Mast cell heterogeneity between two different species of Hoplias sp. (Characiformes: Erythrinidae):...

Fish & Shellfish Immunology 22 (2007) 218e229www.elsevier.com/locate/fsi

Mast cell heterogeneity between two different species ofHoplias sp. (Characiformes: Erythrinidae): Response

to fixatives, anatomical distribution, histochemicalcontents and ultrastructural features

Juliana Silva Rocha, Helio Chiarini-Garcia*

Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais,

Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil

Received 20 February 2006; revised 24 March 2006; accepted 8 May 2006

Available online 20 May 2006

Abstract

Mast cells from two erythrinid species: Hoplias malabaricus and Hoplias lacerdae, were studied in several tissues throughoutthe body using light and electron microscopy. Mast cells were found in all organs studied, but were especially abundant in the gas-trointestinal tract, and were always in association with connective tissue. These cells showed different characteristics between thetwo species studied, like varied morphology, anatomical distribution, density, basophilic/eosinophilic staining and heparin content.In H. malabaricus, the tissues fixed with Helly’s solution contained mast cells that were basophilic, metachromatic and had heparinin their cytoplasmic granules, while the tissues fixed with Karnovsky’s solution contained eosinophilic and orthochromatic mastcells in which heparin was not detected. In H. lacerdae, the use of both fixatives resulted in mast cells that were eosinophilic,orthochromatic, with no identifiable heparin content. Exclusively in H. malabaricus oesophagus, the mast cells were additionallyseen among the epithelial cells. The ultrastructural studies performed in hindgut fixed with Karnovsky’s solution revealed that thecytoplasmic granules seen in H. lacerdae mast cells were better preserved than in H. malabaricus mast cells. The latter had electron-lucent granules that were often merged, forming channels. The present study demonstrated that mast cells from two speciesbelonging to the same genus or even mast cells from the same species but under different fixatives can present heterogeneouscharacteristics, possibly due to their functional properties or to their sensitivity to fixatives.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Mast cell; Basophilic granular cell; Eosinophilic granular cell; Heterogeneity; Fixation; Hoplias sp.; Erythrinid; Teleost

1. Introduction

Mast cells are found mainly in the connective tissue distributed throughout the body, and are more frequently foundin regions that are exposed to the external environment. In these locations, in tegumentary, respiratory, urinary, repro-ductive and gastrointestinal systems, they are strategically positioned as effectors cells, with both positive and

* Corresponding author. Tel.: þ55 31 3499 2807; fax: þ55 31 3499 2780.

E-mail address: [email protected] (H. Chiarini-Garcia).

1050-4648/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.fsi.2006.05.002

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219J.S. Rocha, H. Chiarini-Garcia / Fish & Shellfish Immunology 22 (2007) 218e229

negative effects in many pathologic processes such as defence against parasitic and microbial infections and foodhypersensitivity [1,2].

Mast cells have been observed in all vertebrates, like fish [3e6], amphibians [7], reptiles [8], birds [9] andmammals [10,11]. However, there is some controversy in the literature about their existence in fish. Several reportsdescribe a great amount of granular cells in varied fish families, like salmonids [12,13], cyprinids [14,15] anderythrinids [4,16]. On the other hand, some authors did not find any granular cells in the fish species studied[6,17] or observed only a few granular cells [18,19]. Review papers on mast cells in fish [20,21] have shownthat fish mast cells seem to constitute a heterogeneous cell group. In teleosts, granular cells present heterogeneousmorphology, granular content, sensitivity to fixatives and response to drugs. Concerning this heterogeneity, one ofthe most controversial aspects is related to the staining properties of the mast cell cytoplasmic granules that arefrequently described either as basophilic or eosinophilic. Because of this, different authors have referred tothem in different species as: mast cells, basophilic granular cells or acidophilic/eosinophilic granular cells [reviewin 9,20,22].

Previous studies have shown that in different teleostean species, the mast cells are usually heterogeneous cells,although in two families, salmonids and labrids, the similarities among species were striking [5,23]. For instance,while in labrids the mast cell granules were predominantly eosinophilic; in salmonids these structures seemed to bepredominantly basophilic [5]. This is probably because in fish, the mast cell granules present both eosinophilic andbasophilic contents but one type can be more easily detected than the other depending on the fish family [5,23].Nevertheless, the present study shows that these similarities cannot be seen in all teleostean families since inerythrinids the mast cells of two species belonging to the genus Hoplias (H. malabaricus and H. lacerdae)were heterogeneous regarding their morphology, anatomical position, density, staining properties and granularcontents, as reported herein.

