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Journal of Fish Biology (2005) 66, 729­740 doi:10.1111/j.1095-8649.2005.00635.x, available online at http://www.blackwell-synergy.com

Histology and histochemical enzyme-staining patterns of major immune organs in Epinephelus malabaricus

H.-T. L I N *, H.-Y. L I N

AND

H.-L. Y A N G

*Graduate Institute of Life Sciences, National Defense Medical Center, No. 161, Section 6, Min-Chuan East Road, Taipei 114, Taiwan and Institute of Biotechnology, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan (Received 17 February 2004, Accepted 15 November 2004)

The histological architecture of major immune organs in the Malabar grouper Epinephelus malabaricus was investigated. The novel characteristics such as melanomacrophage centres (MMCs) appeared in the thymus and lymphopoietic tissue formed as foci in the head kidney. Leukocyte distribution in organs was identified by enzyme histochemistry. b-glucuronidase (BG) reactive cells in the cortex region of the thymus were botryoidally aggregated. Both acid phosphatase (AcP) and BG reactive cells concentrated within the specialized lymphopoietic foci in the head kidney, suggesting that the foci might be functional. Primitive histological characters of immunity were observed in the spleen. Although leukocyte aggregation was demonstrated in the spleen, additional enzyme histochemistry indicated that the aggregate might not be the equivalent of white pulp found in other vertebrates. The histological evidence did not support intestinal involvement in the immune system: there was no demonstrable gut associated lymphoid tissue. The limited distribution in the cortex and medulla boundary and the condensed format of the lymphocytes suggests a functional role for the MMC in the thymus of E. malabaricus. The MMC appearance in the thymus of a teleost was # 2005 The Fisheries Society of the British Isles unusual. Key words: enzyme histological staining; histology; immune organs; Malabar grouper; melanomacrophage centres.

INTRODUCTION The adaptive immune system of several teleosts has been explored by either histology or molecular analysis (Agustin et al., 1996; Press & Evensen, 1999). Most of the knowledge about the immune system of teleosts comes from a few species, principally cold water species that are of economic importance such as Atlantic salmon Salmo salar L., Atlantic cod Gadus morhua L., rainbow trout Oncorhynchus mykiss (Walbaum), sea bass Dicentrarchus labrax (L.) and sea bream Sparus aurata L. In contrast, very little is known about the immunology of warm water marine fishes. An example is the Malabar grouper Epinephelus malabaricus (Bloch & Schneider). This species is one of the most common

Author to whom correspondence should be addressed. Tel.: þ886 6 2757575 ext 65600; fax: þ886 6 2766505; email: [email protected]

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farmed fishes in Asia. The present study was undertaken to elucidate the histological structure and function of the major immune organs of the Malabar grouper. Enzyme histochemistry, specifically the staining of leukocyte enzymes, has been used commonly in histological analysis to demonstrate the structure and function of immune organs in higher vertebrates and fish species including S. salar (Ellis, 1977; Press et al., 1994), Cyprinus carpio L. (Secombes et al., 1983), Ictalurus punctatus (Rafinesque) (Petrie-Hanson & Ainsworth, 2000), O. mykiss (Razquin et al., 1990) and Platichthys flesus (L.) (Pulsford et al., 1994). The most common target enzymes used to identify cell types and to determine the leukocyte cytochemical profile characteristic of immune organs are acid phosphatase (AcP), a-naphthyl butyrate esterase (NBE) and b-glucuronidase (BG). These standard techniques were used to obtain a broader knowledge of the Malabar grouper's immune system. MATERIALS AND METHODS

