HELGOL~NDER MEERESUNTERSUCHUNGEN
Helgol~nder Meeresuntersuch. 37, 415-424 (1984)
M e l a n o - m a c r o p h a g e centres in liver and s p l e e n of ruffe
(Gymnocephalus cernua)from the Elbe Estuary
H. Kranz & N. Peters
Zoologisches Institut und Zoologisches Museum, Universit~t Hamburg;
Martin-Luther-King-Platz 3, D-2000 Hamburg 13,
Federal Republic of Germany
ABSTRACT: Macrophages with yellow or black deposits of melanin occur singly or in groups in the
melano-macrophage centres are functionally the primitive analogues of lymph nodes in birds and
mammals. From 1980 to 1982, turnouts and pretumorous tissue changes in ruffe from the Elbe
Estuary were studied. Pathological alterations occur mainly in the liver and spleen. In connection
with both, neoplastic abnormalities in liver and spleen, and fatty degenerative processes in the
liver, an obvious increase in number and size of the melano-macrophage centres was observed. The
colour, structure and some histochemical properties of melano-macrophage centres in the liver
differ somewhat from those in the spleen. The majority of splenic centres were filled with large
amounts of haemosiderin, whereas many hepatic macrophages contained fatty inclusions. Possible
differences in the functions of splenic and hepatic melano-macrophage centres are discussed,
INTRODUCTION
Since fish do not h a v e b o n e m a r r o w or l y m p h nodes, the m a i n sites of h a e m o p o e s i s
are the anterior k i d n e y a n d t h e s p l e e n . Less i m p o r t a n t for the p r o d u c t i o n of b l o o d cells in
teleosts are s p e c i a l areas in the liver, t h e s u b m u c o s a of t h e intestine, a n d the t h y m u s
(Roberts, 1978).
A characteristic f e a t u r e of h a e m o p o e t i c tissue is t h e p r e s e n c e of s o - c a l l e d m e l a n o m a c r o p h a g e centres. T h e s e are n o d u l e s of m a c r o p h a g e cells w h i c h are m o r e or less
coloured by yellow, b r o w n or b l a c k p i g m e n t s , w h i c h are r e f e r r e d to as m e l a n i n . M e l a n i n
is a class of i n s o l u b l e p o l y c y c l i c p o l y m e r s d e f i n e d to i n c l u d e not o n l y t h e p i g m e n t s in
m e l a n o p h o r e s but also r e l a t e d substances, such as l i p o f u s c i n a n d c e r o i d s (Edelstein,
1971). In contrast to m e l a n o c y t e s , m e l a n o - m a c r o p h a g e s are not a b l e to s y n t h e s i z e
m e l a n i n p i g m e n t s , so t h e o r i g i n of this m a t e r i a l m u s t still b e d e t e r m i n e d (Ellis, 1974).
Groups of p i g m e n t e d m a c r o p h a g e s in t h e h a e m o p o e t i c tissue of teleosts a r e often
s u r r o u n d e d by a collar of l y m p h o c y t e s , a n d s o m e of t h e m e l a n o - m a c r o p h a g e s c o n t a i n
i m m u n o g l o b u l i n s on their surface (Ellis, 1980). B e c a u s e of t h e s e characteristics, it is
a s s u m e d that m e l a n o - m a c r o p h a g e c e n t r e s are t h e p r i m i t i v e a n a l o g u e s of the l y m p h
nodes in h i g h e r vertebrates. T h e s p e c i a l function of t h e s e m e l a n o - m a c r o p h a g e s is
p h a g o c y t i c c o n s u m p t i o n of c a t a b o l i c p r o d u c t s a n d f o r e i g n material, i n c l u d i n g i n j e c t e d
carbon particles (Ellis et al., 1976). T h e r e f o r e m e l a n o - m a c r o p h a g e c e n t r e s c a n b e
c o n s i d e r e d as m e t a b o l i c d u m p s (Agius, 1980).