2. Materials and methods

2.1. Tissue collection

Eleven specimens of Hoplias sp. (Characiformes, Erythrinidae) were used: four H. malabaricus (Bloch, 1794),and seven H. lacerdae (Ribeiro, 1908). Sexually mature fish (0.2e1.0 kg of body weight) were caught in Brazilianlakes (Furnas lake and Federal University of Vicosa lake, both in Minas Gerais State) using gill nets and kept ina 500-L tank with aerated water under controlled temperature (26e28 �C). Animals were killed by a sharp blowon the head, followed by the collection of parts of the gastrointestinal tract (oesophagus, stomach, liver, foregut,pyloric caeca, midgut and hindgut), as well as of the skin, gills, spleen, kidney, ovaries and testes. Fish werefasted for two days before the sampling. The animals looked healthy and had no cutaneous wounds or intestinalworms.

2.2. Light microscopy

The tissues were immersed in two different fixative solutions: (1) modified Karnovsky’s fixative (2.5% glutaralde-hyde 2% paraformaldehyde in 0.1 M cacodylate buffer pH 7.2) [24] for 20 h at 4 �C, and (2) Helly’s solution for 20 hat room temperature. All tissue samples were embedded in glycol methacrylate (Historesin, Leica), according to themanufacturer’s instructions.

The following histological/histochemical techniques were carried out in 2e4 mm thick sections:

(A) Staining with 0.5% toluidine blue O containing 1% sodium borate for histological analysis.(B) Haematoxylin and eosin staining for identification of basophilic and eosinophilic contents.(C) Reaction with 1% toluidine blue O at pH 1.5 (hydrochloride/phosphate buffer) and pH 3.0 (citrate/phosphate

buffer) for identification of sulphated and carboxylated glycosaminoglycan contents, respectively.(D) Berberine sulphate (Sigma) reaction at pH 3.5 (0.02% in water acidified with 0.1 M citric acid) for identifica-

tion of heparin content, which is confirmed by yellow fluorescence using 420e480 nm filter [25].

220 J.S. Rocha, H. Chiarini-Garcia / Fish & Shellfish Immunology 22 (2007) 218e229

2.3. Electron microscopy

Fragments of the hindgut were fixed by immersion in modified Karnovsky’s fixative for 20 h at 4 �C. Tissues werepost-fixed in reduced osmium tetroxide [26] for 1.5 h, dehydrated in a graded series of ethanol and embedded in Epon812 (Polysciences). Ultrathin sections were stained using uranyl acetate and lead citrate, and examined in a ZeissEM-10 electron microscope.

The tissues used for the scanning electron microscopy (SEM) studies were from the same animals used for TEM.After fixation in modified Karnovsky’s solution, the samples were post-fixed in three sequential baths of osmiumtetroxide alternated with tannic acid and thiosemicarbazide baths. Then, the tissue fragments were dehydrated ina graded series of ethanol, fractured in liquid nitrogen, dried in a critical point-CO2 dryer (CPD-020, Balzers),mounted on SEM stubs and sputter-coated (BSV-203 unit of the BAF-300 equipment, Balzers). Finally, the gold-coated fragments were examined using a Zeiss DSM-950 scanning electron microscope.

3. Results

3.1. Anatomical distribution

Mast cells/EGC were found in skin, gills, liver, spleen, kidney and gonads, always in association with the connectivetissue encapsulating the organs and extending inwards in the organs and reaching their parenchyma, especially in thevicinity of blood vessels. The anatomical distribution of the mast cells/EGC was apparently the same, independently ofthe fish weight. Examples of sites where mast cells were found are in the secondary lamellae of the gills, in the thin septasurrounding the oocytes in the ovaries and in the interstitial tissue around the seminiferous tubules in the testes. In all theorgans studied, with the exception of the upper gastrointestinal tract, the mast cells presented a similar pattern ofdistribution in both species of erythrinids, although they were apparently in higher density in H. malabaricus.