FISH SAMPLES

The species used in this study, the Malabar grouper, is the most common of several species of cultured groupers in Taiwan. The broodstock purity of the species is uncertain, perhaps representing a hybrid between E. malabaricus and Epinephelus coicoidase (Hamilton). Several batches of locally farmed Malabar grouper weighing 750­900 g (c. 1 year-old juveniles) were purchased in local markets between July 2002 and April 2003. Fish farmers in southern Taiwan donated larvae and early juveniles (4 month-old). The immune-related organs (thymus, kidney, spleen and intestines) were dissected, non-related tissue removed and the organs studied separately. Samples for paraffin embedding were fixed in 10% formalin for 4 h at ambient temperature (22­26 C), washed twice with deionized water and then stored in 70% ethanol. The protocol of Humason (1970) for dehydration, immersion and embedding was followed. Sections 7 mm in thickness were cut using a model RM2135 microtome (Leica Microsystems, Wetzlar, Germany), stained with haematoxylin and eosin, and observed and photographed under a BX51 system light microscope equipped with a DP70 digital camera (both from Olympus, Tokyo, Japan). Samples intended for frozen sectioning were immersed in Optimal Cutting Temperature Tissue (OTC)-Tek (Sakura Fine Technical Co., Tokyo, Japan), frozen in liquid nitrogen and stored at À70 C. Sections (9 mm) were cut using a model CM 1900 cryostat (Leica Microsystems) and stained as described below. Prior to staining, sections were air-dried and optimal serial sections cut from the same block were chosen. Sections of frozen mouse spleen provided positive controls and a standard to optimize the condition used for staining fish tissue.

E N Z Y M E H I S T O C H E M I C A L S T A I N I N G TO ID E N T I F Y LEUKOCYTES

The mammalian cells reactive for the AcP, NBE and BG marker enzymes are listed in Table I. The enzymes were stained with a Lymphocyte Enzyme Kit (Sigma Chemical Co., St Louis, MO, U.S.A.) after the following modification of the staining procedure. The reagents were warmed to 30 C and mixed sequentially just before staining. For AcP staining, the premixed reagents naphthol AS-BI phosphoric acid solution, sodium nitrate solution and Fast Garnet GBC Base Solution, were mixed sequentially and then put into a staining tank. The slides with sections were air-dried and fixed with a citrate-acetoneformaldehyde (CAF) solution (18 mM citric acid, 9 mM sodium citrate, 12 mM sodium chloride, 65 ml of acetone and 8 ml of 37% formaldehyde, adjusted to pH 3Á6) for 30 s at ambient temperature, rinsed in deionized water for 45 to 60 s, placed into the staining tank immediately and then incubated for 1 h at 30 C. For BG staining, the components

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TABLE I. Staining specificities of mammalian leukocytesa Stain specificity Cell type Monocytes Macrophages Neutrophils T cells B cells AcP þ þ þ þ þ NBE þ À À þ À BG À À À þb þ

þ, reacts; À non-reactive; AcP, acid phosphatase; NBE, a-naphthyl butyrate esterase; BG, b-glucuronidase. a Stained using a Lymphocyte Enzyme Kit [Sigma Diagnostics (R), Sigma Chemical Co.] according to manufacturer's instructions. b Mature thymocytes and circulating T cells were reactive in BG stain.

of the staining reaction solution (consisting primarily of naphthol AS-BI B-D-glucuronic acid, pararosaniline solution and sodium nitrate) were mixed sequentially. Following the manufacturer's instructions, the air-dried slides were fixed with the CAF solution for 30 s at ambient temperature, washed in deionized water for 45 s, then placed in the reaction solution before the slides were dried and incubated in the dark for 90 min at 30 C. For NBE staining, the staining pre-mixture (consisting primarily of a-naphthyl butyrate, pararosanilin and sodium nitrate) were mixed according to the manufacturer's instructions. The air-dried slides were fixed with the CAF solution for 10 s at ambient temperature, washed in deionized water for 45 s, put in the reaction solution before the slides were dried and then incubated in the substrate solution for 60 min at 30 C. Harris modified haematoxylin reagent (Fisher Scientific, Pittsburgh, PA, U.S.A.) was used as the counter stain. After staining, the slides were rinsed for at least 2 min in running tap water, dried in air for at least 5 min and mounted in glycerin-gelatin.