From 1980 to 1982, an i n v e s t i g a t i o n w a s carried out to study t u m o r o u s conditions in
© Biologische Anstalt Helgoland, Hamburg
416
H. Kranz & N. Peters
t h e ruffe G y m n o c e p h a l u s cernua L., a p e r c i d fish, from t h e p o l l u t e d E l b e Estuary (FRG).
D u r i n g this study, it b e c a m e i n c r e a s i n g l y o b v i o u s that m e l a n o - m a c r o p h a g e centres in
the s p l e e n a n d l i v e r a u g m e n t e d d u r i n g t h e d e v e l o p m e n t of s e v e r a l h i s t o p a t h o l o g i c a l
a b n o r m a l i t i e s of t h e s e organs, It is the p u r p o s e of this p a p e r to report p r e l i m i n a r y
o b s e r v a t i o n s on o c c u r r e n c e rates and s p e c i a l h i s t o l o g i c a l a n d h i s t o c h e m i c a l characteristics of m e l a n o - m a c r o p h a g e centres in p a t h o l o g i c a l l y a l t e r e d l i v e r a n d s p l e e n of the ruffe
a n d to discuss p o s s i b l e d i f f e r e n c e s in t h e f u n c t i o n of m e l a n o - m a c r o p h a g e s in both
organs.
MATERIAL A N D M E T H O D S
A total of 551 ruffe from the Elbe Estuary b e t w e e n H a m b u r g H a r b o u r a n d its m o u t h
w e r e e x a m i n e d for gross a b n o r m a l i t i e s of t h e l i v e r and spleen. T h e size of the ruffe
s p e c i m e n s t a k e n r a n g e d from 10 to 28 cm. A n i m a l s w e r e k i l l e d in MS 222 (1 g/l) and
dissected. T h e l i v e r a n d s p l e e n w e r e r e m o v e d , a n d the m a c r o s c o p i c a p p e a r a n c e of each
was noted. T h e o r g a n s w e r e fixed in 6 % b u f f e r e d f o r m a l i n or Bouin's solution. Subsamples s e l e c t e d for s e m i - t h i n Epon sections w e r e fixed in 6 % p h o s p h a t e buffered glutarald e h y d e . S o m e l i v e r s p e c i m e n s t a k e n to d e t e r m i n e the p r e s e n c e of g l y c o g e n and fats
w e r e e i t h e r fixed in C a r n o y ' s fluid or d e e p frozen for cryostatic sectioning. Epon sections
1 ~m thick w e r e s t a i n e d w i t h t o l u i d i n e blue, S a m p l e s e m b e d d e d in paraffin blocks w e r e
cut into 5 ~tm sections, w h i c h w e r e s t a i n e d as follows: h e m a t o x y l i n / e o s i n as g e n e r a l
stains; a z a n for i d e n t i f i c a t i o n of c o n n e c t i v e tissue e l e m e n t s ; Prussian b l u e for the
d e m o n s t r a t i o n of ferric iron; a n d Best's c a r m i n e w i t h K e r n e c h t r o t to d e t e r m i n e the
g l y c o g e n content. S u d a n b l a c k w a s u s e d to detect fats in the 10 ~tm cryostat sections
(Romeis, 1948).
S a m p l e s of l i v e r a n d / o r s p l e e n t a k e n from a total of 77 fishes with a p p a r e n t l y
h e a l t h y o r g a n s a n d d i s p l a y i n g various gross lesions w e r e h i s t o l o g i c a l l y e x a m i n e d . From
35 fishes, m e l a n o - m a c r o p h a g e centres of both the liver a n d s p l e e n w e r e m i c r o s c o p i c a l l y
a n a l y s e d for direct comparison.