In the gastrointestinal tract of both species, there was a gradual increase in the density of mast cells from theoesophagus to the hindgut. However, the mast cells appeared more numerous in H. malabaricus in all regions ofthe gastrointestinal tract when compared to H. lacerdae. They were seen mainly in the lamina propria and submucosalayers and were less frequently seen between the circular and the longitudinal smooth muscle layer of the intestine andin the serosa. The most striking difference in the apparent number of mast cells between the two species was seen inthe pyloric caeca. While the submucosa of the pyloric caeca of H. malabaricus was full of mast cells, they were rare inthis region in H. lacerdae.

Moreover, there were clear differences between species related to the distribution of mast cells in the oesophagusand in the stomach. The oesophagus of H. malabaricus contained intraepithelial mast cells (Fig. 1A), but these wereabsent in H. lacerdae (Fig. 1B). The intraepithelial mast cells could be seen scattered within the stratified epitheliumof the oesophagus, from the basal layer to the area close to the lumen. In the stomach, the difference between the twospecies consisted of a distinct distribution of the mast cells in the submucosa. In H. malabaricus the mast cells weremore numerous in two regions: (1) near the base of the gastric glands and (2) near the circular smooth muscle layer(Fig. 1C). This preferential distribution of mast cells in two regions was not observed in H. lacerdae, in which mastcells were seen uniformly distributed throughout the submucosa in the stomach (Fig. 1D).

3.2. Effects of fixatives

The basophilic and/or eosinophilic staining of mast cell cytoplasmic granules was very heterogeneous dependingon the type of the fixative used, the anatomical position of the cells and the species of erythrinid considered. In thelamina propria (loose connective tissue just beneath the epithelium and inside the intestinal villi) of H. malabaricus,the fixation with Helly’s solution rendered the mast cells basophilic after H&E staining (Fig. 2A). On the other hand,with respect to the same intestinal region in the same species, but after fixation with Karnovsky’s solution, the mastcells were eosinophilic (Fig. 2B). Interestingly, in the tissues fixed using Helly’s solution, the submucosa (dense con-nective tissue below the lamina propria and adjacent to the muscular layer) had mainly eosinophilic mast cells. Theremaining areas in that layer contained basophilic as well as eosinophilic mast cells and some mast cells containeda combination of eosinophilic and basophilic granules (Fig. 2C). Contrary to what was found in H. malabaricus,mast cells in H. lacerdae were always eosinophilic, despite the fixation procedure. However, in H. lacerdae, the


mast cells in the tissues fixed with Helly’s solution were weakly eosinophilic (Fig. 2D) when compared with thosefrom tissues fixed with Karnovsky’s solution (Fig. 2E).

Concerning the morphological preservation under light microscopy, the mast cells from H. malabaricus were wellpreserved after Helly’s or Karnovsky’s fixation, since they presented well-delimited cell borders and easily identifi-able cytoplasmic granules (Fig. 2A,B). On the contrary, in H. lacerdae, the mast cells had irregular shape and poorlyvisible cytoplasmic granules after using either fixative (Fig. 2C,D).

3.3. Histochemical contents

The cytoplasmic granules present in mast cells from all organs studied, except the intestines, showed the same his-tochemical pattern, within the species studied and the fixatives used. H. malabaricus mast cells were metachromatic

Fig. 1. Oesophagus and stomach of H. malabaricus (A,C) and H. lacerdae (B,D) after Helly fixation and stained by toluidine blueesodium borate.

Note that in H. malabaricus oesophagus (A), the mast cells (arrow) are in the lamina propria (LP) and also inside the epithelium (E) and in the

lumen (arrowhead). In the H. lacerdae (B), the mast cells (arrow) are seen only in the lamina propria (LP). In the stomach of H. malabaricus (C)

the mast cells are concentrated mainly in two regions of the submucosa (SM): just below the mucosa layer (Mu) and above the muscular layer (M).

In the stomach of H. lacerdae (D), the mast cells are randomly distributed thorough the connective tissue of submucosa. E, epithelium. Bars

represent 50 mm (A,B) and 250 mm (C,D).


