n a l s t u d i e s o n l y one of the p o p u l a t i o n s was used.

Recovery Attachment-time in C u l t u r e % cells recovered Total

recovery

TABLE 1 of Cells (Mean Results, Coverslip 60 m i n 10-30

%

1

Coverslip 6 hours > 15 %

using the s y s t e m we s t a r t e d w i t h was 40 - 60 %. above cell

6 Fish) 2

Coverslip 20 hours

3

> 15 %

40 - 60 %

T o t a l r e c o v e r y of R a i n b o w t r o u t m a c r o p h a g e s in vitro. For each e x p e r i m e n t 106 cells w e r e s e e d e d and the a d h e r e n t cells r e c o v e r e d a f t e r 60 min, 6 hours, a n d 20 hours. R e s u l t s e x p r e s s e d as p e r c e n t of total n u m b e r of cells seeded.

To o b t a i n c u l t u r e s of o p t i m u m d e n s i t y for all d i f f e r e n t time points, d i f f e r e n t n u m b e r s of cells w e r e seeded. F o r 60 m i n u t e s cultures, we u s e d a h i g h e r n u m b e r of cells, (1.5 x 106 )

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because the number ~f immediately adherent cells was relatively low. Less cells (i0 v) could be seeded for later washes. For 20 hours cultures which contained all the adherent cel~s (those from 60 minutes, 6 hours and 20 hours), only 0.75 x i0 v cells were used for seeding. In this way, uniformly dense cell culture were obtained for comparative morphological a n d p h a g o c y t i c activity studies. Cell morphology under various culture conditions. As can be seen from Fig. 3 some of the early adherent cells were quite spread, while others were still round (Fig. 3a). The 6 hours cell-cultures were predominantly slightly spread cells (Fig. 3b). Cell cultures obtained after 20 hours were dominated by more spread cells (Fig. 3c). After 3 days in culture the cells in all three populations had lost any differences and appeared similar morphologically (Fig. 3d,e,f). The majority of cells from the three populations had spread extensively and had a diameter of about 50 ~m by day 3, and 60 - 100 ~ m by day 5. Most cells in all cultures developed thin pseudopods which extended out from the perinuclear area. After 1 - 3 weeks in culture the macrophages showed a tendency to form giant cells, as large as 120 ~m in diameter, with several bean-shaped nuclei, and numerous vacuoles (not shown). Phagocytic activity. The major function by which these cells were recognized was their phagocytic capacity. Macrophages gained by washing 60 minutes, 6 hours and 20 hours after seeding were tested for their phagocytic activity, immediately after wash and after three days in culture. GA-SRBC added to the cultures for 2h, bound to the macrophages in large numbers (Fig. 4a). After attachment, cultures were transferred into new m e d i u m and allowed to ingest the bound particles for 20 hours, and many of the particles were internalized (Fig. 4b,c,d). Early adherent cells had in some cases slightly higher attachment and phagocytosis rates than late adherent cells w h e n teste~ immediately after wash. Culturing the three populations of cells for 3 days gave an enhancement of phagocytic capacity in some experiments, but not in others. In some cases the attached particle could be seen sinking into the phagocytic cell (Fig. 4c). Finally they become completely enclosed by the macrophage membrane, bulging slightly over the surface (Fig. 4d). If the phagocytes were left in culture for a longer time after ingestion, the internalized particle seemed to be slowly broken down (not shown). The protein content of the different macrophage cultures was similar, and somewhat higher w h e n more cells were seeded (Table 2). The ability of phagocytic cells to degrade the ingested particles was evaluated by measuring lysosomal enzyme activity of cell lysates. Acid phosphatase, the enzyme commonly used to monitor lysosomal enzyme activity in mammalian macrophages (7,8,9) as well as in invertebrate phagocytes, (Bertheussen, per. com.) was demonstrated in Rainbow trout phagocytes (Table II). Cultures from 6 different animals were tested and about 35 m units of enzyme activity were observed.

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al ,~&, g

_

-

~"'~'~

~ m

its_

.

r/

V

°

FIG. Phase contrast m±crographs cell p o p u l a t i o n s in vitro.

3 of m o r p h o l o g y

of the d i f f e r e n t

Cell c u l t u r e s w a s h e d a f t e r 60 m i n u t e s (a), 6 h o u r s 20 h o u r s (c) and then f i x e d i m m e d i a t e l y . a) The m a j o r i t y of the cells are c o m p l e t e l y w h i l e some cells are very w e l l spread. b)

The cells a p p e a r to be q u i t e h o m o g e n e o u s , s l i g h t l y spread.

(b),

rounded, r o u n d or

c) A h o m o g e n e o u s culture, w i t h s l i g h t l y m o r e s p r e a d i n g cells than in 6 h o u r s cultures.

of

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Cell cultures fixed after a total of 3 days in culture, following washing at 60 min (d), 6 hours (e), and 20 hours

(f). In d, e, and f all three populations have developed well spread cells, without any obvious differences in morphology. Mag. of all plates x 600.