RESULTS
Ruffe from t h e p o l l u t e d E l b e Estuary d i s p l a y e d t h r e e t y p e s of a b n o r m a l i t i e s w h i c h
w e r e r e m a r k a b l e b e c a u s e of t h e i r h i g h i n c i d e n c e rates. T h e most f r e q u e n t one is the only
n o n - t u m o r o u s condition. It occurs in the liver. In 39 % of t h e ruffe, livers d i s p l a y pale
spots or m a r b l e d p a t t e r n s on t h e surface, w h i c h w e r e h i s t o c h e m i c a l l y s h o w n to be
r e g i o n s c o n t a i n i n g a c c u m u l a t i o n s of l i p o i d c o m p o u n d s (Fig. 1). A r e a s w i t h e x c e s s i v e fat
s t o r a g e w e r e u s u a l l y a r r a n g e d a r o u n d afferent b l o o d vessels. Liver p a r e n c h y m a surFig. 1. Liver tissue with limited region of fatty vacuolation in hepatocytes (paraffin 5.0 ~m;
hematoxylin-eosin; × 270)
Fig. 2. Hepatic melano-macrophage centre with homogeneous, vesicular structure of phagocytic
vacuoles (paraffin 5.0 ~m; hematoxylin; x 680)
Fig. 3. Initial stage of tumorous growth with megalocytic hepatocytes (paraffin 5.0 ~m; hematoxylineosin; × 270)
Fig. 4. Border of neoplastic liver nodule. Melano-macrophage centres in the surrounding tissue,
which is forced aside, are indicated by arrows (paraffin 5.0 ~tm; hematoxylin-eosin; x 195)
~L
©
O
Patchy livers of fish _-->17 cm
Nodular livers (fish > 17 cm)
Liversw.s. of fish _-->17cm
Livers w. s. of fish < 17 cm
Nodular spleens (fish >17 cm)
Spleens w.s. of fish -->_17 cm
S p l e e n s w . s . of fish < 1 7 cm
Organs
84 (19)
100 (27)
64(14)
7(14)
I00 (15)
100 (14)
100(11)
(%)
MMC
occurrence
rate
91 (11)
83 (23)
100 (9)
n.d.
7 (14)
8 (13)
0 (8)
18 (11)
13 {23)
33 (9)
n.d.
14 (14)
38 (13)
25 (8)
0 (11)
4 (23)
0 (9)
n.d,
29 (14)
23 (13)
0 (8)
MMCs
MMCs
MMCs
situated surrounded encapsunear
by
latedby
blood lymphocyte fibrous
vessels
collars
cells
(%)
(%)
(%)
100/0 (11)
83/17 (23)
100/0 (9)
n.d.
0/100 (14)
0/100 (13)
25/75 (8)
9 (11)
9 (23)
33 (9)
n,d,
21 (14)
23 (13)
13 (8)
44 (16)
23 (22)
50 (8)
n,d.
92 (12)
100 (12)
63 (8)
64/36 (11)
48/52 (23)
45/55 (9)
n.d,
0/100 (14)
0/I00 (13)
0/I00 (8)
Yellow/ Presence of Presence
Hom./
brown
black
of
friable
colouration m e l a n i n
ferric
structure of
of
in
iron
vacuolesin
MMCs
MMCs
MMCs
(%1
(%)
(%)
{%)
72 -- 7 (11)
75_+ 6(23)
58 ± 7 (9)
n.d.
174 --+ 17 (14)
129 _+ 18 (13)
84 -k--19 (8)
~ _+ s~of
maximum
diameter of
MMCs
(~m)
238 -- 23 (II)
221_+24{23)
342 ___59 (9)
n.d.
154 "4- 9 (14)
200 -+ 24 (13)
195 -4- 14 (8)
~ _+ s~of
minimum
distance
b e t w e e n centres
of MMCs {~m)
T a b l e 1. C h a r a c t e r i s t i c s of m e l a n o - m a c r o p h a g e
c e n t r e s ( M M C s ) i n p a t h o l o g i c a l l y a l t e r e d a n d s e e m i n g l y n o r m a l l i v e r a n d s p l e e n of ruffe.