Fig. 2. Mast cells of the intestine of erythrinids stained by haematoxylineeosin, comparing their basophilic and eosinophilic properties in be-

tween species (H. malabaricus and H. lacerdae) and after different fixation (Helly and Karnovsky solution). In H. malabaricus, the mast cells

present in the lamina propria were basophilic (arrows) after Helly’s fixation (A) and eosinophilic (arrows) after Karnovsky’s (B). However, in

the submucosa (SM) after Helly’s fixation (C) they were basophilic (Ba), eosinophilic (Eo) or a mixture of staining (arrow). In H. lacerdae,

the mast cells (arrow) were not well preserved and were weakly eosinophilic either after Helly (D) or Karnovsky (E) fixation. E, intestinal

epithelium. Bars represent 15 mm (AeC) and 20 mm (D,E).


under toluidine blue, pH 1.5 and 3.0, and yellow fluorescent under berberine treatment, all these after Helly’s fixation.However, after Karnovsky’s fixation, they were metachromatic only after toluidine pH 3.0 treatment and containedgreen fluorescent granules after berberine reaction. In H. lacerdae, with both fixatives, the mast cells did not reactto toluidine blue pH 1.5 and were weak to moderately metachromatic with toluidine blue pH 3.0. Additionally,they were green fluorescent after berberine treatment.

Specifically in the intestines, the mast cells presented some heterogeneity concerning the glycosaminoglycan con-tent in their cytoplasmic granules. In H. malabaricus, after fixation with Helly’s solution, mast cells in the laminapropria showed strong metachromasia after reaction with toluidine blue pH 1.5 and presented bright yellow fluores-cent cytoplasmic granules after treatment with berberine (Fig. 3A), indicating sulphated glycosaminoglycan andheparin content, respectively. However, in the submucosa, below the lamina propria, the mast cells presented moder-ate metachromasia under toluidine blue pH 1.5 and faint yellow fluorescence after berberine reaction. Conversely,when intestinal fragments from H. malabaricus were fixed with Karnovsky’s solution, the mast cell cytoplasmicgranules showed extreme variation after toluidine blue pH 1.5 and berberine treatments. Mast cells ranged fromnot reactive at all to strongly metachromatic and yellow fluorescent.

In the intestines of the other species, H. lacerdae, all mast cells were orthochromatic after toluidine blue pH 1.5reaction with Helly’s fixation and were either not stained or orthochromatic in the tissues fixed with Karnovsky’ssolution. Despite the fixative used, mast cells were overall negative for the reaction with berberine sulphate(Fig. 3B), although a few cells presented some faintly positive granules.

Independently of the species, of the tissue sample or the fixative used, mast cell contents ranged from moderate tostrongly metachromatic after toluidine blue pH 3.0 reaction.

3.4. Ultrastructural features

The hindgut of both species was analysed using TEM. The fibroblasts present in the connective tissues showedclassical ultrastructural features such as moderately condensed and elongated nuclei, and cytoplasm containing sparseorganelles. Fine tapering fibroblast cytoplasmic extensions could be seen attached by desmosomes forming the typicalmeshwork of this supporting tissue.

The mast cells were concentrated mainly in the lamina propria (Fig. 4A and B) and appeared in smaller numbersbetween the inner circular and outer longitudinal smooth muscle layers and in the loose connective tissue of the serosa.The array of the surrounding cells in the tissue determined the mast cell shape. They were elongated in the region closeto the epithelium, where the bundles of collagen fibres were compactly disposed. On the other hand, they were oval orround in other regions in which the collagen fibres were loosely distributed.

The mast cell nuclei were frequently oval and eccentrically located in the cells. The nucleus presented some het-erochromatin, most of the times distributed along the inner surface of the nuclear envelope. The cytoplasm contained

Fig. 3. Fluorescence reaction to berberine sulphate specific for identifying heparin. In the intestine H. malabaricus (A), the mast cells showed

strong yellow fluorescence (arrows), mainly in the lamina propria. On the other hand, in H. lacerdae (B) the mast cells either present rare

weak yellow fluorescence granules (arrows) or they were green fluorescent (arrowheads), indicating absence of heparin. Bars represent 100 mm.


numerous cytoplasmic granules, that were pressing the nuclear envelope and creating dents in it, while other organ-elles were scarce (Fig. 5A and B).

