ARTICLE IN PRESS

Acta histochemica 109 (2007) 164—168

www.elsevier.de/acthis

SHORT COMMUNICATION

Melano-macrophage centres and endocytic cells in

kidney and spleen of pearl gouramy and platyfish
(Anabantidae, Poeciliidae: Teleostei)
Ingvar Leiv Leknes

Faculty of Engineering and Science, Sogn og Fjordane University College, P.O. Box 133, N-6851 Sogndal, Norway

Received 1 May 2006; received in revised form 28 September 2006; accepted 8 October 2006

KEYWORDS Summary
Lipofuscin;
The structure and putative cellular content of melano-macrophage centres (MMCs)
Haemosiderin;
and single macrophages in kidney and spleen of two teleosts, pearl gouramy
Melano-macro-
Trichogaster leeri (Bleeker) and platyfish Xiphophorus maculatus (Günther), are
phages;
described and compared. The MMCs were confined to the haemopoetic tissue in both
Pearl gouramy;
species, and were markedly more prominent in pearl gouramy than in platyfish. They
Platyfish
were rich in iron-compounds, probably haemosiderin, in the former species, whereas
they contained mainly iron-free pigments, probably lipofuscin, in the latter species.
We suggest that the kidney and spleen MMCs are involved in the regular storage,
relocation and recycling of iron-compounds of effete or damaged red blood cells
in healthy pearl gouramies, whereas they function more or less as a deposit for
undegradable and potentially harmful iron-free cell debris in the corresponding
organs in healthy platies. Numerous single macrophages took up much intraper-
itoneally injected horse ferritin in the kidney and spleen haemopoetic tissue in
platies, whereas these cells contain much iron-containing pigment, probably
haemosiderin, in the control pearl gouramies. We suggest that these cells are able
to play a role in cleansing the circulation of foreign macromolecules and particles in
platies, whereas they are mainly involved in the regular uptake of scavenger iron-
containing compounds in pearl gouramies.
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Introduction teleosts differ between specimens, species and

The morphological appearance, size and cellular
contents of melano-macrophage centres (MMCs) in
E-mail address: ingvar.leknes@hisf.no.
organs (Wolke, 1992; Meseguer et al.,1994;
Haaparanta et al., 1996; Agius and Roberts,
2003). It is still uncertain whether this variation
reflects age, health status or stressful conditions
like starvation or damaging substances in the water