P e r c e n t a g e s r e f e r t o o r g a n s n o t to s i n g l e c e n t r e s ; n u m b e r s i n p a r e n t h e s e s r e p r e s e n t o r g a n s (resp. f i s h e s ) e x a m i n e d ; w . s. = w i t h o u t s i g n s of
d i s e a s e ; h o r n . ~ h o m o g e n e o u s ; n. d. = n o t d e t e r m i n e d
~3
Z
tq
M e l a n o - m a c r o p h a g e centres
419
r o u n d i n g these r e g i o n s often s h o w e d slight signs of d e g e n e r a t i o n , such as dissociation of
the l o b u l a r structure, vascular congestion, a n d s o m e t i m e s also slightly necrotic processes. S o m e w h a t l i m i t e d areas of cells filled with fatty s u b s t a n c e s w e r e often s u r r o u n d e d
by increased n u m b e r s of e n l a r g e d m e l a n o - m a c r o p h a g e centres (Table 1). T h e colour of
the m e l a n i n was light yellow i n all cases, a n d i n c l u s i o n s of b l a c k m e l a n i n seldom
occurred (Fig. 2). The structure of the m a c r o p h a g e phagocytic vacuoles i n the majority of
patchy livers was vesicular or h o m o g e n e o u s . H i s t o c h e m i c a l i n v e s t i g a t i o n s r e v e a l e d that
lipoids p l a y e d the same histological role as in the hepatocytes. Ferric iron was only
d e m o n s t r a t e d to occur in m e l a n o - m a c r o p h a g e centres of less t h a n half of these livers.
The reaction to detect it was weak, a n d it n e v e r a c c u m u l a t e d i n v a c u o l e s b u t r e m a i n e d i n
the cytoplasm or s u r r o u n d e d the outside of the m a c r o p h a g e s .
A second pathological c o n d i t i o n in the liver of the ruffe is the d e v e l o p m e n t of
tumorous nodules. D u r i n g their initial stage, they could be d i a g n o s e d as pale glassy
spots on the organ surface that d e v e l o p into wart-like structures, sometimes with a
papilliform surface. This disease, w h i c h occurred i n 8 % of the entire ruffe p o p u l a t i o n ,
was restricted to larger s p e c i m e n s w h i c h h a d lived in c o n t a m i n a t e d water for at least
four years. Histologically, the liver n o d u l e s d e v e l o p from s m a l l groups of e n l a r g e d
basophilic cells (Fig. 3). D u r i n g process of a d v a n c e d proliferation, the s u r r o u n d i n g liver
tissue is forced aside. The cell structure w i t h i n the n o d u l e gives the i m p r e s s i o n of b e i n g
uniform a n d healthy, but the n u c l e u s to cytoplasm ratios are slightly increased, a n d
larger blood vessels are a b s e n t from the interior. W h e n the d i a m e t e r of the growth r e g i o n
exceeds about 5 ram, the blood supply b e c o m e s insufficient, a n d necrotic processes
b e g i n in the centre. In the vicinity of these n e o p l a s t i c liver nodules, m e l a n o - m a c r o p h a g e
centres were always found to h a v e i n c r e a s e d in n u m b e r a n d size (Fig. 4). T h e cotour of
the m e l a n i n was u s u a l l y light y e l l o w (Table 1), b u t i n some cases, b r o w n p i g m e n t e d
m a c r o p h a g e s also occurred. S i m u l t a n e o u s l y , the p e r c e n t a g e of livers with v e s i c u l a r or
h o m o g e n e o u s p h a g o s o m e s i n the centres decreased. In c o m p a r i s o n to patchy livers
resulting from fat a c c u m u l a t i o n , the a b u n d a n c e of organs with iron storage was decreased.