Mast cells were seen in contact with other mast cells and with fibroblasts, and occasionally, some portions of thecytoplasmic membrane were seen in direct contact with the collagen fibres (Fig. 5C). No basal membrane was seenembracing the mast cells. Virtually all mast cells present in any layer of this organ were seen surrounded by the del-icate cytoplasmic processes of the fibroblasts (Fig. 5D and E). The majority of mast cells seen presented a portion ofthe cytoplasm devoid of granules. This portion was usually located at one pole of the cell and possessed no or rarecytoplasmic granules and few other organelles (Fig. 5D and E).

Some of the ultrastructural features of the mast cells were different when the two species of erythrinids werecompared. The cytoplasmic granules were weakly electron-dense in H. lacerdae (Figs. 4A and 5A, D) and almosttotally electron-lucent in H. malabaricus (Figs. 4B and 5B, E). Using SEM analysis, the intra-granular contentcould be seen with compact or sponge-like appearance (Fig. 5F). Granule fusions were observed in both species,however, they were more frequent in H. malabaricus. Sometimes the granules would form long cytoplasmic chan-nels (Fig. 5G). In H. malabaricus, besides the granule fusions, a granule was seen protruding into another granulenext to it (Fig. 5E).

Fig. 4. Electron micrographs of H. lacerdae (A) and H. malabaricus (B) intestine, after fixation by Karnovsky solution. The mast cells (Arabic

numbers) are seen in the lamina propria (LP), just below the intestinal epithelium (E), between collagen fibrils and cytoplasmic branches of

fibroblasts (arrows). Bars represent 3 mm.

Fig. 5. In A, mast cell of H. lacerdae showing cytoplasmic granules with low electron density content and some granule fusion and in B, mast cellof H. malabaricus, showing electron-lucent cytoplasmic granules and the granule fusions are more frequent. (C) Collagen fibrils can be seen indirect contact with mast cells, depressing their cytoplasmic membranes (arrows) and note absence of basal lamina. In both species, H. lacerdae (D)and H. malabaricus (E), the mast cells presented cytoplasmic projections (Pl) devoid of granules and their cellular surface covered by narrowcytoplasmic process of fibroblasts (arrows). Specifically in H. malabaricus (E), can be seen projections of the mast cell granules inside other gran-ules (arrowheads). In F, details under scanning electron microscopy of the cytoplasm of a fractured mast cell from H. lacerdae showing partiallyempty (arrows) and compact filled (arrowheads) cytoplasmic granules. The presence of intra-cytoplasmic channels (G) due to granule fusion wasfrequent in H. malabaricus. Nu, nucleus; C, collagen fibrils. Bars represent 1 mm, except C that represents 0.5 mm.


4. Discussion

Despite the broad distribution of mast cells throughout the connective tissue in both species studied, these cellswere in general not so abundant in the organs collected, with exception of the gastrointestinal tract, in which the num-ber of mast cells increased progressively from oesophagus to hindgut, as reported previously for salmonids [27], andfor teleosteans in general [28]. Exclusively in H. malabaricus oesophagus, mast cells were seen among the epithelialcells, as described previously in human gingiva [29], in rat jejunum [30] and in intestinal epithelia of the teleosteansfrom the labrid family [6]. It has been suggested that a higher number of mast cells in the connective tissues of theintestinal tract, as well as the presence of intraepithelial mast cells, are related to parasitic infection. The recruitmentof mast cells has been described in different teleostean species, in different organs, as a regular event in persistentinflammatory reactions [21]. In mammals, it has been demonstrated that the mast cells could contribute to many path-ophysiological processes such as allergic and non-allergic inflammatory processes [31]. Recently it was shown thatthe mast cells are also important effector cells for host defence against bacterial [32] and certain viral pathogens[33]. Interestingly, we did not find any sign of parasitic infection or other inflammatory processes in H. malabaricus,which suggests that mast cells could be a normal constituent of the oesophagus epithelium in this species. Neverthe-less, we are not aware of the physiological meaning of these intraepithelial mast cells exclusively in H. malabaricusand why these cells were not seen in the oesophagus of any H. lacerdae here studied.