RESULTS

TH YM US

The thymus of the Malabar grouper is located in the opercular cavity, which is positioned at the superior edge of the gill cover on the supracleithrum bone of the pectoral girdle [Fig. 1(a)]. It presents as a pair of auricular, pale pink lobes. The thymus conformed to the general concepts of fish thymus from previous studies of other teleosts (Press & Evensen, 1999); structural characters such as the trabeculae of blood-thymus barrier and the medulla and cortex delineation were observed [Fig. 1(b)]. Epithelial-reticular cells, the basic components that form the meshwork were easily observable in the medulla region. In addition, some specific cell cysts made of epithelial-reticular cells were also apparent [Fig. 1(c)]. Morphologically, these were similar to Hassall's corpuscles described in higher vertebrates (Leeson & Leeson, 1981). Furthermore, the cell cysts were observed in the larva of the Malabar grouper as early as 60 days after hatching (unpubl. data). The melanomacrophage centre (MMC) was detectable in the thymus as determined from the examination of several sections from different individuals

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(b)

(c)

M

C

FIG. 1. The external and histological morphology of the thymus in the 1 year-old Epinephelus malabaricus. (a) Lateral view. Opercular cavity was partially removed to reveal the thymus ( ). (b) Low magnification, sagittal view of the thymus. The anterior of the thymus is to the left. The cortex (C) and medulla (M) regions are labelled and the boundary is defined by the `white line'. Melanomacrophage centres are shown in the thymus ( ). Haematoxylin and eosin stain, bar ¼ 1 mm. (c) A melanomacrophage centre which is darkly pigmented. A Hassall's corpuscle-like cell cyst is indicated ( ). Haematoxylin and eosin stain, bar ¼ 100 mm.

harvested from different batches over a 1 year-period [Fig. 1(b), (c)]. In addition, MMCs were observed in juveniles as early as 4 months-old (unpubl. data). Most of the MMCs were observed in the medulla region reaching the edge of medulla and cortex boundary [Fig. 1(b)]. When observed elsewhere, MMCs presented with a different morphology; epithelial-reticular cells banded some of the MMCs located in the medulla region. In addition, the cell cysts were involved [Fig. 1(c)]. The MMC appeared to be anchored on a limited region of the thymic parenchyma. The AcP staining revealed two leukocytic distribution patterns. In one pattern, enzyme reactive cells were apparent as single cells in the medulla region. In the other pattern, the cells were clustered in the cortex region [Fig. 2(a)]. Staining of BG, but not NBE, revealed the similar cluster pattern in the cortex [Fig. 2(b)­(d)], suggesting that the lymphocytes form clusters. Melanomacrophage centres reacted to all three stains [Fig. 2(e), (f)], consistent with an abundance of immune reactive cells such as macrophages and leukocytes.

KIDN EY S

Morphological observation of the head kidney revealed a functional specialization for haematopoiesis and lymphopoiesis. A characteristic renal structure

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(a)

(b)

C M C M

(c) (d)

C

M

(e)

(f)

FIG. 2. Histochemical staining of thymus enzymes (1 year-old Epinephelus malabaricus). (a) Low magnification of an AcP-stained thymus, bar ¼ 1 mm. (b) Low magnification of a BG-stained thymus, bar ¼ 1 mm. (c) High magnification of BG-reactive botryoidal cells, bar ¼ 100 mm. (d) Low magnification of an NBEstained thymus, bar ¼ 1 mm. (e) A high magnification of the BG-stained melanomacrophage centre, bar ¼ 100 mm. (f) A high magnification of the BG-stained melanomacrophage centre, bar ¼ 100 mm. C, cortex region; M, medulla region; , melanomacrophage centres. Haematoxylin as counterstain.