The third pathological c o n d i t i o n affects the spleen. In 10 % of the ruffe from the Elbe
Estuary, opalescent or whitish spots a n d e v e n p r o t r u d i n g warts could b e i d e n t i f i e d on the
n o r m a l l y dark red organ. Just as in the case of the liver n o d u l e s , these s p l e n i c n o d e s
were restricted to fishes i n h i g h e r l e n g t h classes (Table 2). As l o n g as the spots are still
relatively small, several of them c a n occur i n the s a m e organ. I n d i v i d u a l spots e n l a r g e
a n d m e r g e into each other. Histologically, these s p l e n i c n o d u l e s p r o v e d to b e i m m e n s e l y
proliferated fibrous capsules of capillaries, w h i c h i n h e a l t h y s p l e e n form the areas called
ellipsoids (Fig. 5). If this proliferated tissue, the origin of w h i c h b e l o n g s to the reticuloendothelial system, m i g h t still be classified as vascular e p i t h e l i u m , the tumorous
condition c a n be called a n a n g i o m a , U s i n g the light microscope, no p h a g o c y t o s e d
substances could b e o b s e r v e d in the proliferated ellipsoid cells. In the vicinity of the
Table 2 Relationship between splenic nodules and fish length
Fish length (cm)
10 11 12 13 14 15 16 17 18 19 20 21 22 23 >23
Frequency of nodules (%) 0 0 0 0 0 0 0 3.2 14.3 8.9 12.5 25.3 20.5 8.3
0
Number offish examined 5 3 23 29 50 46 28 31 35 56 96 91 44 12
2
D
7
Melano-macrophage centres
421
a n g i o m a , h o w e v e r , i n c r e a s e d n u m b e r s of e n l a r g e d m e l a n o - m a c r o p h a g e c e n t r e s w e r e
found (Table 1). In contrast to the l i v e r centres, the colour of m a c r o p h a g e s in the n o d u l a r
s p l e e n w a s a l w a y s d a r k brown, a n d in s o m e cases, a f e w i n c l u s i o n s c o n t a i n e d b l a c k
m e l a n i n . Thus, the structure of m a c r o p h a g e v a c u o l e s w a s n e v e r h o m o g e n e o u s or
vesicular, but r a t h e r coarse g r a i n e d or friable (Fig. 6). T h e m e l a n o - m a c r o p h a g e c e n t r e s
of most of t h e s p l e e n s s h o w e d a strong p o s i t i v e r e a c t i o n for ferric iron. In a f e w cases
only, the m e l a n o - m a c r o p h a g e c e n t r e s w e r e s u r r o u n d e d by a collar of l y m p h o c y t e s , b u t
almost o n e third s e e m e d to b e n e a r l y e n c a p s u l a t e d by fibrous cells.
T h e f o l l o w i n g s t a t e m e n t can b e m a d e a b o u t g e n e r a l d i f f e r e n c e s b e t w e e n m e l a n o m a c r o p h a g e centres in the liver a n d s p l e e n of ruffe s h o w i n g no o b v i o u s h i s t o p a t h o l o g i cal alterations (Table 1). M e l a n o - m a c r o p h a g e c e n t r e s s e l d o m o c c u r r e d in livers of s m a l l
fishes b e t w e e n 10 a n d 17 cm, b u t t h e y did a l w a y s occur in the spleen. W i t h o u t exception,
m e l a n o - m a c r o p h a g e centres w e r e m o r e a b u n d a n t a n d l a r g e r in the s p l e e n t h a n in the
liver of the s a m e fish, e v e n in cases w h e n the liver w a s p a t h o l o g i c a l l y a l t e r e d a n d the
s p l e e n s h o w e d no sign of a p a t h o l o g i c a l reaction. T h e g r e a t e s t p e r c e n t a g e of the h e p a t i c
m e l a n o - m a c r o p h a g e c e n t r e s w a s s i t u a t e d n e a r b l o o d vessels, w h e r e a s in the spleen,
m e l a n o - m a c r o p h a g e s a g g r e g a t e d w i t h i n the p a r e n c h y m a . T h e colour of m a c r o p h a g e s
was u s u a l l y y e l l o w in the l i v e r a n d b r o w n in s p l e e n , w h i c h c o r r e s p o n d s to their v a c u o l a r
structure: often h o m o g e n e o u s in the l i v e r a n d a l w a y s c o a r s e - g r a i n e d or friable in t h e
s p l e e n (Figs 7 a n d 8). Black m e l a n i n in t h e m e l a n o - m a c r o p h a g e c e n t r e s a n d l y m p h o c y t e
collars a r o u n d t h e m w e r e not v e r y a b u n d a n t in e i t h e r o r g a n of t h e ruffe, b u t t h e y w e r e
found s o m e w h a t m o r e often in the s p l e e n t h a n in t h e liver. F u r t h e r m o r e , t h e a c c u m u l a tion of iron s e e m s to b e a c h a r a c t e r i s t i c f e a t u r e of s p l e n i c m e l a n o - m a c r o p h a g e c e n t r e s
but not of those in t h e liver.