0065-1281/$ - see front matter & 2006 Elsevier GmbH. All rights reserved.
doi:10.1016/j.acthis.2006.10.003
 ARTICLE IN PRESS
Macrophages in kidney and spleen of pearl gouramy and platyfish
(Agius and Roberts, 2003; Hur et al., 2006; Fish-
elson, 2006). Usually, the macrophages in these
centres contain mainly lipofuscin and similar iron-
free pigments in healthy teleosts (Herraez and
Zapata, 1991; Ravaglia and Maggese, 1995; Lamas
et al., 1996; Agius and Roberts, 2003). However, in
sea bass (Dicentrarchus labrax) these cells are
filled mainly by melanin (Meseguer et al., 1994).
The MMCs in bony fish may also contain some iron-
containing pigments, probably haemosiderin, par-
ticularly in the spleen after haemolytic anaemia or
prolonged starvation (Agius 1981; Herraez and
Zapata, 1991; Roberts, 2001; Agius and Roberts,
2003). In the present work, we aimed to reveal
the dominant and species-specific pigment type
in kidney and spleen MMCs in species from two
teleost families, pearl gouramy Trichogaster leeri
(Bleeker) and platyfish, Xiphophorus maculatus
(Günther). When this information is established,
these species may be used in further studies to
reveal any species-specific change in the cellular
pigment content of MMCs in response to stressful
conditions, e.g. chemical exposure in polluted
environments, as well as various pathological
conditions. In addition, this study aimed to inves-
tigate a possible blood cleansing role of single
macrophages in kidney and spleen of pearl gouramy
and platyfish.
Material and methods
Twenty-five healthy specimens of pearl gouramy
T. leeri (Bleeker), 1–2 years old, approx. 1.1 g in
weight and 4–5 cm in length, and 25 healthy
specimens of platyfish X. maculatus (Günther),
1–2 years old, approx. 1.5 g in weight and 5–6 cm
in length, were studied. They were kept in well-
aerated glass aquaria (30  35  60 cm3) at 26 1C for
gourmies and 21 1C for platies, and regularly fed
with TetraMin (Tetra Werke, Melle, Germany). The
aquarium water, pH 6–7, contained 0 mg/l nitrite
and ammonium, and up to 0.3 mg/l nitrate. Two-
thirds of the water was exchanged each month with
clean tap water. Twenty specimens of each species
were killed using an overdose of chlorobutanol.
Kidney and spleen were quickly excised and fixed at
4 1C in 4% formaldehyde (made from paraformalde-
hyde within 24 h before use), in phosphate buffer,
pH 7.4 (Leknes, 1980). After washing in buffer, the
tissues were dehydrated through a graded ethanol
series, treated with xylene, embedded in paraffin
wax and 4 mm-thick sections were cut. Dewaxed
and rehydrated sections were then stained using
three different approaches, as follows. (1) stained
in Mayer’s hematoxylin/eosin, neutral red/eosin,
165
toludine blue, Heidenhain’s Azan or Mallory solu-
tions (Grimstone and Skaer, 1972). (2) incubated for
1 min in Schmorl’s solution, composed of 75 ml 1%
ferric chloride, 10 ml 1% potassium ferriccyanide
and 15 ml distilled water, in order to visualize
lipofuscin, and treated with 1% aqueous solution of
neutral red followed by 1% aqueous solution of
eosin (Pearse, 1980). (3) incubated for 15 min in an
acid ferrocyanide solution, made by dissolving 2 g
potassium ferrocyanide in 100 ml 0.75 mol/l hydro-
chloric acid solution, in order to visualize haemo-
siderin iron ions, and treated with 1% aqueous
solution of neutral red followed by 1% aqueous
solution of eosin (Pearse, 1980). Finally, all sections
were dedydrated through a graded ethanol series
and mounted under coverslips in Mountex medium
(Sigma, St. Louis, Mo., USA).
Five specimens of each species were injected
carefully with 0.02–0.03 ml of a 10% solution of
horse-spleen ferritin (Sigma) into the peritoneal
cavity by means of 0.5 ml tuberculin syringes
(Becton Dickinson and Company, New Jersey, NJ,
USA). After 10 or 24 h, fish were killed with an
overdose of chlorobutanol. Kidney and spleen were
fixed, dehydrated and embedded as described
above. Dewaxed sections were incubated with an
acid ferrocyanide solution composed as detailed
above, in order to visualize ferritin iron ions, and
then dehydrated and mounted as before.
All experiments were carried out in accordance
with established national ethical guidelines,
rules and legislations for this type of experiment
(cf. Leknes, 2004).
Results
MMCs were large and numerous in kidney and
spleen of pearl gouramy, whereas they were usually
smaller and less frequently seen in the platyfish
kidney and spleen (Fig. 1a–g). They occurred
mainly in the haemopoietic tissue of these
organs, in close proximity to the vascular system,
i.e. sinusoids in kidney and spleen and ellipsoids in
spleen (Fig. 1a and b). In pearl gouramy, the MMCs
were composed of tightly packed cells and were
normally encapsulated by a thin cellular layer,
whereas they contained more dispersed cells and
were often unencapsulated in platies (Fig. 1a and
d). Macroscopically, MMCs appeared as distinct
yellow-brown spots on the surface of the entire
kidney in pearl gouramies.
The MMCs cells were packed with a dense
pigment, which was grey-yellow in platyfish and
markedly yellow-brown in pearl gouramy. The
pigment displayed no affitinity to either the
 ARTICLE IN PRESS
166 I.L. Leknes

cationic stain toluidine blue or the anionic stain Nearly all cells in the MMCs of kidney and spleen
eosin (Fig. 1 c and f). Some dark, melanin-like from pearl gourmies were tightly packed by deep
granules regularly occurred in these cells, particu- blue precipitates in tissue treated with acid
larly in kidney of platies. ferrocyanide, whereas they displayed a markedly
 ARTICLE IN PRESS
Macrophages in kidney and spleen of pearl gouramy and platyfish 167