was absent [Fig. 3(a)]. The distribution of lymphopoietic cells was different from that typically encountered in other fish species. Specifically, the cells tended to concentrate as foci near vessels instead of assuming the more typically encountered random distribution [Fig. 3(a), (b)]. The possibility that the lymphopoietic foci assumed a functional significance was supported by the results of the enzyme biochemical examinations. The foci housed AcP [Fig. 3(c)] and BG [Fig. 3(d)] reactive cells. The NBE reactive cells, however, were not localized to the foci [Fig. 3(e)]. Melanomacrophage centres were commonly found in the head kidney [Fig. 3(a), (b)], and were typically located near vessels, although they were

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(b)

h

v

s

(c) (d)

lt

v

h v v h v v h

(e)

h v v

FIG. 3. The lymphopoietic foci in the head kidney (1 year-old Epinephelus malabaricus). (a) Low magnification of lymphopoietic foci in the head kidney. The lymphopoietic foci (darker region) around vessels are visible. Melanomacrophage centres (MMC) ( ) can be seen. The black square outlines the region that is enlarged in (b). Haematoxylin and eosin stain, bar ¼ 500 mm. (b) High magnification of an MMC in the head kidney. Lymphopoietic tissue (lt), sinusoid (s), vessel (v), melanomacrophage centre ( ) and nearby haematopoietic tissue (h) are indicated. Haematoxylin and eosin stain, bar ¼ 50 mm. (c), (d), (e) The histochemical staining of AcP, BG and NBE in lymphopoietic foci. The vessels (v), haematopoietic tissue (h), enzyme reactive cells (darker spots) and melanomacrophage centre ( ) are shown. Haematoxylin as counterstain, bar (c), (e) ¼ 100 mm, bar (d) ¼ 1 mm.

occasionally observed in the parenchyma. Histological examination revealed the MMC to be composed mainly of macrophages, whose cytoplasm was filled with yellow-brick phagocytized debris [Fig. 3(b)]. Although the patterns of AcP, BG and NBE enzyme staining were similar to those found in the thymus, the histological characteristics of MMC in the head kidney differed slightly from the thymus. Specifically, thymus MMC was more enriched in melanin pigment. Both renal and haematopoietic structures were histologically apparent in the trunk kidney. Haematopoietic tissue was located in the interstitial region

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surrounding bundles of renal tubules [Fig. 4(a)]. Within this region were haematopoietic cells, macrophages, granular leukocytes, lymphocytes and fibroblastlike cells. The cells in these haematopoietic foci were randomly distributed, with no discernable specialized histological architecture observed. Similarly, randomly distributed staining patterns were revealed for AcP [Fig. 4(b)], BG [Fig. 4(c)] and NBE [Fig. 4(d)]. The reactive signals of enzyme histochemistry were also observed around the renal tubules of the trunk kidney. A comparison of these results with haematoxylin and eosin stains conducted in parallel samples suggested that the signals around the tubule peripheries were a false positive reaction. The MMC is a common structure in trunk kidneys [Fig. 4(a)], with the majority being located on the edge of the haematopoietic foci, although some were located near vessels. Both morphological and enzyme histochemical characteristics of MMC were similar to those found in the head kidney.

SP L E E N

The spleen was filled with spongy cellular reticulum composed of endothelialreticular cells and erythrocytes (the red pulp). A region of aggregated leukocytes (the white pulp) was apparent near the vessels. The primitive morphology and

(a) (b)

v

v

(c)

(d)

v v

FIG. 4. The haematopoietic foci of the trunk kidney (1 year-old Epinephelus malabaricus). (a) An image of haematopoietic foci in the trunk kidney. A blood vessel (v) is shown in the centre of the haematopoietic foci (darker region). The melanomacrophage centres between the haematopoietic foci and renal tubules are shown ( ). Haematoxylin and eosin stain, bar ¼ 100 mm. (b)­(d) The histochemical staining of AcP, BG and NBE in haematopoietic foci in the trunk kidney. The boundary of haematopoietic foci (`white curve'), melanomacrophage centres ( ), vessels (v) and enzyme reactive cells (darker spots) are shown. Haematoxylin as counterstain, bar ¼ 100 mm.