DISCUSSION
M e l a n o - m a c r o p h a g e c e n t r e s are m o r e a b u n d a n t in livers a n d s p l e e n s of ruffe w h e n
they are afflicted with various p a t h o l o g i c a l conditions. T h e y are also rather a b u n d a n t in
s e e m i n g l y n o r m a l livers and s p l e e n s of ruffe from the p o l l u t e d E l b e River. T h e functions
attributed to m e l a n o - m a c r o p h a g e c e n t r e s b a s e d m a i n l y on studies of those in the spleen,
i n c l u d e special i m m u n e r e s p o n s e s (Ellis & Sousa, 1974), b a c t e r i o c i d a l p r o p e r t i e s attrib u t e d to b l a c k m e l a n i n (Roberts, 1975), tissue c a t a b o l i s m e x p e r i m e n t a l l y s h o w n d u r i n g
c o m p l e t e starvation of fishes (Agius & Roberts, 1981), p h a g o c y t o s i s of f o r e i g n p a r t i c l e s in
the blood a n d l y m p h systems (Ellis et al., 1976), a n d s t o r a g e of iron r e l e a s e d by
destruction of e x h a u s t e d erythrocytes (Agius, 1979). In t h e s p l e e n of ruffe from the E l b e
River, b l a c k m e l a n i n s e l d o m a c c u m u l a t e s in the m e l a n o - m a c r o p h a g e centres, w h i c h are
Fig~ 5. Enlarged melano-macrophage centres in the vicinity of a splenic nodule. The proliferated
tissue in the lower part of photography consist of fibrous cells and erythrocytes (paraffin 5,0 ~m;
hematoxylin-eosin; x 195)
Fig. 6. Splenic melano-macrophage centre with darker vacuoles in contrast to Figure 2. Ellipsoids in
this normal splenic tissue are indicated by arrows (paraffin 5.0 ~m; hematoxylin-eosin; x 270)
Fig. 7. Hepatic melano-macrophage centre. Phagocytosed materials consist of mainly lipoid substances displaying a homogeneous structure (Epon 1.0 ~tm; toluidine blue; x 680)
Fig. 8. Border of splenic melano-macrophage centre. Inclusions of macrophages show granular to
friable structures (Epon 1.0 p~m; toluidine blue; X 680)
422
H. Kranz & N. Peters
only rarely s u r r o u n d e d b y a collar of lymphocytes. Because of these observations, it is
a s s u m e d that bacteriocidal a n d other i m m u n o l o g i c a l processes are not the m a i n functions of m a c r o p h a g e centres i n ruffe spleen. O n the other hand, most of the splenic
centres s t a i n positively for iron, w h i c h i n this s t u d y was h i s t o c h e m i c a l l y d e t e r m i n e d as
h a e m o s i d e r i n . This leads to the c o n c l u s i o n that the m a i n function of s p l e n i c m e l a n o m a c r o p h a g e centres is the d e c o m p o s i t i o n of blood cells.