TABLE 2 Cell Protein and Acid Phosphatase Activity of Rainbow Trout Macrophages

Fish

No of Cells Seeded

Mg Protein per Culture

~OD Acid Phosphatase Activity (250 ~i Sample)

m Units Acid Phosphatase per mg Protein

1

106

42

0. i08-0. 114

31.6-33.3

2

106

32

0.083-0.089

32-34

3

106

37

0. i01-0. 105

33.5-34.8

4

106

33

0.090-0.094

33.5-35.0

5

106

33

0.081-0.103

30.2-38.3

6

2x106

57

0.147-0.177

31.7-38.2

7

2x106

51

0.101

24.3

DISCUSSION To study cell-mediated immunity in fish we have developed a method for separating macrophages from the pronephros of Rainbow trout, Salmo ~airdneri Richardson. For the first time, defined culture conditions for maintaining these cells i__nn vitro have been described (i). In this paper we present data indicating that these macrophages are not a homogeneous population of cells. Mononuclear phagocytes of all mammals are a heterogeneous population of cells (9, ii). This is expressed in many of their properties, including the ability to adhere to glass and spread on culture surfaces. The Rainbow trout macrophages collected in band 3 of the discontinuous Percoll gradient (Fig. i) were used to obtain three populations of cells which adhered to glass after different lengths of time in culture. Though these three populations of cells were different in morphology at the beginning of culture, their differentiation in vitro gave rise to very similar cultures, as observed both by phase contrast m i c r o s c o p y and by scanning electron microscopy. The spreading and surface morphology of fish phagocytes resembles that of mammalian monocytes and macrophages (9,11). The majority of the fish macrophages needed a few days in culture for full spreading.

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FIG.

4

Scanning electron microg r a p h s of a t t a c h m e n t a n d p h a g o c y t o s i s of G A - S R B C by R a i n b o w t r o u t m a c r o phages. a) A large n u m b e r of GAS R B C are a t t a c h e d to the macrophage surfaces. x 2300.

b) The c e l l on the l e f t has p a r t i c l e s b o u n d to the s u r f a c e . The cell on the r i g h t (P) has p h a g o c y t o s e d the b o u n d p a r t i c l e s and r o u n d e d up. x 2750.

c) One of the cells obs e r v e d has s t a r t e d ing e s t i o n . An e r y t h r o c y t e is s h o w n s i n k i n g i n t o the p h a g o c y t e (arrow) . x 5500.

d) C l o s e up of a p h a g o -~ cyte w h i c h has i n g e s t e d p a r t i c l e s . The o u t l i n e of the i n g e s t e d p a r t i c l e s is e a s i l y o b s e r v e d (A). x 5000.

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Cells of all three populations of trout phagocytes showed binding and phagocytic activity, both immediately after washing and after some time in culture. The numbers of macrophages which showed binding and phagocytic activity in the different populations varied to some extent. In some cases enhanced phagocytic activity was observed in older cell cultures (3 day), compared to newly seeded cells. In other experiments older cultures did not phagocytose more than their younger counterparts. Early adherent cells sometimes showed slightly higher attachment and phagocytosis rates than late adherent cells. More detailed kinetic studies of phagocytosis are needed to explain this discrepancy. No clear correlation between the early adherence capacity of the cells and their later morphology or phagocytic capacity were observed. Lysosomal enzyme activity measured in fish macrophage cultures makes it clear that these cells not only have the ability to phagocytose various particles in vitro but are also able to degrade organic material after ingestion. Some of the cells which remained in culture for a week or longer, spread out well, and were slightly phagocytic, and appeared to be slightly different from the typical macrophage. These cells were smaller in size, their nucleus more dense, and not clearly beanshaped. They had a smooth surface, and did not show cytoplasmic ruffling in the typical form observed on macrophages when examined by scanning electron microscopy. These cells were most probably thrombocytes, already reported to show some phagocytic activity in vitro (Lester, per. com.).

ACKNOWLEDGMENTS This study was supported by grants from the Norwegian Fisheries Research Council. We thank Prof. E.L. Cooper for editorial advice.

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REFERENCES i.

BRAUN-NESJE, R., BERTHEUSSEN, K., KAPLAN, G., SELJELID, R. Salmonid macrophages. Separation, in vitro culturing and characterization. J. of Fish Diseases 4, 1981 (in press).

2.

ELLIS, A.E. The leucocytes ii, 453, 1977.

of fish. A review. J. Fish 8i0].

.

LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L., RANDALL, R.J. Protein measurement with the folin phenol reagent. O. 8i0]. Chem. 193, 265, 1951.

.

GIANETTO, R., De DUVE, C. Tissue fraction studies. 4 comparative studies of the binding of acid phosphatase B-glucuronidase and cathepsin by rat liver particles. J. 8i0chem. 59, 433, 1955.

.

ELLIS, A.E., MUNROE, A.L.S., ROBERTS, J.J. Defence mechanisms in fish. I. A study of the phagocytic system and the fate of intraperitoneally injected particulate material in the plaice (Pleuronectes platessa L.). J. Fish Bi0]. 8, 67, 1976.

.

CHILLER, J . M . , HODGINS, H.O., CHAMBERS, V.C. WEISER, R.S. Antibody response in Rainbow trout (Salmo gairdneri). I. Immunocompetent cells in the spleen and anterior kidney. J. Immun0]. 102, 1193, 1969.

.

COHN, Z.A., WIENER, E. The particulate hydrolases of macrophages. I. Comparative enzymology, isolation, and properties. J. Expt]. Med. 118, 1009, 1963b.

.

COHN, Z.A., WIENER, E. The particulate hydrolases of macrophages. II. Biochemical and morphological response to particle ingestion. J. Expt]. Med. i18, 1009, 1963b.

.

M~RLAND, B., KAPLAN, G. Macrophage activation in vitro. Exp. Ceil Res. 108, 279, 1977.

in vivo and

i0.

COHN, Z.A., BENSON, B. The differentiation of mononuclear phagocytes. Morphology, cytochemistry, and biochemistry. J. Expt]. Med. 121, 153, 1965.

ii.

COHN, Z.A., HIRSCH, J.G., FEDORKO, M.E. The in vitro differentiation of mononuclear phagocytes. IV. The ultra structure of macrophage differentiation in the peritoneal cavity and in culture. J. Expt]. Med. 123, 747, 1965.

Received January, 1981 Accepted September, 1981