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scanty cellular involvement of the leukocytes was evident [Fig. 5(a)]. The possibility that the white pulp functioned as the primitive germinal centre in the Malabar grouper was assessed by enzyme histochemistry. Acid phosphatase was condensed in the pulp [Fig. 5(b)]. No condensation was noted for BG [Fig. 5(c)] or NBE [Fig. 5(d)] reactive cells. In addition, the typical arteriole structure made of BG and NBE reactive cells as was found in the mouse spleen, that mature B and T lymphocytes formed an annular structure on the periphery of white pulp, was not observed. Melanomacrophage centres were often seen in the spleen sections, and their distribution was highly correlated with arteries [Fig. 5(a)]. Both morphological and enzyme histochemical characteristics of MMC in spleen samples were similar to the pattern observed in the kidneys.

I N T ES T I N E S

Morphological indications of intestinal involvement in the immune system, such as leukocyte aggregation in the lamina propria (Peyer's Patches-like structure) or the gut-associated lymphoid tissue (GALT) were not evident in the proximal, middle [Fig. 6(a)] or distal regions of the intestine. Enzyme histochemical analysis also failed to reveal aggregates. Singly-distributed AcP and BG positive leukocytes cells were seen in the mucosa [Fig. 6(b)] and some regions of lamina propria [Fig. 6(c)]. A few NBE positive cells were found in

(a) (b)

v a

(c) (d)

a v a a

a v a a

a

v

a a

FIG. 5. The spleen of 1 year-old Epinephelus malabaricus. (a) The blood vascular system and the adjacent region. The structure of arterioles (a) and venules (v) is shown. , melanomacrophage centres (MMC). The white pulp (darker area near the vessels) can be seen. Haematoxylin and eosin stain, bar ¼ 100 mm. (b)­(d) The enzyme histochemistry of AcP, BG and NBE in the spleen. MMC ( ), venules (v), arteioles (a) and enzyme reactive cells (dark spots) are shown. Haematoxylin as counterstain, bar ¼ 500 mm.

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(a)

(b)

lu

(c)

(d)

lu

lp lp

FIG. 6. The intestine (middle part) of a 1 year-old Epinephelus malabaricus. (a) A cross-section view of the intestine. Haematoxylin and eosin stain, bar ¼ 1 mm. (b) The enzyme histochemistry of AcP. Enzyme reactive cells are visible ( ). The non-specific stain in the lumen (lu) is `heavy', bar ¼ 100 mm. (c) The histochemical staining of BG. Enzyme reactive cells are visible ( ) lp, lamina propria. Haematoxylin as counterstain, bar ¼ 10 mm. (d) The histochemical staining of NBE in the distal rectal gut of the grouper. The non-specific stain in lumen (lu) is `heavy'. lp, lamina propria. Haematoxylin as counterstain, bar ¼ 10 mm.

the lamina propria, but none were evident in the mucosa [Fig. 6(d)]. The MMC was often observed at the base of villi; morphological and enzyme histochemistry patterns were similar to those found in head and truck kidneys and the spleen. DISCUSSION The thymus, kidney and spleen are regarded as being the major immune organs in fishes, albeit with slightly variant roles between species. In a preliminary study using RT-PCR to analyse the mRNA expression of immune related genes, the thymus and the head and trunk kidneys of the Malabar grouper expressed rag-1 (recombination activating gene 1), a gene that is a marker of an earlier lymphopoiesis stage expression, but the gene was not expressed in the spleen (unpubl. data). These observations provide evidence that the involvement of the spleen in the whole immune system might be limited. The present study provides histological observations, which differ from those obtained with O. mykiss (Hansen, 1997). Compared to the degeneration of the spleen, the head kidney in the Malabar grouper seems to be more specialized. This finding could help to define the role that various organs like the spleen and kidney play in the immune system, and so provide the groundwork for subsequent studies.