A special r e l a t i o n s h i p seems to exist b e t w e e n tumorous n o d u l e s of ruffe s p l e e n and
a n i n c r e a s e in n u m b e r a n d size of s p l e n i c m e l a n o - m a c r o p h a g e centres. T h e investigations of Ellis (1974) a n d F e r g u s o n (1976) o n the r e t i c u l o - e n d o t h e l i a l system of flatfish
s h o w e d that m e l a n o - m a c r o p h a g e centres i n the s p l e e n originate from cells of the
ellipsoids. M a c r o p h a g e s filled with i n g e s t e d s u b s t a n c e s from the blood circulation
m i g r a t e from the area a r o u n d the arterial capillaries to the white or red pulp of the
spleen, w h e r e they form a g g r e g a t e s of m e l a n o - m a c r o p h a g e s . That m e a n s that the
s p l e n i c a n g i o m a s of ruffe, w h i c h consist of h i g h l y proliferated ellipsoid ceils, might be a
t u m o r c o n s i s t i n g of precursor ceils of m e l a n o - m a c r o p h a g e s . In most tumorous spleens,
m e l a n o - m a c r o p h a g e centres c o n t a i n h a e m o s i d e r i n . O n l y those in the i m m e d i a t e vicinity
of the n o d u l e s s o m e t i m e s lack t r i v a l e n t iron, e v e n if they c o n t a i n dark b r o w n p i g m e n t .
T h e i r p h a g o c y t i c activities could b e directed a g a i n s t the t u m o r cells.
M e l a n o - m a c r o p h a g e centres i n the liver of the ruffe have different characteristics.
T h e y are g e n e r a l l y less a b u n d a n t a n d s m a l l e r t h a n i n the spleen, e v e n w h e n their
n u m b e r a n d size h a v e b e e n i n c r e a s e d d u e to p a t c h y pathological fat a c c u m u l a t i o n a n d
d u r i n g d e v e l o p m e n t of n e o p l a s t i c h e p a t i c nodules. I n c r e a s e d a b u n d a n c e of m e l a n o m a c r o p h a g e centres i n fish liver has b e e n reported to b e a s y m p t o m of several diseases.
Elarifi (1982) s t u d i e d the d e v e l o p m e n t of h e p a t i c m e l a n o - m a c r o p h a g e centres after
n e m a t o d e s h a d infested the organ. Smith (1979) m e n t i o n e d a n increase of m e l a n o m a c r o p h a g e s i n s a l m o n suffering from lipoid liver d e g e n e r a t i o n , a n d Pierce et al. (1978)
stated that m e l a n o - m a c r o p h a g e centres i n c r e a s e d i n a b u n d a n c e w h e n h e p a t o m a s occurred i n the livers of Parophrys vetulus.
A n i n c r e a s e i n the a b u n d a n c e of h e p a t i c m e l a n o - m a c r o p h a g e centres has also b e e n
recorded w h e n the water was polluted. A g a i n Pierce et al. (1980) reported that the starry
f l o u n d e r (Platichthys stellatus) from the p o l l u t e d D u w a m i s h River h a d more hepatic
m e l a n o - m a c r o p h a g e centres t h a n those from u n p o l l u t e d reference areas. H a e n s l y et al.
(1982) f o u n d that one l o n g - t e r m effect of the "Amoco Cadiz" crude oil spill was a n
i n c r e a s e i n the n u m b e r of m e l a n o - m a c r o p h a g e centres in livers of plaice from highly
c o n t a m i n a t e d areas.