The heterogeneity observed in mammalian mast cells, which is widely described in the literature [2], is probablydue, at least in part, to the nature of the chemical constituents of the cytoplasmic granules and their preservation bydifferent fixative solutions. Several authors consider fixation a critical step in the evaluation of fish mast cells[3,20,27,34]. Fish mast cells are better preserved with alcoholic fixative and staining solutions (not watery)[20,35] as well as by coagulating fixative solutions, like Helly’s solution [20,28]. In an extensive review of granularcells in fish, Reite [20] explored the controversy in the literature about the existence of mast cells in fish and con-cluded that the nature of the fixative used and of the staining solutions applied are determinant in preserving thesecells and that watery solutions should be avoided. Additionally, Reite [20] assumes that fixative solutions containingmercury dichloride as, for instance, Helly’s solution, make the granular content of these cells more resistant to his-tological processing. In fact, previous studies [4,34] confirmed Helly’s solution as a good fixative for mast cells in fish.The same was true for the present study, although curiously only in one of the erythrinid species studied eH. malabaricus.

One unexpected finding of the present study was the demonstration of the presence of heparin in the mast cell gran-ules of only one of the species studied, H. malabaricus, and exclusively when the fixative used was Helly’s solution.We could not detect heparin in the mast cells of H. lacerdae, despite the fixative used, or in mast cell granules ofH. malabaricus, in tissues fixed with Karnovsky’s solution. There is some controversy about the presence of heparinin fish granule cells, which is reported to be absent in some cases [7] and in other cases, to be present [4,36]. Our studyprovides some clues that the fixation is a really important step because in the same species, H. malabaricus, the hep-arin was detected or not, depending on the fixative solution used. The apparent absence of heparin in mast cells ofH. lacerdae may represent a higher susceptibility of its mast cells to both fixatives used or that the proteoglycan con-tent is other than heparin, and it was not detected by the berberine method applied. In mammals, for instance, the mastcells in the connective tissues throughout the body have heparin in their cytoplasmic granules while those present inthe intestinal mucosa contain little or no heparin [37].

There are several reports describing in the literature describing granular cells in fish, and even today there is someconfusion about the occurrence of mast cells in this group of vertebrates. Some granular cells are referred to as PAS-positive cells [38e40], acidophilic or eosinophilic granule cells (EGC) [3,12,16,41e44] and as mast cells[3,4,20,27,45e47]. The EGCs are reported to be similar to mast cells because of their morphology and location inconnective tissues and their degranulation in response to compound 48/80 [12] and substance P [48]. Regardingthe terms EGC or mast cell in fish, authors usually base their nomenclature on the observation whether the granularcell stains with eosin (acid dye), so then they call it EGC or with haematoxylin (basic dye) and the granular cell isreferred to as mast cell. In the present study we observed that in H. malabaricus, the mast cells were basophilicwhen the tissues were fixed with Helly’s solution and eosinophilic when the method of fixation was Karnovsky’s so-lution. Of note, it seems evident that it was the Helly’s solution that preserved the mast cell heparin content in thatspecies. This demonstrates that the same type of cell can be eosinophilic or basophilic depending on the fixative so-lution utilized. Former studies have reported similar results in teleostean mast cells, which stained basophilically after


fixation with alcoholic solutions and eosinophilically after the use of watery solutions [23,35]. Additionally, in thetissues from H. malabaricus fixed with Helly’s solution, we found mast cells with heterogeneous staining character-istics. The cells in the lamina propria were strongly basophilic while the cells present in the submucosa ranged frombasophilic to eosinophilic, with intermediate colours. This finding raises the following possibilities: (1) the polyanioncontent of the mast cells is different and, because of that, it was diversely preserved or (2) there are different types ofmast cells in this species; (3) the lamina propria and the submucosa represent two distinct microenvironments influ-encing the functions of the mast cells and consequently altering their morphology and granular content, or (4) thereare, in the same area of connective tissue, true basophilic mast cells coexisting with eosinophilic granulocytes thatmigrated from the blood. It is known for mammals that the molecules released by the mast cells influence the gastro-intestinal secretion, absorption, and motility through paracrine effects [49]. However, these effects are differentdepending on the location of these cells since there are different morphological and functional mast cell subtypesin the gastrointestinal wall [50]. In fact, it was demonstrated in mice that if mast cells from the connective tissueare transplanted to a mucosal region of the intestine, they change their morphology, granular content and functionto a mucosal-like mast cell, the same happens in the opposite direction [51]. Because in H. malabaricus the presenceof mast cells containing eosinophilic or basophilic granules was directly dependent on the mucosal region, it ispossible that the microenvironment has influence on the mast cell phenotype.