fainter and mainly blue-green colour in tissue
treated with Schmorl’s solution (Fig. 1 d, e and
g). In platies, few or none of the MMCs contained
blue precipitates in the ferrocyanide-treated tissue
either in kidney or in spleen, whereas nearly all of
them displayed a deep blue colour when treated
with the Schmorl’s stain (Fig. 1a–c). Some of the
kidney and spleen single macrophages displayed a
similar staining reaction as the surrounding MMCs
(Fig. 1a and b).
In ferritin-injected platies, large numbers of
single macrophages were filled by deep blue
precipitates in kidney and spleen incubated with
acid ferrocyanide, whereas the corresponding
macrophages containing no such precipitates in
the control tissues (Fig. 1c and h). In kidney and
spleen from ferritin-injected pearl gouramies,
numerous macrophages displayed deep blue pre-
cipitates in tissue incubated with acid ferrocya-
nide, but a number of blue-stained cells were also
noted in the control tissues from this species
(Fig. 1e and i).
Discussion
The macrophages in teleosts take up scavenger
molecules, worn-out or damaged cells, coagulated
blood and damaged connective tissue fibrils, i.e.
these cells have a regular uptake of various
scavenger materials and act to clean up the tissue
after infection and mechanical damage (Wolke,
1992; Agius and Roberts, 2003). Such materials are
usually degraded within the macrophage vacuoles
and re-used in the synthesis of new substances.
Undegradable tissue debris may gradually accumu-
late in these cells (Wolke, 1992; Meseguer
et al.,1994; Agius and Roberts, 2003; Passantio
et al., 2005). The latter material appears as dense,
grey or faintly yellow macrophage pigment termed
lipofuscin, which is suggested to be composed
mainly of polyunsaturated fatty acids denaturated
by strongly oxidative substances (Agius and
Roberts, 2003). The present study shows that the
platyfish kidney and spleen macrophages—singly or
in MMCs—react strongly with Schmorl’s stain for
lipofuscin, and very faintly or not at all with acid
ferrocyanide for haemosiderin (Pearse, 1980).
Thus, these cells contain large amounts of iron-
free pigments, probably lipofuscin, i.e. they may
play a significant role as a deposit for undegrad-
able, denaturated lipoproteins, whereas they seem
to play no significant role in the handling of iron-
containing pigments like haemosiderin in healthy
platies. In pearl gouramy, however, the situation is
quite different: the MMCs and numerous single
macrophages in kidney and spleen react strongly
with acid ferrocyanide and usually weakly with
Schmorl’s stain. This observation suggests that
these cells are normally involved in the regular
storage, relocation and recycling of iron-com-
pounds, probably from effete or damaged red
blood cells; whereas they may play no significant
role as a deposit for lipofuscin in healthy pearl
gouramies. The MMCs in the platyfish liver often
contain both haemosiderin and lipofuscin-like pig-
ments, i.e. these liver structures may play a role in
the recycling of iron-compounds in this species
(Leknes, 2004). Recent studies in our laboratory
have shown, however, that MMCs in the liver in
healthty pearl gouramies often contained signifi-
cantly more iron-free pigments, probably lipofus-
cin, than iron-containing pigments. We suggest that
the storage and recycling of the blood iron-
compounds are maintained mainly by the kidney
and spleen in this species.
Iron-recycling functions have previously been
suggested for splenic macrophages in several
teleosts (Gabrielle and McMillan, 1984; Agius,
1985; Herraez and Zapata, 1991). The close spatial
relationship between macrophages and the vascu-
lar system in the kidney and spleen may enhance
the uptake of scavengers from the blood stream in
both species, i.e. mainly iron-containing cell debris
in pearl gourmies and mainly iron-free cell debris in
platies.
Numerous single macrophages associated with
the kidney and spleen vascular system may play a
significant role in the clearance of scavenger and

Figure 1. (a, b) Melano-macrophage centres (MMCs) (arrows) in kidney and spleen, respectively, of platyfish,
X. maculatus, located in close proximity to a venule in kidney and ellipsoid in spleen. The tissues were treated with
Schmorl’s solution. A single macrophage displaying the same staining reaction as the MMCs is shown in both
micrographs. (c) MMC (arrow) treated with acid ferrocyanide in the spleen of platyfish. (d, e) MMCs (arrows) in spleen
and kidney, respectively, of pearl gouramy, T. leeri, the tissues were treated with Schmorl’s solution or acid
ferrocyanide, respectively. (f, g) MMCs (arrows) in kidney and spleen, respectively, of pearl gouramy, the tissue in (g)
was treated with Schmorl’s solution. (h, i) Numerous single macrophages, tightly packed by Prussian blue precipitates,
in kidney of platyfish and spleen of pearl gouramy, respectively; the tissues were treated with acid ferrocyanide and are
from specimens injected intraperitoneally with horse-spleen ferritin 10 and 24 h before sacrifice, respectively. Staining
method (a–i): Neutral red followed by eosin. Scale bar: 20 mm.
 ARTICLE IN PRESS
168
foreign substances from the circulation in teleosts
(Tsujii and Seno, 1990; Smedsrød et al. 1993;
Meseguer et al. 1994; Dalmo et al., 1996; Press
and Evensen, 1999). The present study reveals that
large numbers of single macrophages are capable of
taking up intraperitoneally injected horse-ferritin
in the kidney and spleen haemopoetic tissue in
platyfish. We suggest that the macrophages in
kidney and spleen in platies are able to play a role
in the cleansing of the circulation of foreign
macromolecules and particles, whereas most of
these cells are mainly involved in the regular
uptake of scavenger iron-containing compounds in
pearl gouramies.
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