T h e differences i n structure a n d colour of m e l a n o - m a c r o p h a g e centres indicate
dissimilarities i n their functions i n s p l e e n a n d liver of ruffe. As i n the splenic centres,
b l a c k m e l a n i n a n d l y m p h o c y t e s are only occasionally a c c u m u l a t e d in a n d a r o u n d
h e p a t i c m e l a n o - m a c r o p h a g e centres. This is e v i d e n c e a g a i n s t a bacteriocidal function. If
h e p a t i c centres stain positively for iron, w h i c h occurred i n a m i n o r i t y of the organs
e x a m i n e d , the reaction is very weak, a n d s o m e t i m e s only s i n g l e m a c r o p h a g e s c o n t a i n
trace a m o u n t s of iron. Therefore, it is a s s u m e d that d e c o m p o s i t i o n of blood cells is not
the m a i n f u n c t i o n of h e p a t i c m e l a n o - m a c r o p h a g e centres. Most of the m a c r o p h a g e
centres in both d i s e a s e d a n d n o r m a l livers are p a l e yellow. In m a n y cases, the phagocytic v a c u o l e s show a v e s i c u l a r or h o m o g e n e o u s structure i n light microscope preparations. Histochemically, they w e r e s h o w n to c o n t a i n fatty substances. Such lipoid-filled
Melano-macrophage
centres
423
m a c r o p h a g e s d o n o t a p p e a r o n l y i n l i v e r s w h i c h s h o w s i g n s of f a t t y d e g e n e r a t i o n i n
s u r r o u n d i n g h e p a t o c y t e s a n d m a n y of t h o s e w i t h t u m o r o u s g r o w t h , b u t a l s o i n t h o s e
w i t h o u t a n y s i g n s of p a t h o l o g y . P i e r c e e t al. (1978) r e p o r t e d s i m i l a r v a c u o l a t i o n t o o c c u r
i n r n e l a n o - m a c r o p h a g e c e n t r e s of o r g a n s w i t h h e p a t o c e l l u l a r f a t a c c u m u l a t i o n i n f i s h e s
f r o m t h e p o l l u t e d D u w a m i s h River. L i p o i d d e g e n e r a t i o n is a h i s t o c h e m i c a l r e s p o n s e t o
s p e c i a l t o x i c a n t s , a n d fat s o l u b l e t o x i c a n t s a r e o f t e n d e m o n s t r a t e d to b e p a r t i c u l a r l y
a b u n d a n t i n t h e l i v e r . T o x i c c h e m i c a l s c a n t h e r e f o r e b e s u s p e c t e d to c o n c e n t r a t e i n t h e
melano-macrophage centres and thus be eliminated from metabolic systems.
I n 1980, A g i u s s h o w e d t h a t a t d i f f e r e n t p h y l o g e n e t i c l e v e l s , t h e l i v e r a n d s p l e e n
change their relative importance in developing melano-macrophage
centres. In the
e v o l u t i o n f r o m A g n a t h a to C h o n d r i c h t h y e s a n d o n to a d v a n c e d T e l e o s t e i , t h e l i v e r l o s e s
a n d t h e k i d n e y a n d s p l e e n g a i n i m p o r t a n c e a s s i t e s of m a c r o p h a g e c e n t r e s . T h i s
d e v e l o p m e n t p r o c e e d s p a r a l l e l to t h e e v o l u t i o n of t h e l y m p h o i d a n d r e t i c u l o - e n d o t h e l i a l
systems in fishes.
I n t h i s i n v e s t i g a t i o n , it w a s c o n f i r m e d t h a t t h e s p l e e n is t h e m a i n o r g a n for
producing melano-macrophage
c e n t r e s . U n d e r c o n d i t i o n s of p o l l u t i o n , f o l l o w e d b y
certain histopathological reactions, familiar immunological functions in these centres
a r e c o m p l e t e d i n t h e s p l e e n , a n d s e e m to b e c o n v e r t e d i n t h e l i v e r : w h i l e s p l e n i c m a c r o p h a g e c e n t r e s a r e s p e c i a l i z e d for d e c o m p o s i t i o n of p o s s i b l y i n c r e a s e d r a t e s of e f f e t e
b l o o d cells, t h o s e i n t h e l i v e r s e e m to h a v e t a k e n o n i m p o r t a n c e for s t o r i n g a n d
eventually eliminating toxic substances.
Acknowledgements. The authors wish to t h a n k Dr. G. Peters, W. Schmidt, M. B r u m m a n d J.
Gercken for helpful assistance d u r i n g p r e p a r a t i o n of ruffe. This investigation was supported b y
Forschungsbereich " U m w e l t f o r s c h u n g u n d U m w e l t g e s t a l t u n g ' , Universit~it H a m b u r g .
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