At the ultrastructural level, the mast cells from both species studied showed cytoplasmic granules with low electrondensity associated with a finely granulated electron-lucent matrix, as opposed to the normally higher electron densityobserved in mammals [52] and in some teleostean species [12,48]. In H. malabaricus, the granules seemed practicallyempty and were merging, forming cytoplasmic channels. Similar findings were described in rainbow trout after intra-peritoneal injections of Aeromonas salmonicida extracellular products, Compound 48/80 and Concanavalin A. Thesemolecules promote degranulation of the eosinophilic granule cells, and the fusion of membranes seen between adja-cent granules led to the formation of cytoplasmic vacuoles, labyrinthine channels and extrusion of debris [12].Although the H. malabaricus mast cells presented cytoplasmic vacuoles forming labyrinthine channels, no openpores and extracellular debris were observed. It is established that the presence of large electron-lucent cytoplasmicvacuoles is suggestive of exocytosis after mast cell stimulation, although partial degranulation can also occur in non-stimulated cells or in stimulated cells not showing signs of cellular rupture and granular extrusion [48]. Our findings inH. malabaricus suggest that the intestinal mast cells could be effector cells in some particular physiological processesin this species, even though the mast cells had no classical degranulation appearance. Under TEM analysis, mast cellsfrom H. lacerdae proved once more to be morphologically different from the ones in H. malabaricus. The H. lacerdaemast cells presented a more homogeneous and electron-dense granular matrix, additionally, cytoplasmic vacuoleswere not frequently seen. The implications of these differences between these two species of erythrinids cannot beexplained by the methods here applied.

In both erythrinids studied, mast cells were not involved by a basal membrane, had no microplicae and presenteda few long cytoplasmic processes depleted of organelles. These characteristics, taken together with the absence offormation of cellular junctions with the neighbouring cells, suggest that the mast cells are mobile cells in the connec-tive tissues of the intestine. Moreover, a clear and tight apposition of cellular membranes was observed between mastcells and fibroblast processes. The same was reported previously in rainbow trout eosinophilic cells [42,53], and thesefibroblasts were named as ‘‘ensheathing cells’’. Nevertheless, even though in our study the mast cells are in contactwith long fibroblast extensions, we do not believe that they are completely enclosed by fibroblast processes. In fact, ithas been described in rainbow trout that eosinophilic cells can migrate into the lamina propria after injections of exo-toxins [12]. Besides the possibility that mast cells use the fibroblasts and the collagen fibrils as a support to migrate, webelieve that this cellular association could also represent some functional collaboration. It is known that fibroblasts caninfluence mast cell functions like motility and proliferation [10,54], conversely, the histamine produced by mousemast cells can stimulate fibroblasts to synthesize collagen and, additionally, mast cell number was found to beincreased in fibrosis conditions [55].

In the present study we have shown that in Hoplias sp., the mast cells were seen in all organs investigated, alwaysassociated with connective tissue and that each of the fixatives used preserved mast cells in a different manner in thespecies studied. This study reinforces the idea that the controversy encountered in the literature concerning fish mastcells may probably be due, at least in part, to the nature of the fixatives or other solutions used in the tissue processing,additionally it could also represent differences among mast cell subtypes or microenvironments. We have also shownthat although the mast cell characteristics can be very similar in the same fish family, for erythrinids this general


concept does not apply. Although the implication of this is not clear, the present study could be a good model toevaluate, by functional approaches, the biology of fish mast cells.

Acknowledgements

We wish to thank CODEVASF, Furnas Centrais Eletricas, Federal University of Vicosa and GM Alevinos forkindly providing the fish used in this study. The ultrastructural studies were developed at the Electron MicroscopyCenter (CEMEL) of the Institute of Biological Sciences at UFMG. JSR was supported by CAPES and FAPEMIGand HC-G was in part supported by CNPq.

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Mast cell heterogeneity between two different species of Hoplias sp. (Characiformes: Erythrinidae):...

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