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The MMC is thought to be a scavenger structure in lower vertebrates, but its role in the immune system is equivocal. Immunization to foreign pathogens correlates with the number or size change of MMC both dependently (Herraez & Zapata, 1986) and independently (Ellis et al., 1976; Herraez & Zapata, 1987; Tsujii & Seno, 1990). The suggestion that MMCs are primitive germinal centres (Agius, 1980; Lamers & de Haas, 1985) is not supported by the findings of an ultrastructural analysis (Herraez & Zapata, 1991), which found that the core of MMC contained degenerate macrophages, not immature lymphocytes. The chronological correlation between pathogen inoculation, the time of antigen condensation in the MMC, and the time of rise in the adaptive immune system has not been established. Antigen trapping in MMC of Carassius auratus (L.) appears as late as the time of immunoglobulin decrease in the blood (Herraez & Zapata, 1987). The appearance of MMCs in immune organs, and the presence of both antigen presenting cells and leukocytes in MMCs (Agius, 1980; Press et al., 1994; Abelli et al., 1996; Scapigliati et al., 1996; Fournier-Betz et al., 2000), however, strongly suggests that MMCs play a role in the immune system. The MMC present in the thymus of teleosts have been rarely described (Gorgollon, 1983). Several lines of evidence are consistent with an immunologically functional role of MMC in the thymus. Most of the MMCs are located in the medulla and cortex boundary (the region thought to be a transient site of thymocyte maturation), with both macrophages and lymphocytes being condensed nearby. Furthermore, the MMC appearance in the thymus is observed not only in the Malabar grouper, but in Sicyases sanguineus Muller & Troschel ¨ (Gorgollon, 1983). While these two species are distantly related (as is confirmed histologically), they occupy a similar ecological niche. The appearance of MMC in the thymus might thus be the result of analogous evolution due to the natural selective force of the coral-reef ecosystem (Paine & Palmer, 1978; Heemstra & Randall, 1993). In situ hybridization, immunohistochemistry (IHC) and enzyme histochemistry are commonly used to detect specific cell types. For comparisons based on target gene cloning and the production of anti-specific immune relative proteins antibodies, the preparations before experiments are less necessary when enzyme histochemistry is utilized. The histological characterization of a novel species such as the Malabar grouper can thus provide meaningful data for future comparative studies. In addition, the fact that some of these enzyme reactions have been conserved in evolution will help to integrate the current data with information gleaned from diverse species. Such a comparison reveals similarities and differences. Using the same method, the distribution patterns of enzyme reactive cells in thymus noted presently in E. malabaricus is different from I. punctatus (Petrie-Hanson & Ainsworth, 2000). The spleen is also different between these two species; the arteriole circulate structure made of BG and NBE reactive cells shown in I. punctatus is not present in the Malabar grouper, consistent with the idea of a functional role of the white pulp of the spleen in I. punctatus. The functional difference could be intensified when using the same method. The specificity and accuracy of enzyme histochemistry is a recognized concern. Indeed, all three kinds of enzyme reaction are incapable of identifying the single specific cell type. Furthermore, the accuracy in this system was hard to

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assess rigorously in the absence of a convenient and specific lymphocytic cell line as a control. The BG reactive cells representative of lymphocytes in blood, however, could be confirmed in a preliminary examination of smears (unpubl. data). The suitably of the NBE enzyme reaction for examination of the intestine was a point of specific debate. T lymphocytes can be observed in the intestinal mucosa of D. labrax (Abelli et al., 1997) using IHC and in I. punctatus using NBE enzyme histochemistry (Hebert et al., 2002). It was not possible, however, to achieve similar results in this system based on NBE reactive cells. Thus, the identity of NBE reactive cells as T lymphocytes requires further investigation.

The National Science Council, Taipei, Taiwan, Republic of China funded this research.

References

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Histology and histochemical enzymestaining patterns of major immune organs in Epinephelus malabaricus